| What is claimed is: 1 . A method for predicting the clinical sensitivity of a diffuse large B-celi lymphoma (DLBCL) to treatment with lenaiidomide comprising: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 3; (c) comparing the l evel of expression of the one, two, three, four, five or more of the genes identified in Table 3 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having a DLBCL, wherein the DLBCL in the second patient is clinically insensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof and wherein the differential expression of the one, two, three, four, five or more of the genes in the first biological sample relati ve to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the DLBCL in the first patient will be clinical sensitive to treatment with lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof. 2. A method for predicting the clinical sensitivity of a DLBCL to treatment with lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 4; (c) comparing the l evel of expression of the one, two, three, four, five or more of the genes identified in Table 4 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having a DLBCL, wherein the DLBCL in the second patient is clinically insensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and wherein the differential expression of the one, two, three, four, five or more of the genes in the first biologi cal sample relati ve to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the DLBCL in the first patient will be clinical sensitive to treatment with lenaiidomide. - 176 - 226269-228501 / 12827-501-228 SDI-60022 l07vl A method for predicting the clinical sensitivity of a DLBCL to treatment with Ienalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1 ; and (c) comparing the l evel of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having a DLBCL, wherein the DLBCL cancer in the second patient is clinically insensitive to Ienalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and wherein a higher level of expression of the one, two, three, four, five or more of the genes in the first biological sample relati ve to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the DLBCL in the first patient will be clinical sensitive to treatment with Ienahdomide, or pharmaceutically acceptable salts, solvates or isomers thereof. A method for predicting the clinical sensitivity of a DLBCL to treatment with Ienalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2; and (c) comparing the l evel of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having a DLBCL, wherein the DLBCL in the second patient is clinically insensitive to Ienalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and wherein a lower level of expression of the one, two, three, four, five or more of the genes in the first biologi cal sample relati ve to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the DLBCL in the first patient will be clinical sensitive to treatment with Ienahdomide, or pharmaceutically acceptable salts, solvates or isomers thereof. - 177 - 226269-228501 / 12827-501-228 SDI-60022 107vl A method for predicting the clinical sensitivity of a DLBCL to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising: (a) obtaining a first tumor sample from a first patient having a DLBCL; (b) measuring the proportion of dendritic ceils in the first tumor sample; and (c) comparing the proportion of dendritic cells in the first tumor sample with the proportion of dendritic cells in a second tumor sample from a second patient having a DLBCL, wherein the second patient's DLBCL is clinically insensitive to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and wherein a higher proportion of dendritic cells in the first tumor sample relative the proportion of dendritic cells in the second tumor sample indicates that the DLBCL in the first patient will be clinical sensitive to treatment with ienalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof. A methods for predicting the clinical sensitivity' of a DLBCL to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising: (a) obtaining a first tumor sample from a first patient having a DLBCL; (b) measuring the proportion of plasma cells in the first tumor sample; and (c) comparing the proportion of plasma cells in the first tumor sample with the proportion of plasma ceils in a second tumor sample from a second patient having a DLBCL, wherein the second patient's DLBCL is clinically insensitive to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and wherein a higher proportion of plasma ceils in the first tumor sample relative the proportion of plasma cells in the second tumor sample indicates that the DLBCL in the first patient will be clinical sensitive to treatment with ienalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof A method for predicting the clinical sensitivity of a DLBCL to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising: (a) obtaining a first biological sample from a first patient having a DLBCL (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1, 2, 3, or 4 in the first biological sample; and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having DLBCL which are clinically sensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having DLBCL which are clinically insensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having DLBCL which are clinically sensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, indicates that the DLBCL in the first patient will be clinical sensitive to treatment with lenaiidomide, and a gene expression profile for the genes or subset of genes in first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having DLBCL which are clinically insensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, indicates that the DLBCL of the first patient will be clinically insensitive to treatment with lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof. 8. A method for predicting the clinical sensitivity of a DLBCL to treatment with lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, comprising; (a) obtaining a first tumor sample from a first patient having a hematological cancer; (b) measuring the proportion of immune cells in the first tumor sample; and (c) comparing the proportion of the immune cel ls in the first tumor sample to (i) the proportion of the same immune cells in tumor samples from patients having DLBCL which are clinical ly sensitive to lenaiidomide, or pharmaceutical ly acceptable salts, solvates or isomers thereof, and (ii) the proportion of the same immune cells in tumor samples from patients having DLBCL which are clinically insensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, wherein a proportion of the immune cells in the first tumor sample similar to the proportion of the same immune cells in tumor samples from patients having DLBC L which are clinically sensitive to lenaiidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, indicates that the DLBCL in the first patient will be clinical sensitive to treatment - 179 - 226269-228501 / 12827-501-228 SDI-60022 107vl with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and a proportion of the immune cells in the first tumor sample similar to the proportion of the same immune cells in tumor samples from patients having DLBCL which are clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, mdicates that the DLBCL in the first patient will be clinical insensitive to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof. 9. A method for managing or treating a DLBCL comprising: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the le vel of expression of one, two, three, four, five or more of the genes identified in Table 3; (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 3 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having a DLBCL, wherein the DLBCL in the second patient is clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof! to the first patient if the one, two, three, four, five or more of the genes in the first biological sample are differentially expressed relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. 10. A method for managing or treating a DLBCL comprising: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, fi ve or more of the genes identified in Table 4; (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 4 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having a DLBCL, wherein the DLBCL, in the second patient is clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof,; and (d) administering lenalidomide, or pharmaceutical]}'' acceptable salts, solvates or isomers thereof, to the first patient if the one, two, three, four, five or more of the genes in the - 180 - 226269-228501 / 12827-501-228 SDI-60022 107vl first biological sample are differentially expressed relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, A method for managing or treating a DLBCL comprising; (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1 ; (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having a DLBCL, wherein the DLBCL in the second patient is clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, to the first patient if a higher level of expression of the one, two, three, four, five or more of the genes in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. A method for managing or treating a DLBCL comprising; (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2; (c) comparing the level of expression of the one, two, three, four, fi ve or more of the genes identified in Table 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having a DLBCL, wherein the DLBCL in the second patient is clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, to the first patient if a lower level of expression of the one, two, three, four, five or more of the genes in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. 13. A method for managing or treating a DLBCL: (a) obtaining a first biological sample from a first patient having a DLBCL; (b) measuring the expression of a certain subset of genes set forth in Table 1 , 2, 3 or 4, or any combination thereof in the first biological sample, (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profi le of the subset of genes in tumor samples from patients having DLBCL which are clinically sensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and (ii) the gene expression of the subset of genes in tumor samples from patients having DLBCL which are clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, to the first patient if: (i) the gene expression profile for the subset of genes in the first biological sample is similar to the gene expression profile for the subset of genes in tumor samples from patients having DLBCL which are clinically sensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and (ii) the gene expression profile for the subset of genes in first biological sample is not similar to the gene expression profile for the subset of genes in tumor samples from patients having DLBCL which are clinically insensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof. 14. A method for managing or treating a DLBCL comprising: (a) obtaining a first tumor sample from a first patient having a DLBCL; (b) measuring the proportion of dendritic cells in the first tumor sample; (c) comparing the proportion of dendritic ceils in the first tumor sample with the proportion of dendritic cells in a second tumor sample from a second patient having a DLBCL, wherem the second patient's DLBCL is clinically insensitive to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, to the first patient if a higher proportion of dendritic cells in the first tumor sample is measured relative the proportion of dendritic cells in the second tumor sample, - 182 - 226269-228501 / 12827-501-228 SDI-60022 107vl 15. A method for managing or treating a DLBCL comprising: (a) obtaining a first tumor sample from a first patient having a DLBCL; (b) measuring the proportion of plasma cells in the first tumor sample; (c) comparing the proportion of plasma cells in the first tumor sample with the proportion of plasma cells in a second tumor sample from a second patient having a DLBCL, wherem the second patient's DLBCL is clinically insensitive to treatment with lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, to the first patient i f a higher proportion of plasma ceils in the first tumor sample is measured relative the proportion of plasma cells in the second tumor sample. 16. A method for managing or treating a DLBCL comprising; (a) obtaining a first tumor sample from a first patient having a DLBC L; (b) measuring the proportion of immune cells in the first tumor sample; and (c) comparing the proportion of the immune cells in the first tumor sample to (i) the proportion of the same immune ceils in tumor samples from patients having DLBCL which are clinically sensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and (ii) the proportion of the same immune cells in tumor samples f om patients having DLBCL which are clinically insensitive to the lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof; and (d) administering lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, to the first patient if the proportion of the immune cells in the first tumor sample is (i) similar to the proportion of the same immune cells in tumor samples from patients having DLBCL which are clinically sensitive to lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof, and (ii) not similar to the proportion of the same immune cel ls in tumor samples from patients having DLBC L which are clinically insensitive to the lenalidomide, or pharmaceutically acceptable salts, solvates or isomers thereof. 17. The method of any one of claims 1 to 16, wherein the DLBCL is refractory. 18. The method of any one of claims 1 to 16, wherein the DLBCL is relapsed in the first patient. - 183 - 226269-228501 / 12827-501-228 SDI-60022 107vl The meihod of any one of claims 1 to 16, wherein the DLBCL is a germinal center B-cell- like subtype. The method of any one of claims 1 to 16, wherem the DLBCL is an activated B-cell-like subtype. - 184 - 226269-228501 / 12827-501-228 SDI-60022 107vl |
1. FIELD
[0Θ01] Provided herein are biomarkers for use in predicting the clinical sensitivity of hematologic cancers, such as non-Hodgkin's lymphoma, and a patient's response to treatment with an immiinomodulatory agent, such as 3-(4~amino-l-oxo-l ,3-dihydro-isoindoi-2-yi)--piperidine-2,6- dione, which is also known as lenalidomide or Revlimid®. In one aspect, provided herein are methods of treating or managing non-Hodgkin's lymphomas, including but not limited to, diffuse large B-cell lymphoma (DLBCL), using prognostic factors.
2. BACKGROUND
2.1 Pathobiology of Cancer
[0002] Cancer is characterized primarily by an increase in the number of abnormal ceils derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, or lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). Clinical data and molecular biologic studies indicate that cancer is a multistep process that begins with minor pre-neo lastic changes, which may under certain conditions progress to neoplasia. The neoplastic lesion may evolve clonally and develop an increasing capacity for invasion, growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance. Roitt, I., Brostoff, J and Kale, D., Immunology, 17.1-17.12 (3rd ed., Mosby, St. Louis, Mo., 1993).
[0003] There is an enormous variety of cancers which are described in detail in the medical literature. Examples include cancers of the lung, colon, rectum, prostate, breast, brain, blood and intestine. The incidence of cancer continues to climb as the general population ages, as new cancers develop, and as susceptible populations (e.g., people infected with AIDS or excessively exposed to sunlight) grow. However, options for the treatment of cancer are limited. For example, in the case of blood cancers (e.g., mul tiple myeloma), few treatment options are available, especially when conventional chemotherapy fails and bone-marrow transplantation is not an option. A tremendous demand therefore exists for new methods and compositions that can be used to treat patients with cancer.
[0004] Many types of cancers are associated with new blood vessel formation, a process known as angiogenesis. Several of the mechanisms involved in tumor-induced angiogenesis have been elucidated. The most direct of these mechanisms is the secretion by the tumor cells of cytokines with angiogenic properties. Examples of these cytokines include acidic and basic fibroblastic growth factor (a,b-FGF), angiogenin, vascular endothelial growth factor (VEGF), and TNF-a. Alternatively, tumor cel ls can release angiogenic peptides through the production of proteases and the subsequent breakdown of the extracellular matrix where some cytokines are stored (e.g., b-FGF). Angiogenesis can also be induced indirectly through the recruitment of inflammatory cel ls (particularly macrophages) and their subsequent release of angiogenic cytokines (e.g., TNF-a, b-FGF).
[00Θ5] Lymphoma refers to cancers that originate in the lymphatic system. Lymphoma is characterized by malignant neoplasms of lymphocytes— B lymphocytes and T lymphocytes (i.e., B- cells and T-cells). Lymphoma generally starts in lymph nodes or col lections of lymphatic tissue in organs including, but not limited to, the stomach or intestines. Lymphoma may involve the marrow and the blood in some cases. Lymphoma may spread from one site to other parts of the body.
[0006] The treatment of various forms of lymphomas are described, for example, in U.S. patent no. 7,468,363, the entirety of which is incorporated herein by reference. Such lymphomas include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activated B-cell lymphoma, DLBCL, mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocyti c lymphoma of intermediate di fferentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocyte lymphoma, diffuse small-cleaved cell lymphoma ( DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma and mantle zone lymphoma and low grade follicular lymphoma.
[0007] The non-Hodgkin lymphomas (NHLs) are a diverse group of blood cancers that include any kind of lymphoma except Hodgkin's lymphomas. Types of NHL vary significantly in their severity, from indolent to very aggressive. Less aggressive non-Hodgkin lymphomas are compatible with a long survival while more aggressive non-Hodgkin lymphomas can be rapidly fatal without
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SDI-60022 107vl treatment, they can be formed from either B-cells or T-cells. B-cell non-Hodgldn lymphomas include Burkitt lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma
(CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, imrnunoblastic large cell lymphoma, precursor B-Iymphoblastic lymphoma, and mantle cell lymphoma. T-cell non-Hodgkin lymphomas include mycosis fungoides, anaplastic large cell lymphoma, and precursor T- lymphoblastic lymphoma. Prognosis and treatment depend on the stage and type of disease.
[0Θ08] Diffuse large B-cell lymphoma (DLBCL) accounts for approximately one-third of non- Hodgkin 's lymphomas. While some DLBCL patients are cured with traditional chemotherapy, the remainder die from the disease. Anticancer drugs cause rapid and persistent depletion of lymphocytes, possibly by direct apoptosis induction in mature T and B cells. See K. Stahnke, et al. Blood 2001, 98:3066-3073. Absolute lymphocyte count (ALC) has been shown to be a prognostic factor in follicular non-Hodgkin's lymphoma and recent results have suggested that ALC at diagnosis is an important prognostic factor in diffuse large B-cell lymphoma.
[0009] The diffuse large-B-cell lymphomas (DLBCL) can be divided into distinct molecular subtypes according to their gene profiling patterns: germinal-center B-cell-like DLBCL (GCB- DLBCL), activated B-cell-like DLBCL (ABC-DLBCL), and primary mediastinal B-cell lymphoma (PMBL) or unclassified type. These subtypes are characterized by distinct differences in survival, chemo-responsiveness, and signaling pathway dependence, particularly the NF-κΒ pathway. See D. Kim et al.. Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No. 18S (June 20 Supplement), 2007: 8082. See Bea S, et al,, Blood 2005; 106: 3183-90; Ngo V.N. et al,, Nature 201 1; 470: 115-9. Such differences have prompted the search for more effective and subtype-specific treatment strategies in DLBCL,
[0010] Leukemia refers to malignant neoplasms of the blood-forming tissues. V arious forms of leukemias are described, for example, in U.S. patent no. 7,393,862 and U.S. provisional patent application no. 60/380,842, filed May 17, 2002, the entireties of which are incorporated herein by reference. Although viruses reportedly cause several forms of leukemia in animals, causes of leukemia in humans are to a large extent unknown. The Merck Manual, 944-952 (1 7 ed. 1999). Transformation to malignancy typically occurs in a single cell through two or more steps with subsequent proliferation and clonal expansion. In some leukemias, specific chromosomal translocations have been identified with consistent leukemic cell morphology and special clinical features (e.g., translocations of 9 and 22 in chronic myelocytic leukemia, and of 15 and 17 in acute
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SDI-60022 107vl promyelocvtic leukemia). Acute leukemias are predominantly undifferentiated cel l populations and chronic leukemias more mature cell forms.
[001 ί J Acute leukemias are divided into lymphoblastic (ALL) and non-lymphobiastic (AN LL) types. The Merck Manual, 946-949 (17 th ed. 1999). They may be further subdivided by their morphologic and cytochemical appearance according to the French- merican-British (FAB) classification or according to their type and degree of differentiation. The use of specific B- and T- ceil and myeloid-antigen monoclonal antibodies are most helpful for classification, ALL is predominantly a childhood disease which is established by laboratory findings and bone marrow examination. ANLL, also known as acute myelogenous leukemia or acute myeloid leukemia (AML), occurs at all ages and is the more common acute leukemia among adults; it is the form usually associated with irradiation as a causative agent.
[0012] Chronic leukemias are described as being lymphocytic (CLL) or myelocytic (CML). The Merck Manual, 949-952 (17* ed. 1999). CLL is characterized by the appearance of mature lymphocytes in blood, bone marrow, and lymphoid organs. The hal lmark of CLL is sustained, absolute lymphocytosis (> 5,000/μΤ) and an increase of lymphocytes in the bone marrow. Most CLL patients also have clonal expansion of lymphocytes with B-cell characteristics. CLL is a disease of middle or old age. In CML, the characteristic feature is the predominance of
granulocytic cells of all stages of differentiation in blood, bone marrow, liver, spleen, and other organs. In the symptomatic patient at diagnosis, the total white blood cell (WBC) count is usually about 200,000/μί, but may reach Ι,ΟΟΟ,ΟΟΟ/μί. CML is relatively easy to diagnose because of the presence of the Philadelphia chromosome.
[0Θ13] Bone marrow stromal cells are well known to support CLL disease progression and resistance to chemotherapy. Disrupting the interactions between CLL cells and stromal cel ls is an additional target of CLL chemotherapy.
[0014] In addition to the acute and chronic categorization, neoplasms are also categorized based upon the cells giving rise to such disorder into precursor or peripheral. See e.g., U.S. patent publication no. 2008/0051379, the disclosure of which is incorporated herein by reference in its entirety. Precursor neoplasms include ALLs and lymphoblastic lymphomas and occur in lymphocytes before they have differentiated into either a T- or B-ceil. Peripheral neoplasms are those that occur in lymphocytes that have differentiated into either T- or B-cells. Such peripheral neoplasms include, but are not limited to, B-ceil CLL, B-celi prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, extranodal marginal
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SDI-60022 107vl zone B-cel! lymphoma of mucosa-associated iymphoid tissue, nodai marginal zone lymphoma, splenic marginal zone lymphoma, hairy cell leukemia, plasmacytoma, diffuse large B-eell lymphoma and Burkitt lymphoma. In over 95 percent of CLL cases, the clonal expansion is of a B cell lineage. See Cancer: Principles & Practice of Oncology (3rd Edition) (1989) (pp. 1843-1847). In less than 5 percent of CLL cases, the tumor cells have a T-ceil phenotype. Notwithstanding these classifications, however, the pathological impairment of normal hematopoiesis is the hallmark of all leukemias.
[0015] Multiple myeloma (MM) is a cancer of pl asma cells in the bone marrow . Normal ly, plasma cells produce antibodies and play a key role in immune function. However, uncontrolled growth of these cells leads to bone pain and fractures, anemia, infections, and other complications. Multiple myeloma is the second most common hematological malignancy, although the exact causes of multiple myeloma remain unknown. Multiple myeloma causes high levels of proteins in the blood, urine, and organs, including but not limited to M-protein and other immunoglobulins (antibodies), albumin, and beta-2-microglobulin. M-protein, short for monoclonal protein, also known as paraprotein, is a particularly abnormal protein produced by the myeloma plasma cells and can be found in the blood or urine of almost all patients with multiple myeloma.
[001 ] Skeletal symptoms, including bone pain, are among the most clinically significant symptoms of multiple myeloma. Malignant plasma cells release osteoclast stimulating factors (including IL-1 , IL-6 and TNF) which cause calcium to be leached from bones causing lytic lesions; hypercalcemia is another symptom. The osteoclast stimulating factors, also referred to as cytokines, may prevent apoptosis, or death of myeloma ceils. Fifty percent of patients have radiologically detectable myeloma-related skeletal lesions at diagnosis. Other common clinical symptoms for multiple myeloma include polyneuropathy, anemia, hyperviscosity, infections, and renal
insufficiency,
[0017] Bone marrow stromal cells are well known to support multiple myeloma disease progression and resistance to chemotherapy. Disrupting the interactions between multiple myeloma cells and stromal cells is an additional target of multiple myeloma chemotherapy.
[0018] Further, rituximab is known to deplete normal host B cells. M. Aklilu et al., Annals of Oncology 15: 1 109-1114, 2004. The long-term immunologic effects of B cell depletion with rituximab and the characteristics of the reconstituting B cell pool in lymphoma patients are not well defined, despite the widespread usage of this therapy. See Jennifer H. Anolik et a/., Clinical Immunology, vol. 122, issue 2, February 2007, pages 139-145,
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SDI-60022 107vl [0019] The approach for patients with relapsed or refractor}' disease relies heavily on experimental treatments followed by stem cell transplantation, which may not be appropriate for patients with a poor performance status or advanced age. Therefore, a tremendous demand exists for new methods that can be used to treat patients with NHL.
[0020] The incidence of cancer continues to climb as the general population ages, as new cancers develop, and as susceptible populations (e.g., people infected with AIDS or excessively exposed to sunlight) grow. A tremendous demand therefore exists for new methods and
compositions that can be used to treat patients with cancer including NHL.
2,2, Methods of Treatment
[0001] Current cancer therapy may involve surgery, chemotherapy, hormonal therapy and/or radiation treatment to eradicate neoplastic cells in a patient (see, for example, Stockdale, 1998, Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). Recently, cancer therapy could also involve biological therapy or immunotherapy. All of these approaches pose significant drawbacks for the patient. Surgery, for example, may be contraindicated due to the health of a patient or may be unacceptable to the patient. Additionally, surgery may not completely remove neoplastic tissue. Radiation therapy is only effective when the neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue. Radiation therapy can also often elicit serious side effects. Hormonal therapy is rarely given as a single agent. Although hormonal therapy can be effective, it is often used to prevent or delay recurrence of cancer after other treatments have removed the majority of cancer cells. Biological therapies and immunotherapies are limited in number and may produce side effects such as rashes or swellings, flu-like symptoms, including fever, chilis and fatigue, digestive tract problems or allergic reactions.
[0002] With respect to chemotherapy, there are a variety of chemotherapeutic agents available for treatment of cancer. A majority of cancer chemotherapeutics act by inhibiting DNA synthesis, either directly, or indirectly by inhibiting the biosynthesis of deoxyribon cleotide triphosphate precursors, to prevent DNA replication and concomitant cell division. Oilman et al., Goodman and Gilman 's: The Pharmacological Basis of Therapeutics, Tenth Ed. (McGraw Hill, New York).
[0003] Despite availability of a variety of chemotherapeutic agents, chemotherapy has many drawbacks. Stockdale, Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10, 1998. Almost all chemotherapeutic agents are toxic, and chemotherapy causes significant, and often
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SDI-60022 107vl dangerous side effects including severe nausea, bone marrow depression, and immunosuppression. Additionally, even with administration of combinations of chemotherapeutic agents, many tumor cells are resistant or develop resistance to the chemotherapeutic agents. In fact, those cells resistant to the particular chemotherapeutic agents used in the treatment protocol often prove to be resistant to other drugs, even if those agents act by different mechanism from those of the drugs used in the specific treatment. This phenomenon is referred to as pleiotropic drag or multidrug resistance. Because of the drug resistance, many cancers prove refractory to standard chemotherapeutic treatment protocols.
[0Θ04] Still, there is a significant need for safe and effective methods of treating, preventing and managing cancer, particularly for tumors that are refractor}' to standard treatments, such as surgery, radiation therapy, chemotherapy and hormonal therapy, while reducing or avoiding the toxicities and/or side effects associated with the conventional therapies. Moreover, there remains a need for the ability to predict and monitor response to cancer therapy in order to increase the quality of care for cancer patients, avoid unnecessary treatment and to increase the success rate in cancer therapy in clinical practice.
3. SUMMARY
[0021 J The present invention is based, in part, on the finding that certain genes are differentially expressed in DLBCL patients responsive to the immunomodulatory therapy lenalidomide
(Revlimid®) relative to DLBCL patients unresponsive to lenalidomide. In addition, the present invention is based, in part, on the finding that the cellular composition (e.g., immune cell composition) of the tumor of a DLBCL patient may be indicative of whether the patient tumor will respond to an immunomodulatory therapy, such as lenalidomide, including its pharmaceutically acceptable salts, solvates or isomers.
[0022] In one aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 3, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 3 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first
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SDI-60022 107vl patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and wherein the differential expression of the one, two, three, four, five or more of the genes in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the
immunomodulatory therapy. In a specific embodiment, the hematological cancer is DLBCL. in certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient, in a specific embodiment, the DLBCL is an activated B-ceil-like subtype. In another specific embodiment, the DLBCL is a germinal center B- cell-like subtype. The immunomodulatory therapy can compri se the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide,
[0Θ23] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 4, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 4 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and wherein the differential expression of the one, two, three, four, five or more of the genes in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the
immunomodulatory therapy. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B- cell-like subtype. The immunomodulatory therapy can comprise the administration of an
226269-228501 / 12827-501-228
SDI-60022 107vl immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0024] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, and (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample with the level of expression of the same genes in a second bioiogical sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to the immunomodulatory therapy, and wherein a higher level of expression of the one, two, three, four, fi ve or more of t he genes in the first biological sample rel ativ e to the level of expression of the one, two, three, four, five or more of the genes in the second bioiogical sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the
immunomodulatory therapy. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is refractory relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-Iike subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide,
[0025] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2, infra, and (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in
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SDI-60022 107vl Table 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to the immunomodulatory therapy, and wherein a lower level of expression of the one, two, three, four, fi ve or more of the genes in the first biological sample rel ative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the
immunomodulatory therapy, in a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B- cell-like subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0Θ26] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1 , infra, and measuring the level of expression one, two, three, four, fi ve or more of the genes identified in Table 2, infra, and (c) comparing the level of expression of the genes identified in Tables 1 and 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to the immunomodulatory therapy, and wherein (i) a higher level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, and (ii) a lower level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, indicates that the hematological cancer in the first patient
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SDI-60022 107vl will be clinical sensitive to treatment with the immunomodulator therapy. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0G27] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1 , 2, 3 or 4, or any combination thereof in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profi le of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, and a gene expression profile for the genes or subset of genes in first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the sam e type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 15 or more of the genes in Table 1, 2, 3 or 4, or any combination thereof. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 1 , 2, 3 or 4, or any combination thereof. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-
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SDI-60022 107vl cell-like subtype. The immunomodulatory therapy can compri se the administration of an immunomodulatory compound, such as ienalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise ienalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is Ienalidomide.
[0Θ28] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells in the first tumor sample, and (c) comparing the proportion of dendritic cells in the first tumor sample with the proportion of dendritic cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of dendritic cells in the first tumor sample relative the proportion of dendritic cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy. In a specific
embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-ceil-like subtype. The
immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as Ienalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can comprise Ienalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is Ienalidomide,
[0029] in another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of plasma cells in the first tumor sample, and (c) comparing the proportion of plasma cells in the first tumor sample with the proportion of plasma cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and
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SDI-60022 107vl wherein a higher proportion of plasma cells in the first tumor sample relative the proportion of plasma cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL. is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The
immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can comprise leiialidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0030] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells and plasma cells in the first tumor sample, and (c) comparing the proportion of dendritic cells and plasma cells in the first tumor sample with the proportion of dendritic cells and plasma cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of dendritic cells and plasma cells in the first tumor sample relative the proportion of dendritic cells and plasma cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy. In a specific embodiment, the hematological cancer is DLBCL, In certain embodiments, the DLBCL is refractor}' to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The
immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
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SDI-60022 107vl [0031] in another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune ceils in the tumor sample, and (c) comparing the proportion of the immune ceils in the first tumor sample to (i) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an
immunomodulatory therapy and (ii) the proportion of the same immune ceils in tumor samples from pati ents having the same type of hematological cancer which are clinically insensi tive to the immimomodulatoiy therapy, wherein a proportion of the immune cells in the first tumor sample simi lar to the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, and a proportion of the immune cells in the first tumor sample similar to the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulator therapy indicates that the hematological cancer in the first patient will be clinical insensitive to treatment with the immunomodulatory therapy. In some embodiments, the immune cel ls are subset of immune cells, such as subset of B cells. In certain embodiments, the immune cells are dendritic cells, in some embodiments, the immune cells are plasma cells. In certain embodiments, the immune cells are monocytes, in some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cells are T cells. In some embodiments, the immune cells are B cells. In certain embodiments, the immune cells are NK ceils. In some embodiments, the immune cells are two, three or more subsets of immune cells, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cells in the tumor samples from pati ents having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatoiy therapy. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like
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SDI-60022 107vl subtype, in another specific embodiment, the DLBCL is a germinal center B-cell-iike subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as Ienalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can compri se Ienalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatoiy therapy is Ienalidomide.
[0Θ32] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 3, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 3 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodul atory therapy to the first patient if the one, two, three, four, five or more of the genes in the first biological sample are differentially expressed relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In a specific embodiment, the hematological cancer is DLBCL, In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-ceil-like subtype. The
immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as Ienalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can comprise Ienalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is Ienalidomide,
[0Θ33] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Tabl e 4, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 4 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same
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SDI-60022 107vl type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if the one, two, three, four, five or more of the genes in the first biological sample are differentially expressed relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractor}' to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-ceil-fike subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The
immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0034] in another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, (c) comparing the level of expression of the one, two, three, four, fi ve or more of the genes identified in Table 1 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodul atory therapy to the first patient if a higher level of expression of the one, two, three, four, five or more of the genes in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In a specific embodiment, the hematological cancer is DLBCL. in certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can compri se
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SDI-60022 107vl !enalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0035] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodulatorv' therapy to the first patient if a lower level of expression of the one, two, three, four, fi ve or more of the genes in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In certain embodiments, the immunomodulatory therapy is not administered or additional assays are conducted if the level of expression of one, two, three, four, five or more of the genes are not lower in the first biological sample than in the second biological sample. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0036] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, and measuring the level of expression one, two, three, four, five or more of the genes identified in Table 2, supra, (c) comparing the level of expression of the genes identified in Tabl es 1 and 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of
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SDI-60022 107vl hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the
immunomodulatory to the first patient if (i) a higher level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, and (ii) a lower level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The
immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide,
[0Θ37] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of a certain subset of genes set forth in Table 1 , 2, 3 or 4, or any combination thereof in the first biological sample, and (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if: (i) the gene expression profile for the subset of genes in the first biological sample is similar to the gene expression profi le for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the gene expression profile for the subset of genes in first biological sample is not similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically
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SDI-60022 107vl insensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 1, 2, 3 or 4, or any combmation thereof. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 1, 2, 3 or 4, or any combination thereof. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0038] in another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells in the first tumor sample, (c) comparing the proportion of dendritic cells in the first tumor sample with the proportion of dendritic ceils in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an
immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of dendritic cells in the first tumor sample is measured relative the proportion of dendritic ceils in the second tumor sample. In a specific embodiment, the
hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0039] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of plasma cells in the first tumor sample, (c) comparing the proportion of plasma cel ls in the first tumor sample with the proportion of plasma ceils in a second tumor sample from a second patient having the same type of hematological cancer, wherein the
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SDI-60022 107vl second patient's hematological cancer is clinically insensitive to treatment with an
immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patien t if a higher proportion of plasma cells in the first tumor sample is measured relative the proportion of plasma cells in the second tumor sample, in a specific embodiment, the
hematological cancer is DLBCL. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtvpe. In another specific embodiment, the DLBCL is a germinal center B-cell-like subtype. The immunomodulator therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenal idomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide.
[0040] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells and plasma cells in the first tumor sample, (c) comparing the proportion of dendritic cells and plasma cells in the first tumor sample with the proportion of dendritic cells and plasma cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of dendritic cells and plasma cells in the first tumor sample is measured relative the proportion of dendritic ceils and plasma cells in the second tumor sample. In a specific embodiment, the hematological cancer is DLBCL,. In certain embodiments, the DLBCL is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL is a germinal center B- cell-like subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An immunomodulatory therapy of the embodiments of the methods provided herein can comprise lenalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is lenalidomide,
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SDI-60022 107vl [0041] in another aspect, provided herein are for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune cells in the first tumor sample, and (c) comparing the proportion of the immune cells in the first tumor sample to (i) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the proportion of the same immune ceils in tumor samples from patients having the same type of hematological cancer which are clinical ly insensitive to the immunomodulatory therapy, and (d) administering the
immunomodulatory therapy to the first patient if the proportion of the immune cells in the first tumor sample is (i) similar to the proportion of the same immune cell s in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy, and (ii) not similar to the proportion of the same immune cell s in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy. In some embodiments, the immune cells are subset of immune cells, such as subset of B cells. In certain embodiments, the immune cells are dendritic ceils. In some embodiments, the immune cells are plasma cells. In certain embodiments, the immune cel ls are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cells are T cells. In some embodiments, the immune cells are B cells. In certain embodiments, the immune cel ls are NK cells. In some embodiments, the immune cells are two, three or more subsets of immune cells, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cel ls in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy. In a specific embodiment, the hematological cancer is DLBCL. In certain embodiments, the DLBCL. is refractory to certain therapies, such as chemotherapy. In some embodiments, the DLBCL is relapsed in a patient. In a specific embodiment, the DLBCL is an activated B-cell-like subtype. In another specific embodiment, the DLBCL. is a germinal center B-cell-like subtype. The immunomodulatory therapy can comprise the administration of an immunomodulatory compound, such as lenalidomide, or its pharmaceutically acceptable salts, solvates or isomers. An
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SDI-60022 107vl immunomodulatory therapy of the embodiments of the methods provided herein can comprise Ienalidomide as immunomodulatory compound, or its pharmaceutically acceptable salts, solvates or isomers. In another specific embodiment, the immunomodulatory therapy is Ienalidomide.
[0042] In accordance with the methods described herein, the biological sample can be any sample obtained from the patient. In certain embodiments, the biological sample is a cell sample, in other embodiments, the biological sample is whole blood sample, peripheral blood mononuclear ceil sample, or tissue sample. In specific embodiments, the biological sample is a tumor sample. See Section 5.8, infra, regarding biological samples.
[0043] In accordance with the methods described herein, the level of expression of one, two, three, four, five or more of the genes in Table 1 and/or Table 2 and/or Table 3 and/or Table 4, infra, can be measured at the RNA and/or protein levels. In certam embodiments, the level of expression of the genes are measured at the RNA (e.g. , mRNA) level. In other embodiments, the level of expression of the genes are measured at the protein level.
[0044] In another aspect, provided herein are kits useful for predicting the likelihood of an effective patient tumor response. In certain embodiments, the kit comprises a solid support, and a means for detecting the protein expression of at least one biomarker in a biological sample. Such a kit may employ, for example, a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical fiber. The solid support of the kit can be, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide. In some embodiments, the kit comprises a solid support, nucleic acids contacting the support, where the nucleic acids are complementary to at least 20, 50, 100, 200, 350, or more bases of mRNA, and a means for detecting the expression of the mRN A in a biological sample.
[0045] In certain embodiments, the kits provided herein employ means for detecting the expression of a biomarker by quantitative real-time PCR (QRT-PCR), microarray, flow cytometry or immunofluorescence. In other embodiments, the expression of the biomarker is measured by ELISA-based methodologies or other similar methods known in the art.
4, BRIEF DESCRIPTION OF THE FIGURES
[0046] FIG. 1 : Hierarchical clustering (Eucli dean distance; Ward linkage) of rel ative gene expression across 21 lenalidomide/Revlimid®-arm FF profiles, as represented by A. 1018 genes deemed significantly differentially regulated at FD 5%, and B. A subset of those genes deemed
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SDI-60022 l07vl significantly differentially regulated at FDRJ %, between discrete best-response categories. Gene expressions standardized to zero mean and unit standard variance across all profiles. Bars below dendrogram display: DLBCL cell of origin sub-type {GCB (white), ABC/Other (black), as determined by IHC at screen}; Investigator defined best response of patients in the Revlimid arm, {CR,PR,SD} (black) vs. {PD,Death} (white).
[0047] FIG. 2: Decomposition of 21 Ienalidomide/Revlimid®-arm profiles derived from FF samples. Each box lot represents estimated proportion (y-axis) of corresponding cell phenotype (x- axis) across two discrete Investigator defined best-response categories, {CR,PR,8D} (grey) and {PD,death} (white). Cell-types on the x-axis are: T-helper cells (Th); Activated T -helper ceils (Th act); T-celis (Tc); Activated T-celfs (Tc act); B-cells (B); Activated B-cells (B act); BCR-ligated B- cells (B aigM); IgG Memory B-cells (Mem IgG); IgM Memory B-cells (Mem IgM); Plasma cells (PC); Natural Killer cells (NK); Activated Natural Killer cells (N act); Monocytes (mono);
Activated Monocytes (mono act); Dendritic Cells (DC); Activated Dendritic cells (DC act);
Neutrophils (neutro). Phenotypic cell types defined in (Abbas et al., PLoS One, 2009).
[0048] FIG. 3: S ummed estimated proportion of resting and activated dendritic cells (y-axis, left) across 21 lmalidomide/Revlimid®-arm profiles derived from FF samples (x-axis; triangles, ordered by descending PFS). PFS (y-axis, right; unit weeks) overlaid as line-connected points, with censor events denoted by a cross.
[0049] FIG. 4: Summed estimated proportion of BCR-ligated B-cells (y-axis, left) across 21 lenaIidomide/ ' Revlimid®-arm profiles derived from FF samples (x-axis; triangles, ordered by descending PFS). PFS (y-axis, right; unit weeks) overlaid as line-connected points, with censor events denoted by a cross.
[0050] FIG, 5: Bar plot of difference in estimated proportion of BCR-ligated B-cells and plasma cells (y-axis, left), derived from ienafidomide Revfimid®-arm FF profiles (one profile per bar, x-axis; sorted in order of increasing difference between BCR-ligated B-cell/plasma-cell proportions. PFS (y-axis, right; unit weeks) overlaid as line-connected points, with censor events denoted by a cross. Dashed line represents median PFS in the two groups defined by estimated BCR-ligated B-cell proportion being greater or less than estimated plasma cell proportion.
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SDI-60022 107vl 5. DETAILED DESCRIPTION 5.1 Terminology
[0051 J As used herein, and unless otherwise specified, the terms "treat," "treating" and
"treatment" refer to an action that occurs while a patient is suffering from the specified cancer, which includes the reduction in the severity of the cancer, reduces tumor size, or retards or slows the progression of the cancer.
[0052] The term "sensitivity" and "sensitive" when made in reference to treatment with compound is a relative term which refers to the degree of effectiveness of the compound in lessening or decreasing the progress of a tumor or the disease being treated.
[0053] As used herein, and unless otherwise specified, the term "effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a cancer, or to delay or minimize one or more symptoms associated with the presence of the cancer. An effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the cancer. The term "effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of cancer, or enhances the therapeutic efficacy of another therapeutic agent.
[0054] As used herein, an "effective patient tumor response" refers to any increase in the therapeutic benefit to the patient. An "effective patient tumor response" can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the rate of progress of the tumor. An "effective patient tumor response" can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the physical symptoms of a cancer. An "effective patient tumor response" can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the size of a tumor. An "effective patient tumor response" can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the physical symptoms of a cancer. An "effective patient tumor response" can also be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% , or 200%, or more increase in the response of the patient, as measured by any suitable means, such as gene expression, cell counts, assay results, etc.
[0055J The term "likelihood" generally refers to an increase in the probability of an event. The term "likelihood" when used in reference to the effectiveness of a patient tumor response generally
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SDI-60022 107vl contemplates an increased probability that the rate of tumor progress or tumor cell growth will decrease. The term 'likelihood" when used in reference to the effectiveness of a patient tumor response can also generally mean the increase of indicators, such as mRN A or protein expression, that may evidence an increase in the progress in treating the tumor.
[0056] The term "predict" generally means to determine or tell in advance. When used to "predict" the effectiveness of a cancer treatment, for example, the term "predict" can mean that the likelihood of the outcome of the cancer treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially .
[0057] An improvement in the cancer or cancer-related disease can be characterized as a complete or partial response, "Complete response" refers to an essential absence (or absence) of clinically detectable disease with normalization of any previously abnormal radiographic studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements.
"Partial response" refers to at least about a 5%, 10%, 15%, 20%, 25%, 30%, 40%», 50%, 60%,, 70%, 80%, or 90% decrease in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured bulk of tumor masses or the quantity of abnormal monoclonal protein) in the absence of new lesions. The term "treatment" contemplates both a complete and a partial response.
[0058] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Neoplastic," as used herein, refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Thus, "neoplastic cells" include malignant and benign cells having dysregulated or unregulated cell growth.
[0059] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, blood-boroe tumors (e.g., multiple myeloma, lymphoma and leukemia), and solid tumors.
[0060] The term "refractory or resistant" refers to a circumstance where patients, even after intensive treatment, have residual cancer cells {e.g., leukemia or lymphoma cells) in their lymphatic system, blood and/or blood forming tissues (e.g., marrow).
[0061 J As used herein the terms "polypeptide" and "protein" as used interchangeably herein, refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogues,
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SDI-60022 107vl oligopeptides and the like. The term polypeptide as used herein can also refer to a peptide. The amino acids making up the polypeptide may be naturally derived, or may be synthetic. The polypeptide can be purified from a biological sample.
[0062] An mRNA that is "upregulated" is generally increased upon a given treatment or condition. An mRNA that is "downregulated" generally refers to a decrease in the level of expression of the mRNA in response to a given treatment or condition. In some situations, the mRNA level can remain unchanged upon a given treatment or condition.
[0063] An mRNA from a patient sample can be "upregulated" when treated with an
immunomodulatory therapy, as compared to a control. This upregulatioii can be, for example, an increase of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 600%, 700%, 800%, 900%, 1,000%, 1,500%, 2,000%, 2,500%, 3,00%, 3,500%, 4,000%, 4,500%, 5,000% or more of the comparative control mRNA level.
[0064] Alternatively, an mRNA can be "downregulated", or expressed at a lower level, in response to administration of certain immunomodulatory therapies or other therapies. A
downregulated mRNA can be, for example, present at a level of about 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%», 20%, 15%, 10%, 5%, 3%, 1% or less of the comparative control mRNA level.
[0065] Similarly, the level of a polypeptide or protein biomarker from a patient sample can be increased when treated with an immunomodulatory therapy, as compared to a non-treated control. This increase can be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1,000%, 1 ,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, 5,000% or more of the comparative control protein level.
[0066] Alternatively, the level of a protein biomarker can be decreased in response to administration of certain immunomodulatory therapies or other agents. This decrease can be, for example, present at a level of about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%», 20%, 10%, 5%, 3%, 1 % or less of the comparative control protein level.
[0067] The terms "determining", "measuring", "evaluating", "assessing" and "assaying" as used herein generally refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. " Assessing the presence of can include determining the amount of something present, as well as determining wiiether it is present or absent.
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SDI-60022 107vl [0068] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotid.es or
ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. As used herein in the context of a polynucleotide sequence, the term "bases" (or "base") is synonymous with
"nucleotides" (or "nucleotide"), i.e., the monomer subunit of a polynucleotide. The terms
"nucleoside" and "nucleotide" are intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrrolidines, acyiated purines or pyrrolidines, alkylated riboses or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxy! groups are replaced with halogen atoms or aliphatic groups, or are functionaiized as ethers, amines, or the like. "Analogues" refer to molecules having structural features that are recognized in the literature as being mimetics, derivatives, having analogous structures, or other like terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2 '-modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide that has added substituent groups, such as protecting groups or linking moieties.
[0069] The terms "isolated" and "purified" refer to isolation of a substance (such as mRNA or protein) such that the substance comprises a substantial portion of the sample in which it resides, i.e. greater than the substance is typically found in its natural or un-isolated state. Typically, a substantial portion of the sample comprises, e.g., greater than 1 %, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 50%, or more, usually up to about 90%- 100% of the sample. For example, a sample of isolated mRNA can typical ly comprise at least about 1% total mRNA. Techniques for purifying polynucleotides are well known in the art and include, for example, gel electrophoresis, ion-exchange chromatography, affinity
chromatography, flow sorting, and sedimentation according to density.
[0070] The term "sample" as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, containing one or more components of interest.
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SDI-60022 107vl [0071] "Biological sample" as used herein refers to a sample obtained from a biological subject, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo or in situ, A biological sample also includes samples from a region of a biological subject containing precancerous or cancer cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions and cells isolated from a subject. Exemplary biological samples include but are not limited to cell lysate, a cell culture, a cell line, a tissue, oral tissue, gastrointestinal tissue, an organ, an organelle, a biological fluid, a blood sample, a urine sample, a skin sample, and the like.
Preferred biological samples include but are not limited to whole blood, partially purified blood, PBMCs, tissue biopsies, and the like.
[0072] As used herein, the terms "patient" and "subject" refer to an animal, such as a mammal, in a specific embodiment, the patient is a human, in other embodiments, the patient is a non-human animal, such as a dog, cat, farm animal {e.g., horse, pig, or donkey), chimpanzee, or monkey.
[0073] A biological marker or "biomarker" is a substance whose detection indicates a particular biological state, such as, for example, the presence of cancer. In some embodiments, biomarkers can either be determined individually, or several biomarkers can be measured simultaneously.
[0G74] A "biomarker" can indicate a change in the level of mRNA expression that may correlate with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. In some embodiments, the biomarker is a nucleic acid, such as a mRNA or cDNA.
[0075] A "biomarker" can also indicate a change in the level of polypeptide or protein expression that may correlate with the risk, susceptibility to treatment, or progression of a disease. The biomarker can be a polypeptide or protein, or a fragment thereof. The relative level of specific proteins can be determined by methods known in the art. For example, antibody based methods, such as an immunoblot, enzyme-linked immunosorbent assay (ELISA), or other methods can be used.
[0Θ76] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" encompasses non-toxic acid and base addition salts of the compound to which the term refers. Acceptable non-toxic acid addition salts include those derived from organic and inorganic acids or bases know in the art, which include, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, iiiethanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, embolic acid, enanthic acid, and the like.
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SDI-60022 107vl [0077] Compounds that are acidic in nature are capable of forming salts with various pharmaceutically acceptable bases. The bases that can be used to prepare pharmaceutically acceptable base addition salts of such acidic compounds are those that form non-toxic base addition salts, i.e., salts containing pharmacologically acceptable cations such as, but not limited to, alkali metal or alkaline earth metal salts and the calcium, magnesium, sodium or potassium salts in particular. Suitable organic bases include, but are not limited to, N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine (N-methylglucamine), lysine, and procaine,
[0Θ78] As used herein and unless otherwise indicated, the term "solvate" means a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the sol vate is a hydrate.
[0079] As used herein and unless otherwise indicated, the term "stereomerically pure" means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure composition of a compound having two chiral centers will be substantial ly free of other diastereomers of the compound, A typical stereomerically pure compound comprises greater than about 80% by weigh t of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and most preferably greater than about 97%> by weigh t of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound. As used herein and unless otherwise indicated, the tenn "stereomerically enriched" means a composition that comprises greater than about 60% by weight of one stereoisomer of a compound, preferably greater than about 70% by weight, more preferably greater than about 80% by weight of one stereoisomer of a compound. As used herein and unless otherwise indicated, the term "enantiomerically pure" means a stereomerically pure composition of a compound having one chiral center. Similarly, the tenn "stereomerically enriched" means a stereomerically enriched composition of a compound having one chiral center,
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SDI-60022 107vl [0080] it should be noted that if th ere is a discrepancy between a depicted structure and a name given that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it.
[0081] The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology, which are within the skill of those working in the art. Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook et al. (1989) Molecular Cloning; A Laboratory Manual (2d ed.); D.N. Glover, ed. (1985) DNA Cloning, Volumes I and II; M.J. Gait, ed. (1984) Oligonucleotide Synthesis; B.D. Hames & SJ. Higgins, eds. (1984) Nucleic Acid Hybridization; B.D. Hames & S.J. Higgins, eds. (1984) Transcription and Translation; R.I. Freshney, ed. (1986) Animal Cell Culture; Immobilized Cells and Enzymes (IRL Press, 1986); immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes (1987) Protein Purification: Principles and Practice (2d ed.; Springer Verlag, N.Y.); and D.M. Weir and C. C. Blackwell, eds. (1986) Handbook of Experimental Immunology , Volumes I- IV.
5.2 Methods for Predicting Clinical Sensitivity of a
Hematological Cancer By Measuring Gene Expression
[0082] In one aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identi fied in Table 3 or 4, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 3 or 4 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and wherein the differential expression of the one, two, three, four, five or more of the genes in the first biologicai sample rel ative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sampl e indi cates that the
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SDI-60022 107vl hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[0083] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, and (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to the immunomodulatory therapy, and wherein a higher level of expression of the one, two, three, four, five or more of the genes in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the
immunomodulatory therapy.
[0Θ84] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2, infra, and (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to the immunomodulatory therapy, and wherein a lower level of expression of the one, two, three, four, five or more of the genes in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the
imm unomodul atory therapy .
[0085] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising; (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, and
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SDI-60022 107vl measuring the level of expression one, two, three, four, five or more of the genes identified in Table 2, infra, and (c) comparing the level of expression of the genes identified in Tables 1 and 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to the immunomodulatory therapy, and wherein (i) a higher level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, and (ii) a lower level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[0086] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomod ulatory therapy comprising: (a) obtaining biological samples from patients having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 3, infra, (c) assessing expression levels of the selected genes, either individually, conjointly, or via a functional transformation thereof, and (d) using of the expression levels to predict patients as sensitive or insensitive to an immunomodulatory therapy, via similarity to expression phenotypes displayed across the same genes by patients with the same indication and already known to be sensitive or insensitive to that therapy.
[0087] In one aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomod ulatory therapy comprising: (a) obtaining biological samples from patients having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 4, infra, (c) assessing expression levels of the selected genes, either individually, conjointly, or via a functional transformation thereof, and (d) using of the expression levels to predict patients as sensitive or insensitive to an immunomodulatory therapy, via similarity to expression phenotypes displayed across the same genes by patients with the same indication and already known to be sensitive or insensitive to that therapy.
[0088] In one aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining
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SDI-60022 107vl biological samples from patients having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, (c) assessing expression levels of the selected genes, either individually, conjointly, or via a functional transformation thereof, and (d) using of the expression levels to predict patients as sensitive or insensitive to an immunomodulatory therapy, via similarity to expression phenotypes displayed across the same genes by patients with the same indication and already known to be sensitive or insensitive to that therapy.
[0089] In one aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining biological samples from patients having a hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2, infra, (c) assessing expression levels of the selected genes, either individually, conjointly, or via a functional transformation thereof, and (d) using of the expression levels to predict patients as sensitive or insensitive to an immunomodulatory therapy, via similarity to expression phenotypes displayed across the same genes by patients with the same indication and already known to be sensitive or insensitive to that therapy.
[0090] in another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of a certain subset of genes set forth in Table 3 in the first biological sample, and (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from pati ents having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the subset of genes in the first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, and a gene expression profile for the subset of genes in first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the
- jj -
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SDI-60022 107vl immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 3, In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 3.
[0091] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (h) measuring the expression of a certain subset of genes set forth in Table 3 in the first biological sample, and (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profi le for the subset of genes in the first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 3. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 3.
[0092] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of a certain subset of genes set forth in Table 3 in the first biological sample, and (c) comparing the gene expression profi le of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the subset of genes in first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of
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SDI-60022 107vl hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 3. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 3.
[0093] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 4 in the first biological sampl e, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profi le of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in Table 4 in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profi le for the genes or subset of genes in the first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, and a gene expression profile for the subset of genes in first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 14, 15 or more of the genes in Table 4. In some embodiments, the subset of genes comprises 2-5, 5-10, or 10-15 of the genes in Table 4.
[0Θ94] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the gen es or a certain subset of genes set forth in Table 4 in the first biological sample, and (e) comparing the gene expression profi le of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples
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SDI-60022 107vl from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in Table 4 in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy mdicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, in certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 4. In some embodiments, the subset of genes comprises 2-5, 5-10, or 10-15 of the genes in Table 4.
[0095] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 4 in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor sam ples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in Table 4 in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the subset of genes in first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 15 or more of the genes in Table 4. In some embodiments, the subset of genes comprises 2-5, 5-10, or 10-15 of the genes in Table 4.
[0096] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1 in the first biological
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SDI-60022 107vl sampl e, and (c) comparing the gene expression profile of the genes or s ubset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinical ly insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the subset of genes in tumor sampl es from patients having the same type of hematological cancer whi ch are clinical ly sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, and a gene expression profile for the genes or subset of genes in first biological sample similar to the gene expression profi le for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 1. In some
embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 1.
[0097] in another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1 in the first biological sampl e, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulator therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinical ly sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy. In certain
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SDI-60022 107vl embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 15 or more of the genes in Table 1. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 1.
[0098] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1 in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the imniunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table I . In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15- 20, 20-25 or 25-30 of the genes in Table 1.
[0099] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 2 in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor sam ples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are
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SDI-60022 107vl clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy, and a gene expression profi le for the genes or subset of genes in first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 2. In some
embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 2.
[00100] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 2 in the first biological sampl e, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinical ly insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in the first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 2, In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 2.
[00101] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first biological sample from a first patient having a hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 2 in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first
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SDI-60022 107vl biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a gene expression profile for the genes or subset of genes in first biological sample similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immimomoduiatoiy therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 14, 15 or more of the genes in Table 2. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15- 20, 20-25 or 25-30 of the genes in Table 2.
[00102] In accordance with the methods described herein, the biological sample can be any sample obtained from the patient. In certain embodiments, the biological sample is a cell sample. In other embodiments, the biological sample is whole blood sample, peripheral blood mononuclear ceil sample, or tissue sample. In specific embodiments, the biological sample is a tumor sample. See Section 5.8, infra, regarding biological samples.
[00103] In accordance with the methods described herein, the hematological cancer can be any hematological cancer. Examples of hematological cancers can be found in Section 5.5, infra, in a specific embodiment, the hematological cancer is a lymphoma. In another specific embodiment, the hematological cancer is a non-Hodgkm's lymphoma. In yet another embodiment, the hematological cancer is a diffuse large B-cell lymphoma (DLBCL). In certain embodiments, the DLBCL is a germinal center B-cell-like DLBCL. In other embodiments, the DLBCL. is an activated B-cell-like DLBCL.
[00104] In accordance with the methods described herein, the level of expression of one, two, three, four, five or more of the genes in Table 1 , Table 2, Table 3, and/or Table 4, infra, can be measured at the RNA and/or protein levels. In certain embodiments, the level of expression of the genes are measured at the RNA {e.g., mRNA) level. In other embodiments, the level of expression of the genes are measured at the protein level.
[00105] Techniques known to one skilled in the art may be used to measure the amount of an RNA transcript(s). In some embodiments, the amount of one, two, three, four, five or more RNA transcripts is measured using deep sequencing, such as ILLLIMINA® RNASeq, ILLLIMINA® next
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SDI-60022 107vl generation sequencing (NGS), ION TORRENT IM RNA next generation sequencing, 454™ pyrosequencing, or Sequencing by Oiigo Ligation Detection (SOLID™), In other embodiments, the amount of multiple RNA transcripts is measured using a microarray and/or gene chip, such as described in Section 6, infra. In certain embodiments, the amount of one, two, three or more RNA transcripts is determined by RT-PCR , In other embodiments, the amount of one, two, three or more RNA transcripts is measured by RT-qPCR. Techniques for conducting these assays are known to one skilled in the art. See Section 5.9, infra, for examples of assays to measure RNA transcripts.
[00106] in some embodiments, a statistical analysis or other analysis is performed on data from the assay utilized to measure an RNA transcript or protein. In certain specific embodiments, p value of those RNA transcripts or proteins differential [y expressed is 0.1 , 0.5, 0.4, 0.3, 0.2, 0.01, 0.05, 0.001 , 0.005, or 0.0001. In specific embodiments, a false discovery rate (FDR) of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less is selected.
[00107] Techniques known to one skilled in the art may be used to measure the amount of a protein. For example, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay- based methodologies (ELISA) and similar assays known in the art. See Section 5.10, infra, for examples of assays to measure protein.
[001 8] in accordance with the methods described herein, the immunomodulatory therapy can be any therapy that modulates the immune system or immune response. Examples of
immunomodulatory therapies are provided in Section 5.6, infra. In a specific embodiment, the immunomodulatory therapy is lenalidomide (Revlimid©).
5,3 Methods for Predicting Cli ical Sensitivity of a
Hematological Cancer By Measuring Proportion of Cells
[00109 J In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells in the first tumor sample, and (c) comparing the proportion of dendritic cells in the first tumor sample with the proportion of dendritic ceils in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of dendritic cells in the first tumor sample relative the proportion of
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SDI-60022 107vl dendritic cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[00110] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of plasma cells in the first tumor sample, and (c) comparing the proportion of plasma ceils in the first tumor sample with the proportion of plasma ceils in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of plasma cel ls in the first tumor sample relative the proportion of plasma cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[00111] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment, with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells and plasma cells in the first tumor sample, and (c) comparing the proportion of dendritic cells and plasma cells in the first tumor sample with the proportion of dendritic cells and plasma cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of dendritic cells and plasma cells in the first, tumor sample relative the proportion of dendritic cells and plasma cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment, with the immunomodulatory therapy.
[00112 J in another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of B ceils in the first tumor sample, and (c) comparing the proportion of B cells in the first tumor sample with the proportion of B cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a decreased proportion of B cells in the first tumor sample relative the proportion of B cells in the second tumor
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SDI-60022 107vl sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[00113] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of Natural Killer (NK) cells in the first tumor sample, and (c) comparing the proportion of NK cells in the first tumor sample with the proportion of NK cells in a second tumor sample from a second patient having the same type of hematological cancer, wherem the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of NK cells in the first tumor sample relative the proportion of NK cells in the second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[00114] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of tumor infiltrating immune cells in the first tumor sample, and (c) comparing the proportion of tumor infiltrating immune cells in the first tumor sample with the proportion of tumor infiltrating immune ceils in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of tumor infiltrating immune cells in the first tumor sample relative the proportion of tumor infiltrating immune cells in the second tumor sam le indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulator therapy.
[00115] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of monocytes in the first tumor sample, and (c) comparing the proportion of NK cells in the first tumor sample wi th the proportion of monocytes in a second tumor sampl e from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with the immunomodulatory therapy, and wherein a higher proportion of monocytes in the first tumor sample relative the proportion of monocytes in the
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SDI-60022 107vl second tumor sample indicates that the hematological cancer in the first patient will be clinical sensitive to treatment with the immunomodulatory therapy.
[00116] In certain embodiments of the foregoing paragraphs in this section, th e second patient is a single patient, in other embodiments of the foregoing paragraphs in this section, the second patient is a population of patients. In specific embodiments, the population comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 225, 250, 300 or more patients.
[00117] in another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune ceils in the tumor sample, and (c) comparing the proportion of the immune cells in the first tumor sample to (i) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an
immunomodulatory therapy and (ii) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a proportion of the immune cells in the first tumor sample similar to the proportion of the same immune cel ls in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient wi ll be clinical sensitive to treatment with the immunomodulatory therapy. In some embodiments, the immune cells are subset of immune cells, such as subset of B cells. In certain embodiments, the immune cells are dendritic cells. In some embodiments, the immune cells are plasma ceils. In certain embodiments, the immune ceils are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cel ls are T cells. In some embodiments, the immune cells are B ceils. In certain embodiments, the immune cells are NK cells. In some embodiments, the immune cel ls are two, three or more subsets of immune cells, such as two more types of T cel ls (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune ceils in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same
populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy,
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SDI-60022 107vl [00118 J In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune ceils in the tumor sample, and (c) comparing the proportion of the immune cells in the first tumor sample to (i) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an
immunomodulatory therapy and (ii) the proportion of the same immune ceils in tumor samples from pati ents having the same type of hematological cancer which are clinically insensi tive to the immunomodulatory therapy, wherein a proportion of the immune cells in the first tumor sample simi lar to the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In some embodiments, the immune cells are subset of immune cells, such as subset of B cells. In certain embodiments, the immune ceils are dendritic cells. In some embodiments, the immune cells are plasma cells. In certain embodiments, the immune cells are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cel ls. In certain embodiments, the immune cells are T cel ls. In some embodiments, the immune cells are B cells. In certain embodiments, the immune cells are NK cells. In some embodiments, the immune cells are two, three or more subsets of immune cel ls, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy.
[0Θ119] In another aspect, provided herein are methods for predicting the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune ceils in the tumor sample, and (c) comparing the proportion of the immune cell s in the first tumor sample to (i) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an
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SDI-60022 107vl immunomodulatory therapy and (ii) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, wherein a proportion of the immune cel ls in the first tumor sample similar to the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy indicates that the hematological cancer in the first patient, and a proportion of the immune cells in the first tumor sample similar to the proportion of the same immune cells in tumor samples from pati ents having the same type of hematological cancer which are clinically insensi tive to the immunomodulatory therapy indicates that the hematological cancer of the first patient will be clinically insensitive to the treatment with the immunomodulatory therapy. In some embodiments, the immune cells are subset of immune cells, such as subset of B cells. In certain embodiments, the immune cells are dendritic cells. In some embodiments, the immune cells are plasma cells. In certain embodiments, the immune cells are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cells are T cells. In some embodiments, the immune cells are B cells. In certain embodiments, the immune cells are NK. ceils. In some embodiments, the immune cells are two, three or more subsets of immune cells, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy.
|00120| In addition to measuring the cells (e.g., dendritic cells, plasma cells, B cells, monocytes, and infiltrating immune cells) in a tumor sample from a patient, levels of expression of genes (e.g. , one, two, three, four, five or more of the genes in Table 1 and/or Table 2) may be assessed. In specific embodiments, the methods set forth in Section 5.1, supra, are combined with the methods set forth in this Section 5.2 to predict the clinical sensitivity of a hematological cancer to treatment with an immunomodulatory therapy.
[00121] Techniques known to one skilled in the art may be used to measure the proportion of cells in a tumor sample. In certain embodiments, the proportion of cells is measured by flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay-based methodologies
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SDI-60022 107vl (ELISA) and similar assays known in the art. See Section 5,8, infra, regarding techniques for measuring and distinguishing cell types. In other embodiments, the proportion of cells is measured by inference from gene expression profiles.
[00122] In accordance with the methods described herein, the hematological cancer can be any hematological cancer. Examples of hematological cancers can be found in Section 5.5, infra. In a specific embodiment, the hematological cancer is a lymphoma. In another specific embodiment, the hematoiogical cancer is a non-Hodgkin's lymphoma. In yet another embodiment, the hematological cancer is a diffuse large B-cell lymphoma (DLBCL). In certain embodiments, the DLBCL is a germinal center B-cell-iike DLBCL. In other embodiments, the DLBCL is an activated B-cell-iike DLBCL.
[00123] In accordance with the methods described herein, the immunomodulatory therapy can be any therapy that modulates the immune system or immune response. Examples of
immunomodulatory therapies are provided in Section 5.6, infra. In a specific embodiment, the immunomodulatory therapy is lenalidomi.de (Revlimid.®).
5.4 Methods for Managing and Treating Hematological Cancer
[00124] In one aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 3 or 4, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 3 or 4 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same ty pe of hematological cancer as the first patient, wherein the hematoiogical cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if the one, two, three, four, five or more of the genes in the first biological sample are differentially expressed relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In certain embodiments, the immunomodulatory therapy is not administered or additional assays are conducted if the level of expression of one, two, three, four, five or more of the genes are not higher in the first biological sample than in the second biological sample.
[00125] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
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SDI-60022 107vl hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 1, infra, (c) comparing the level of expression of the one, two, three, four, fi ve or more of the genes identified in Table 1 in the first biological sample with the level of expression of the same genes in a second biological sample from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher level of expression of the one, two, three, four, five or more of the genes in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In certain embodiments, the immunomodulator therapy is not administered or additional assays are conducted if the level of expression of one, two, three, four, five or more of the genes are not higher in the first biological sample than in the second biological sample,
[00126] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of the genes identified in Table 2, infra, (c) comparing the level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a lower level of expression of the one, two, three, four, five or more of the genes in the first biological sample is measured relative to the level of expression of the one, two, three, four, fi ve or more of the genes in the second biological sample. In certain embodiments, the immunomodulator therapy is not administered or additional assays are conducted if the level of expression of one, two, three, four, five or more of the genes are not lower in the first biological sample than in the second biological sample. In certain embodiments, the immunomodulatory therapy is not administered or additional assays are conducted if the level of expression of one, two, three, four, five or more of the genes are not higher in the first biological sample than in the second biological sample.
[00127] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the level of expression of one, two, three, four, five or more of
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SDI-60022 107vl the genes identified in Table 1 , infra, and measuring the level of expression one, two, three, four, five or more of the genes identified in Table 2, infra, (c) comparing the level of expression of the genes identified in Tables 1 and 2 in the first biological sample with the level of expression of the same genes in a second biological sample is from a second patient having the same type of hematological cancer as the first patient, wherein the hematological cancer in the second patient is clinically insensitive to an immunomodulatory therapy, and (d) administering the
immunomodulatory to the first patient if (i) a higher level of expression of the one, two, three, four, five or more of the genes identified in Table 1 in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample, and (ii) a lower level of expression of the one, two, three, four, five or more of the genes identified in Table 2 in the first biological sample is measured relative to the level of expression of the one, two, three, four, five or more of the genes in the second biological sample. In certain embodiments, the immunomodulatory therapy is not administered or additional assays are conducted if the level of expression of one, two, three, four, fi ve or more of the genes are not higher in the first biological sample than in the second biological sample.
[ΘΘ128] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of a certain subset of genes set forth in Table 3, infra, in the first biological sample, and (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensiti ve to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulator therapy, and (d) administering the immunomodulatory therapy to the first patient if the gene expression profile for the subset of genes in the first biological sample is similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 14, 15 or more of the genes in Table 3. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15- 20, 20-25 or 25-30 of the genes in Table 3.
[00129] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
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SDI-60022 107vl hematological cancer, (b) measuring the expression of a certain subset of genes set forth in Tabl e 3, infra, in the first biological sample, and (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if the gene expression profile for the subset of genes in the first biological sample is not similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 3, In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20-25 or 25-30 of the genes in Table 3.
[00130] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of a certain subset of genes set forth in Table 3, infra, in the first biological sample, and (c) comparing the gene expression profile of the subset of genes in the first biological sample to (i) the gene expression profile of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatoiy therapy, and (d) administering the immunomodulatory therapy to the first patient if: (i) the gene expression profile for the subset of genes in the first biological sample is similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatoiy therapy and (ii) the gene expression profile for the subset of genes in first biological sample is not similar to the gene expression profile for the subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory, in certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 15 or more of the genes in Table 3. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, .15- 20, 20-25 or 25-30 of the genes in Table 3.
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SDI-60022 107vl [00131 J In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 4, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or a subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodul atory therapy to the first patient if the gene expression profile for the genes or subset of genes in the first biological sample is similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically sensitive to the immunomodulatoiy therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 4. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 4.
[00132] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of t he genes or a certain subset of genes set forth in Table 4, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from pati ents having the same type of
hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) admini stering the immunomodulator therapy to the first patient if the gene expression profile for the genes or subset of genes in the first biological sample is not similar to the gene expression profil e for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 15 or more of the genes in Table 4. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 4.
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SDI-60022 107vl [00133] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 4, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if: (i) the gene expression profile for the genes or subset of genes in the first biological sample is similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the gene expression profile for the genes or subset of genes in first biological sample is not similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 4. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15- 20, 20-25 or 25-30 of the genes in Table 4.
[00134] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1 , infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or a subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if the gene expression profile for the genes or subset of genes in the first biological sample is similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically sensitive to the immunomodulatory therapy. In certain
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SDI-60022 l07vl embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 15 or more of the genes in Table 1. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 1.
[00135] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table I, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes i in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if the gene expression profile for the genes or subset of genes in the first biological sample is not similar to the gene expression profil e for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 14, 15 or more of the genes in Table 1. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 1.
[00136] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 1 , infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if: (i) the gene expression profile for the genes or subset of genes in the first biological sample is similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the
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SDI-60022 107vl gene expression profile for the genes or subset of genes in first biological sample is not similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 1. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15- 20, 20-25 or 25-30 of the genes in Table 1.
[0Θ137] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 2, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or a subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulator therapy, and (d) administering the immunomodulatory therapy to the first patient if the gene expression profile for the genes or subset of genes in the first biological sample is similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically sensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or more of the genes in Table 2. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 2.
[00138] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 2, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes i in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if the gene expression profile for
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SDI-60022 107vl the genes or subset of genes in the first biological sample is not similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 15 or more of the genes in Table 2. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15-20, 20- 25 or 25-30 of the genes in Table 2.
[0Θ139] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first biological sample from a first patient having a
hematological cancer, (b) measuring the expression of the genes or a certain subset of genes set forth in Table 2, infra, in the first biological sample, and (c) comparing the gene expression profile of the genes or subset of genes in the first biological sample to (i) the gene expression profile of the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the gene expression of the genes or subset of genes in tumor samples from patients having the same type of
hematological cancer which are clinically insensitive to the immunomodulator therapy, and (d) administering the immunomodulatory therapy to the first patient if: (i) the gene expression profile for the genes or subset of genes in the first biological sample is similar to the gene expression profile for the genes or subset of genes in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the gene expression profile for the genes or subset of genes in first biological sample is not similar to the gene expression profile for the genes or subset of genes in tumor sampl es from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory. In certain embodiments, the subset of genes comprises 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 14, 15 or more of the genes in Table 2. In some embodiments, the subset of genes comprises 2-5, 5-10, 10-15, 15- 20, 20-25 or 25-30 of the genes in Table 2.
[00140] In accordance with the methods described herein, the biological sample can be any sample obtained from the patient. In certain embodiments, the biological sample is a cell sample. In other embodiments, the biological sample is whole blood sample, peripheral blood mononuclear cell sample, or tissue sample. In specific embodiments, the biological sample is a tumor sample. See Section 5.8, infra, regarding biological samples.
[00141] In accordance with the methods described herein, the level of expression of one, two, three, four, five or more of the genes in Table I and/or Table 2 and/or Table 3 and/or Table 4, infra,
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SDI-60022 107vl can be measured at the RNA and/or protein levels. In certain embodiments, the level of expression of the genes are measured at the RNA (e.g., mRNA) level. In other embodiments, the level of expression of the genes are measured at the protein level.
[00142] Techniques known to one skilled in the art may be used to measure the amount of an RNA transcript(s). In some embodiments, the amount of one, two, three, four, five or more RNA transcripts is measured using deep sequencing, such as ILLUMINA® RNASeq, ILLUMINA® next generation sequencing (NGS), ION TORRENT™ RNA next generation sequencing, 454™ pyroseq encing, or Sequencing by Oligo Ligation Detection (SOLID™). In other embodiments, the amount of multiple RNA transcripts is measured using a mieroarray and/or gene chip, such as described in Section 6, infra. In certain embodiments, the amount of one, two, three or more RNA transcripts is determined by RT-PCR. In other embodiments, the amount of one, two, three or more RNA transcripts is measured by RT-qPCR. Techniques for conducting these assays are known to one skilled in the art. See Section 5.9, infra, for examples of assays for measuring RNA transcripts.
[00143] In some embodiments, a statistical analysis or other analysis is performed on data from the assay utilized to measure an RNA transcript or protein. In some specific embodiments, p value of those RNA transcripts or proteins differentially expressed is 0.1, 0.5, 0.4, 0.3, 0.2, 0.01, 0.05, 0,001, or 0,0001 , In specific embodiments, a false discovery rate (FDR) of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1% or less is selected.
[ 00144] Techniques known to one skill ed in the art may be used to measure the amount of a protein. For example, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay- based methodologies (ELISA) and similar assays known in the art. See Section 5.9, infra, for examples of assays for measuring RNA transcripts.
[00145] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells in the first tumor sample, (c) comparing the proportion of dendritic cells in the first tumor sample with the proportion of dendritic cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an
immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of dendritic cells in the first tumor sample is measured relative the proportion of dendritic cells in the second tumor sample.
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SDI-60022 107vl [00146] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of plasma cells in the first tumor sample, (c) comparing the proportion of plasma cells in the first tumor sample with the proportion of plasma cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an
immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first pati ent if a higher proportion of plasma cells in the first tumor sample is measured relative the proportion of plasma cells in the second tumor sample,
[00147] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtammg a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of dendritic cells and plasma cells in the first tumor sample, (c) comparing the proportion of dendritic cells and plasma cells in the first tumor sample with the proportion of dendritic cells and plasma cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of dendritic cel ls and plasma cells in the first tumor sample is measured relative the proportion of dendritic ceils and plasma cells in the second tumor sample.
[00148] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of B cells in the first tumor sample, (c) comparing the proportion of B cells in the first tumor sample with the proportion of B cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a decreased proportion of B ceils in the first tumor sample is measured relative the proportion of B cells in the second tumor sample.
[00149] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of tumor infiltrating immune cells in the first tumor sample, (c) comparing the proportion of tumor infiltrating immune cells in the first tumor sample with the
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SDI-60022 107vl proportion of tumor infi ltrating immune cel ls in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with an immimomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of tumor infiltrating immune cells in the first tumor sample is measured rel ative the proportion of tumor infiltrating immune cells in the second tumor sample.
[0Θ150] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of NK cells in the first tumor sample, (c) comparing the proportion of NK cells in the first tumor sample with the proportion of NK cells in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment with, an immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of NK. cells in the first tumor sample is measured relative the proportion of NK cells in the second tumor sample.
[0Θ151] In another aspect, provided herein are methods for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having the hematological cancer, (b) measuring the proportion of monocytes in the first tumor sample, (c) comparing the proportion of monocytes in the first tumor sample with the proportion of monocytes in a second tumor sample from a second patient having the same type of hematological cancer, wherein the second patient's hematological cancer is clinically insensitive to treatment wit an
immunomodulatory therapy, and (d) administering the immunomodulatory therapy to the first patient if a higher proportion of monocytes in the first tumor sample is measured relative the proportion of monocytes in the second tumor sample.
[00152] In certain embodiments of the foregoing paragraphs in this section, the second patient is a single patient. In other embodiments of the foregoing paragraphs in this section, the second patient is a population of patients. In specific embodiments, the population comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, .125, 150, 200, 225, 250, 300 or more patients.
[00153] In another aspect, provided herein are for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune ceils in the first tumor sample, and (c) comparing the
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SDI-60022 107vl proportion of the immune cells in the first tumor sample to (i) the proportion of the same immune ceils in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the
immunomodulatory therapy to the first patient if the proportion of the immune ceils in the first tumor sample is similar to the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the
immunomodulatory therapy. In some embodiments, the immune cells are subset of immune ceils, such as subset of B cel ls, in certain embodiments, the immune cells are dendritic cells, in some embodiments, the immune cells are plasma cells. In certain embodiments, the immune cells are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cells are T cells. In some embodiments, the immune cells are B cells. In certain embodiments, the immune cel ls are NK cells. In some embodiments, the immune cells are two, three or more subsets of immime cells, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cel ls in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy.
[00154] In another aspect, provided herein are for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immime cel ls in the first tumor sample, and (c) comparing the proportion of the immune cells in the first tumor sample to (i) the proportion of the same immune cel ls in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the
immunomodulatory therapy to the first patient if the proportion of the imm une cells in the first tumor sample is not similar to the proportion of the same immune cel ls in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the
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SDI-60022 l07vl immunomodulatory therapy. In some embodiments, the immune cells are subset of immune cells, such as subset of B cells. In certain embodiments, the immune ceils are dendritic ceils. In some embodiments, the immune cel ls are plasma cells. In certain embodiments, the immune cel ls are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cells are T cel ls. In some embodiments, the immune ceils are B cells. In certain embodiments, the immune cells are NK cells. In some embodiments, the immune cells are two, three or more subsets of immune cells, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cel ls in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy.
[00155] In another aspect, provided herein are for managing or treating a hematological cancer comprising: (a) obtaining a first tumor sample from a first patient having a hematological cancer, (b) measuring the proportion of immune cells in the first tumor sample, and (c) comparing the proportion of the immune cel ls in the first tumor sample to (i ) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to an immunomodulatory therapy and (ii) the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy, and (d) administering the
immunomodulatory therapy to the first patient if the proportion of the immune ceils in the first tumor sample is (i) similar to the proportion of the same immune cells in tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy, and (ii) not similar to the proportion of the same immune cells in tumor sampl es from patients having the same type of hematological cancer whi ch are clinical ly insensitive to the immunomodulatory therapy. In some embodiments, the immune cells are subset of immune cells, such as subset of B cells. In certain embodiments, the immune ceils are dendritic cells. In some embodiments, the immune cells are plasma cells. In certain embodiments, the immune cells are monocytes. In some embodiments, the immune cells are tumor infiltrating immune cells. In certain embodiments, the immune cells are T cells. In some embodiments, the immune cells are B cells. In certain embodiments, the immune cells are NK cells. In some
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SDI-60022 107vl embodiments, the immune cells are two, three or more subsets of immune cells, such as two more types of T cells (e.g., CD4+ and CD8+ T cells). In some embodiments, the proportion of different populations of immune cells in the first tumor sample are compared to (i) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically sensitive to the immunomodulatory therapy and (ii) the proportion of the same populations of immune cells in the tumor samples from patients having the same type of hematological cancer which are clinically insensitive to the immunomodulatory therapy.
[0Θ156] In addition to measuring the cells (e.g., dendritic cells and plasma cells) in a tumor sample from a patient, levels of expression of genes (e.g., one, two, three, four, five or more of the genes in Table 1 and/or Table 2 and/or Table 3 and/or Table 4) may be assessed. In specific embodiments, the methods set forth for measuring gene expression supra, are combined with the methods set forth for measuring a proportion of cells to determine if an immunomodulatory therapy is to be administered to a patient with, a hematological cancer.
[00157] Techniques known to one skilled in the art may be used to measure the proportion of ceils in a tumor sample. In certain embodiments, the proportion of cells is measured by flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay-based methodologies (ELISA) and similar assays known in the art. In other embodiments, the proportion of cells is measured by inference from gene expression profiles.
[00158] In accordance with the methods described herein, the immunomodulatory therapy can be any therapy that modulates the immune system or immune response. Examples of
immunomodulatory therapies are provided in Section 5.6, infra. In a specific embodiment, the immunomodulatory therapy is lenalidomi.de (Revlimid.®).
[00159] In specific embodiments, an immunomodulatory therapy is administered to a
hematological cancer patient in the form of a pharmaceutical composition. In a specific
embodiment, an pharmaceutical composition administered to a hematological cancer patient comprising an immunomodulatory therapy and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition may comprise an additional therapy, such as described in Section 5.7, infra. The dosage form of the pharmaceutical composition will vary depending upon the route of administration. The immunomodulatory therapy or a pharmaceutical composition thereof may be administered by any route of administration, such as oral, mucosal, parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular), topical, transdermal, or
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SDI-60022 107vl transcutaneous. In a specific embodiment, the immunomodulatory therapy or a pharmaceutical composition thereof is orally administered to a hematological cancer patient.
[00160] [00160] In accordance with the methods described herein, the dose of an
immunomodulatory therapy administered to a patient varies depending on a variety of factors, such as the health and age of the patient. In certain embodiments, in accordance with the methods described herein the patient is administered a dose of 0.01 mg to 1000 mg of an immunomodulatory therapy. In certain embodiments, in accordance with the methods described herein the patient is administered a dose of 0,01 mg to 500 mg of an immunomodulatory therapy. In certain
embodiments, in accordance with the methods described herein the patient is administered a dose of 0.01 mg to 100 mg of an immunomodulatory therapy. In some embodiments, in accordance with the methods described herein the patient is administered a dose of 0.1 mg to 500 mg of an immunomodulatory therapy. . In some embodiments, in accordance with the methods described herein the patient is administered a dose of 0.01 mg to 500 mg of an immunomodulatoiy therapy. In some embodiments, in accordance with the methods described herein the patient is administered a dose of 1 mg to 500 mg of an immunomodulatory therapy. In certain embodiments, in accordance with the methods described herein the patient is administered a dose of 0.1 mg to 100 mg of an immunomodulatory therapy. In certain embodiments, in accordance with the methods described herein the patient is administered a dose of 1 mg to 100 mg of an immunomodulatory therapy. In some embodiments, in accordance with the methods described herein the patient is administered a dose of 1 mg to 50 mg of an immunomodulatoiy therapy. In some embodiments, in accordance with the methods described herein the patient is admmistered a dose of 1 mg to 100 mg of an
immunomodulatory therapy, in some embodiments, in accordance with the methods described herein the patient is administered a dose of 1 mg to 500 mg of an immunomodulatory therapy. In some embodiments, in accordance with the methods described herein the patient is administered a dose of 1 mg to 1000 mg of an immunomodulatoiy therapy. The dose of the immunomodulatory therapy can be administered once, twice or three times per day. The dose of the immunomodulatory therapy can be administered every other day, every two days, every three days, every four days, every five days, every six days, or once per week. Specific doses per day include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg per day. The dose of the immunomodulatory therapy is administered in accordance with the label for the therapy. The immunomodulatory therapy can be lenalidomide (Revlimid®), or its pharmaceutically acceptable
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SDI-60022 107vl salt, solvate, hydrate or stereoisomer, and it is administered at a dose of 1 to 50 mg per day, or anything in between, or 25 mg per day.
[00161] In accordance with the methods described herein, the hematological cancer can be any hematological cancer. Examples of hematological cancers can be found in Section 5.5, infra. In a specific embodiment, the hematological cancer is a lymphoma. In another specific embodiment, the hematological cancer is a non-Hodgkm's lymphoma. In yet another embodiment, the hematological cancer is a diffuse large B-celi lymphoma (DLBCL). In certain embodiments, the DLBCL is a germinal center B-cell-like DLBCL. In other embodiments, the DLBCL is an activated B-cell-like DLBCL.
[00162] in certain embodiments, the methods of managing or treating a hematological cancer involve the administration of another therapy. In some embodiments, the other therapy is to alleviate pain or one or more other symptoms associated with the hematological cancer. Examples of other therapies that may be used in combination with an immunomodulatory therapy are disclosed in Section 5.7, infra. In certain embodiments, one or more of the following additional active ingredients are administered in combination with an immunomodulatory therapy in accordance with the methods described herein: oblimersen, melphalan, G-CSF, GM-CSF, EPO, a co -2 inhibitor, topotecan, pentoxifylline, ciprofloxacin, taxotere, iritotecan, dexamethasone, doxorubicin, vincristine, IL 2, IFN, dacarbazine, Ara-C, vinorelbine and/or isotretinoin. In a specific embodiment, chemotherapeutic agents, such as cyc!ohexarnide, hydroxy daunorubicin, Oncovin, and prednisone (CHOP), are used in combination with an immunomodulatory therapy, such as lenalidomi.de, in accordance with the methods described herein. In another specific embodiment, rituximab is used in combination with an immunomodulatory therapy, such as lenalidomide,. In another specific embodiment, CHOP and rituximab are used in combination with an immunomodulatory therapy, such as lenalidomide.
5.5 Hematological Cancers
[001 3] In some embodiments, the hematological cancer is a lymphoma. In other embodiments, the hematological cancer is a leukemia. In one embodiment, the hematological cancer is multiple myeloma. In another embodiment, the hematological cancer is chronic lymphocytic leukemia (CLL). In another embodiment, the hematological cancer is myelodysplastic syndrome, an acute leukemia, e.g., acute T cell leukemia, acute myelogenous leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia (Burkitt's
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SDI-60022 107vl lymphoma), or acute biphenotypic leukemia; a chronic leukemia, e.g., chronic myeloid lymphoma, chronic myelogenous leukemia (CML), chronic monocytic leukemia, small lymphocytic lymphoma, or B-celi prolymphocytic leukemia; hair}' cell lymphoma; T-cell prolymphocytic leukemia; or a lymphoma, e.g, histiocytic lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom rnacroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasm (e.g., plasma cell myeloma, plasmacytoma, a monoclonal immunoglobulin deposition disease, or a heavy chain disease), extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal N /T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sezary syndrome), a primary cutaneous CD30-positive T cell
lymphoproliferative disorder {e.g., primary cutaneous anaplastic large cell lymphoma or
lymphomatoid papulosis), angioimmunoblastic T cell lymphoma, peripheral T cel l lymphoma, unspecified, anaplastic large cell lymphoma, a Hodgkin's lymphoma or a nodular lymphocyte- predominant Hodgkin's lymphoma.
[00164] In a specific embodiment, the hematological cancer is DLBCL. In another specific embodiment, the hematological cancer is an activated B-ee!l-like DLBCL. In another specific embodiment, the hematological cancer is a germinal center B-cell-like DLBCL.
5.6 Immunomodulatory Therapies
[00165] Immunomodulatory therapies described in the methods provided herein include compounds known as "IMiDs* (Ceigene Corporation), a group of compounds that can be useful to treat several types of human diseases, including certain cancers.
[00166] As used herein and unless otherwise indicated, the v rms "immunomodulatory compound", "immunomodulatory agent" and "immunomodulator therapy" are used
interchangeably, and can encompass certain small organic molecules that inhibit LPS induced monocyte TNF-a, IL-1B, IL-12, IL-6, MIP-la, MCP-1 , GM-CSF, G-CSF, and COX-2 production. These compounds can be prepared synthetically, or can be obtained commercially.
[00167] Exemplary immunomodulating compounds include but are not limited to N-{[2-(2,6- dioxo(3-piperidyi)-l,3-dioxoisomdolin-4-yl.]met ' hyl}cyclopropyl-carboxamide; 3-[2-(2,6-dioxo-
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SDI-60022 107vl piperidin-3-yl)-l ,3-dioxo-2,3-dihydro-lH-iso (-)-3-(3,4- Dimethoxy-phenyl)-3-(l-oxo-l ,3-dihydro-isoindol-2-yl)-propionamide; (+)-3-(3,4-Dimethoxy- phenyl)-3-(l -oxo-l ,3-dihydro-isoindol-2-yl)-propionamide; (-)- {2-[.l -(3-ethoxy-4-m.ethoxyphenyl)- 2-methylsulfony lethyl]-4-acetylaminoisomdoline- 1 ,3-dione} ; (+)- (2-[ 1 -(3-ethoxy-4- m.ethoxypheny3)-2-methyisu3 onylethyi]-4-acetylaminoisoindoime-l. ,3-dione} ; Difluoro-methoxy SelCIDs; l-phthalimido-l -(3,4-diethoxyphenyl)ethane; 3-(3,4-dimethoxyphenyl)-3-(3,5- dimethoxyphenyl)acrylo nitrile; 1 -oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; 1 ,3-dioxo- 2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; 4-amino-2-(3-methyl-2,6-dioxo-piperidine-3-yl)- isoindole- 1 ,3-dione; 3 -(3 -acetoarnidophthalimido)-3 -(3 -ethoxy-4-methoxyphenyl)-N- hydroxypropionamide; l -oxo-2-(2,6-dioxopiperidrn-3-yi)-4-methylisorndolme; Cyclopropyi-N- {2- [( 1 S)- 1 -(3 -ethoxy-4-methoxypheny l)-2-(methylsulfonyl)ethyl] -3 -oxoisoindoline-4-yl } car boxamide; Substituted 2-(3-hydroxy-2,6- dioxopiperidin-5-yl) isoindoline; N-[2-(2,6-Dioxo-piperidin-3-yl)- l ,3-dioxo-23-dihydro-l H-isoindol-5-ylmethyl]-4-trifluoromethoxybenzamide; (S)-4-chloro-N-((2- (3-methyi-2,6-dioxopiperidin-3-yi)-l ,3-dioxoisomdo3.in-5-yl)raethyl) benzamide; Pyri.dine-2- carboxyHc acid [2-[(3S)-3-methyl-2,6-dioxo-piperidin-3-yl]-l ,3-dioxo-2,3-dihydro-lH-isoindol-5- yimethyl]-amide; (S)-N-((2-(3-methyl-2,6-dioxopiperidin-3-yl)-13-dioxoisoindo lin^
(trifluoromethy3 )benzamide; 3-(2,5-dimethyi-4-oxo-4H-q inazoirn-3-yi)-piperidme-2,6-dione, and the like. In a specific embodiment, the immunomodulatory compound is 3 -(4-amino- 1 -oxo- 1,3- dihydro-isoindol-2-yl)-piperidine-2,6-dione, or a salt, solvate or hydrate thereof.
[00168] Immunomodulatory compounds disclosed herein may enhance the degradation of TNF-a mRNA. Immunomodulatory compounds disclosed herein may also be potent co-stimulators of T cells and increase cell proliferation dramatically in a dose dependent manner. Immunomodulatory compounds disclosed herein may also have a greater co-stimuiatory effect on the CD8+ T cell subset than on the CD4+ T cell subset. Immunomodulatory compounds disclosed herein may be capable of acting both indirectly through cytokine activation and directly on Natural Killer ("NK") cel ls and Natural Killer T ("NKT") cells, and increase the NK cells' abi lity to produce beneficial cytokines such as, but not limited to, IFN-γ, and to enhance NK and NKT cell cytotoxic activity.
[00169] Specific examples of immunomodulatory compounds include cyano and carboxy derivatives of substituted styrenes such as those disclosed in U.S. patent no. 5,929, 1 17; l-oxo-2- (2,6-dioxo-3-f3.uoropiperi.din-3yl) isoindolines and l ,3-dioxo-2-(2,6-dioxo-3-fiuoropiperid.ine-3-yi) isoindolines such as those described in U.S. patent nos. 5,874,448 and 5,955,476; the tetra substituted 2-(2,6-dioxopiperdin-3-yl)-l-oxoisoindolines described in U.S. patent no. 5,798,368; I-
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SDI-60022 107vl oxo and l,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines (e.g., 4-meth.yl derivatives of thalidomide), substituted 2-(2,6-dioxopiperidm-3-yi) phthalimides and substituted 2-(2,6- dioxopiperidin-3-yI)- 1 -oxoisoindoles including, but not limited to, those disclosed in U.S. patent nos. 5,635,517, 6,281,230, 6,316,471, 6,403,613, 6,476,052 and 6,555,554; i - oxo and 1 ,3- dioxoisoindolmes substituted in the 4~ or 5-position of the indoline ring (e.g., 4-(4-amino-l,3- dioxoisoindoline-2-yl)-4-carbamoylbutanoic acid) described in U.S. patent no. 6,380,239;
isoindoiiiie-l-one and isoindoline-l,3-dione substituted in the 2-position with 2,6-dioxo-3- hydroxypiperi.din-5-yl (e.g., 2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoind olin-l- one) described in U.S. patent no. 6,458,810; a class of non-polypeptide cyclic amides disclosed in U.S. patent nos. 5,698,579 and 5,877,200; and isoindofe-imide compounds such as those described in U.S. patent publication no. 2003/0045552 published on March 6, 2003, U.S. patent publication no. 2003/0096841 published on May 22, 2003, and international Application No. PCT/US01/50401 (International Publication No. WO 02/059106). US patent publication no. 2006/0205787 describes 4-armno~2-(3~methy l-2,6-dioxopiperidin~3-y l)-isoindole- 1 ,3-dione compositions. US patent publication no. 2007/0049618 describes isoindole-imide compounds. The entireties of each of the patents and patent applications identified herein are incorporated by reference. In one embodiment, immunomodulatory compounds do not include thalidomide.
[0Θ170] Various immunomodulatory compounds disclosed herein contain one or more chiral centers, and can exist as racemic mixtures of enantiomers or mixtures of diastereomers. Thus, also provided herein is the use of stereomerically pure forms of such compounds, as well as the use of mixtures of those forms. For example, mixtures comprising equal or unequal amounts of the enantiomers of a particular immunomodulatory compounds may be used. These isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., Jacques, J., et αί, Enantiomers, Racemates and Resolutions
(Wiley-Interscience, New York, 1981); Wilen, S. H., et al, Tetrahedron 33:2725 (1977); Eiiel, E. L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H,, Tables of Resolving Agents and Optical Resolutions p. 268 (E . Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).
[00171] Immunomodulatory compounds provided herein include, but are not limited to, 1 -oxo- and 1 ,3 dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines substituted with amino in the benzo ring as described in U.S. Patent no. 5,635,517 which is incorporated herein by reference.
[00172] These compounds have the structure I:
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SDI-60022 107vl
I
in which one of X and Y is C=0, the other of X and Y is C=0 or CH 2 , and R 2 is hydrogen or lower alkyl, in particular methyl. Specific immunomodulatory compounds include, but are not limited to:
l-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;
1 -dioxo-2-(2,6-dioxopiperidin-3-yl)-4-ammoisoindolme; and
1 ,3-dioxo~2-(3-rnethyi~2,6-dioxopiperidm-3~yi)~4~ammoisoindoi e, and optically pure isomers thereof.
[00173] The compounds can be obtained via standard, synthetic methods (see e.g.. United States Patent No. 5,635,517, incorporated herein by reference). The compounds are also available from Celgene Corporation, Warren, NJ.
[00174] Other specific immunomodulatory compounds belong to a cl ass of substituted 2~(2,6~ dioxopiperidin-3-yl) phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)- 1 -oxoisoindoles, such as those described in U.S. patent nos. 6,281,230; 6,316,471 ; 6,335,349; and 6,476,052, and
International Patent Application No. PCT/US97/13375 (International Publication No. WO
98/03502), each of which is incorporated herein by reference. Representative compounds are of formula:
- 67 -
226269-228501 / 12827-501-228
SDI-60022 107vl
in which:
one of X and Y is C O and the other of X and Y is C=0 or CH 2 ;
(i) each of R 1 , R z , R ~ , and R 4 , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , and R 4 is -NHR 5 and the remaining of R 3 , R " l R', and R 4 are hydrogen;
R "' is hydrogen or alkyl of 1 to 8 carbon atoms;
R 6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or halo;
provided that R° is other than hydrogen if X and Y are C=0 and (i) each of R 1 , R 2 , R 3 , and R 4 is fluoro or (ii) one of R 3 , R", R', or R 4 is amino.
[0Θ175] Compounds representative of this class are of the formulas:
wherein R is hydrogen or methyl. In a separate embodiment, provided herein is the use of enantiomerically pure forms (e.g. optically pure (R) or (8) enantiomers) of these compounds.
[00176] Still other specific immunomodulatory compounds disclosed herein belong to a class of isoindole-imides disclosed in U.S. Patent No. 7,091 ,353, U.S. Patent Publication No.
2003/0045552, and International Application No. PCT/USOl/50401 (International Publication No. WO 02/059106), each of which are incorporated herein by reference. Representative compounds are of formula II:
I I
- 68 -
226269-228501 / 12827-501-228
SDI-60022 107vl and pharmaceutical fy acceptable salts, hydrates, solvates, cfathrates, enantiomers, diastereomers, racemates, and mixtures of stereoisomers thereof, wherein:
one of X and Y is C :: O and the other is CH 2 or C :::: 0;
R 1 is H, (Ci-Cg )alkyl, (C3--C 7 )cycloalkyl, (C 2 ~C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (Co- C4)alkyKC i ~C6)heterocycloaikyi, (C 0 -C 4 )alkyl-(C 2 -C5)heteroaryl, C(G)R 3 , C(S)R 3 , C(0)OR , (C C 8 )alkyl---N(R 6 )2, (C r C 8 )alkyl--QR 5 , (C C 8 )alkyl-C(0)OR 5 , C(0)NHR 3 , C(S)NHR 3 , C(0)NR R 3' C(S)NR R 3 ' or (Cj-C 8 )alkyl--0(CO)R 5 ;
R 2 is H, F, benzyl, (Ci-Cgjalkyl, (C 2 -Cs)alkenyi, or (C 2 -C 8 )alkynyl;
R 3 and R" are independently (Cj-C 8 )alkyl, (C3-C 7 )cycloalkyl, (C 2 -C 8 )alkeiiyl, (C 2 -C 8 )alkynyl, benzyl, aryi, (Co-C4)alkyl---(C C 6 )heterocycloaikyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, {( ' „-< \ }a Iky I N(R 6 )2, (C)-C 8 )alkyl-OR 5 , (Ci-C 8 )alkyl-C(0)OR 5 , (Ci-C 8 )alkyl-0(CO)R 5 , or C(0)OR 5 ;
R 4 is (Ci~C 8 )aikyi, (C 2 -C 8 )alkenyl, (C 2 -C 8 )al.kynyl, (Ci -C 4 )alky l-OR 5 , benzyl, and, (C 0 -C 4 )alkyl- (C i -C6)heterocycloalkyl, or (Co-C 4 )alkyl-(C ? .-C 5 )heteroaryl;
R "' is (Cj-C 8 )alkyl, (C 2 ~C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, or (C 2 ~C5)heteroaryI;
each occurrence of R 6 is independently H, (Ci~C 8 )alkyl, (C?~C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 2 -Cs)heteroaryl, or (Co-C 8 )alkyl-C(0)0-R 5 or the R 6 groups can join to form a
heterocycloalkyl group;
n is 0 or 1 ; and
* represents a chiral-carbon center.
[00177] In specific compounds of formula II, when n is 0 then R 1 is (C 3 -C 7 )cycloalkyl, (C 2 - C 8 )alkenyi, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 0 -C )alkyl-(C 1 -C6)heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 - Csjheteroaryl, C(0)R 3 , C(0)OR 4 , (CVC 8 )aIkyI-N(R 6 ) 2 , (Ci-C 8 )alkyl-OR 5 , ((VC, ;a!ky! C«})OR\ C(S)NHR 3 , or (C C s )aikyi-0(CO)R 5 ;
R *? is H or (Ci-C 8 )alkyl; and
R 3 is (Ci-C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl (C 0 -C 4 )alkyl- (Ci -C 6 )heterocycloaikyi, (O C alkyl {( ' -C \}hcieroaryi. (C 5 -C 8 )aikyi-N(R 6 )2 ; (C 0 -C 8 )alkyl- NH-C(0)Q-R 5 ; (C C 8 )alkyl~-OR 5 , (C C 8 )aIkyI~-C(0)OR 5 , (C 1 -C 8 )alkyI~-0(CO)R 5 , or C(G)OR 5 ; and the other variables have the same definitions.
[00178] In other specific compounds of formula II, R 2 is H or (CrC^alkyl.
[00179] In other specific compounds of formula II, R 1 is (Ci-C 8 )alkyl or benzyl. 180 In other specific compounds of formula Π, R ' is H, (C 1 -Cs)aikyl, benzyl,€Ή 2 0Ο¾,
[00181] in another embodiment of the compounds of formula II, R is
wherein Q is O or S, and each occurrence of R' is independently H,(Ci-€ 8 )alkyl, (C 3 -
C ? )cyeloalkyl, (Cr-CVjalkenyl, (C?-- 8 )alkynyl, benzyl, aryl, halogen, (Co~C4)alk l~(C i- C 6 )heterocycloalkyl, (Co-C 4 )alkyl-(C2-C5)heteioai l, (Co-C 8 )alkyl-N( 6 )2, (Ci-C 8 )alkyl-OR 5 ,
(Cr-C 8 )alkyl--C(0)OR 5 , (Ci-C 8 )alkyl-0(C0)R 5 , or C(0)OR 5 , or adjacent occurrences of R 7 can b taken together to form a bicyclic alkyl or aryl ring,
[00182] In other specific compounds of formula II, R ! is C(0)R 3 .
[0Θ183] In other specific compounds of formula II, R 3 is (C 0 -C 4 )alkyl-(C 2 -C5)heteroaryl, (C \ - C 8 )alkyl, aryl, or (C,. r C , )a] ky] OR .
[00184] In other specific compounds of formula II, heteroaryl is pyridyl, furyl, or thienyl.
[00185] In other specific compounds of formula II, R 1 is C(0)OR 4 .
[00186] In other specific compounds of formula II, the H of C(0)NHC(0) can be replaced with (Cj~C 4 )aikyi, and, or benzyl,
[00187] Further examples of the compounds in this class include, but are not limited to: [2 -(2,6 dioxo-piperidin-3-yl)-l ,3-dioxo-2,3-dihydro-lH-isoindol-4-ylmethyl]-amide; (2-(2,6-dioxo- piperidin-3-yl)-l ,3-dioxo-2,3-dihydro-l H-isoindo]-4-ylmethyl)-carbamic acid tert-butyi ester; 4- (aminomethyl)-2~(2,6-dioxo(3-piperidyl))-isomdoline-I ,3-dione; iV-(2-(2,6-dioxo-piperidin-3-yl)- l ,3-dioxo-2,3-dihydro-lH-isoindo{~4-ylmethyl)-acetamide; N- {(2-(2,6-dioxo(3-piperidyI)- 1 ,3- dioxoisoindolin-4-yl)methyl} cyclopropyl-carboxamide; 2-chIoro-A L {(2-(2,6-dioxo(3-piperidyI))- 1 ,3-dioxoisoindolin-4-y l)methyl} acetamide; A-(2-(2,6-dioxo(3-piperidyl))- 1 ,3-d.ioxoisoindolin-4- yl)-3-pyridylcarboxamide; 3- { l-oxo-4-(benzylamino)isomdoIin-2-yI}piperidine-2,6-dione; 2-(2,6- dioxo(3-piperidyl))-4-(benzylammo)isomdolme-l ,3-dione; iV-{(2-(2,6-dioxo(3-piperidyl))-l ,3- dioxoisomdolin-4-yl)methyl} ropanamide; N- {(2-(2,6-dioxo(3-piperidyl))- 1 ,3-dioxoisoindolin-4- yl)methyl} -3-pyridylcarboxamide; N-{(2-(2,6-dioxo(3-piperidyl))-l,3-dioxoisoindolin-4- yl)methy 1 } heptanamide; JV- {(2-(2,6-dioxo(3-piperidyi))- 1 ,3-dioxoisoindolm-4-yl)methyl } -2- furylcarboxamide; {N-(2-(2,6-dioxo(3-piperidyl))- l ,3-dioxoisoindolin-4-yl)carbamoyl}methyl acetate; N-(2-(2,6-dioxo(3-piperidyl))-l^ N-(2-(2,6~dioxo(3- piperidyl))-1 -dioxoisoindoliii-4-yl)-2 hienylcarboxamide; N- {[2-(2,6-dioxo(3-piperidyl))-l ,3- dioxoisoindolin-4- lj methyl} (butylamino)carboxamide; N-{[2-(2,6-dioxo(3~piperidyl))-l ,3- dioxoisoindolin-4-yl] methyl} (octylamino)carboxamide; and N- {[2-(2,6-dioxo(3-piperidyl))- l ,3- dioxoisoindolm-4-yl] methyl} (benzylamino)carboxamide.
[00188] Still other specific immunomodulatory compounds disclosed herein belong to a class of isomdole-imides disclosed in U.S. Patent Application Publication Nos. US 2002/0045643, International Publication No, WO 98/54170, and United States Patent No. 6,395,754, each of which is incorporated herein by reference. Representative compounds are of formula III:
and pharmaceutically acceptable salts, hydrates, solvates, clathrates, enantiomers, diastereomers, racemates, and mixtures of stereoisomers thereof, wherein:
one of X and Y is 0=0 and the other is C¾ or C=0;
R is H or CH 2 OCOR';
(i) each of R 1 , R , R J , or R 4 , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R f , R 2 , R 3 , or R 4 is nitro or -NHR 3 and the remaining of R l , R 2 , R 3 , or R 4 are hydrogen;
R 5 is hydrogen or alkyl of 1 to 8 carbons
R 6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;
R' is R 7 ~CHR i0 ~N(R 8 R 9 );
R' is m-phenylene or p-phenylene or -(CnH2n)- in which n has a value of 0 to 4;
each of R 8 and R 9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or
R 8 and R taken together are tetramethylene, pentamethylene, hexamethylene,
or ··( 1 1 'CI I : X ! ( H . <( 1 1 ' in which X 1 is -0-, -S-, or -NH-;
R 1 is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and
* represents a chiral-carbon center.
[00189J Other representative compounds are of formula:
- 71 -
226269-228501 / 12827-501-228
SDI-60022 107vl
wherein:
one of X and Y is C O and the other of X and Y is C=0 or CH 2 ;
(i) each of R l , R 2 , R 3 , or R , independently of the others, is halo, aikyi of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R% R 3 , and R 4 is ~NHR J and the remaining of R ¾ , R 2 , R', and R 4 are hydrogen;
R 5 is hydrogen or alkyl of 1 to 8 carbon atoms;
R" is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;
R' is m-phenylene or p-phenylene or -(CnH2n)~ in which n has a value of 0 to 4;
each of R 8 and taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8 and R 9 taken together are tetrametliylene, pentametiivlene, hexaniethylene, or - CH 2 CH 2 X f CH 2 CH 2 - in which X f is -0-, -S-, or -NH-; and
R 10 is hydrogen, aikyi of to 8 carbon atoms, or phenyl.
[00190] Other representative compounds are of formula:
in which
one of X and Y is C=0 and the other of X and Y is C=0 or CH 2 ;
each of R 1 , R 2 , R J , and R 4 , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , K 2 , R 3 , and R 4 is nitro or protected amino and the remaining of R ¾ , R 2 , R 3 , and R 4 are hydrogen; and
R° is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.
[00191] Other representative compounds are of formula:
in which :
one of X and Y is C=0 and the other of X and Y is C=0 or CH 2 ;
(i) each of R 1 , % R J , and R 4 , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R " , R 3 , and R 4 is -NHR 5 and the remaining of R 1 , R , R J , and R 4 are hydrogen;
R 3 is hydrogen, alkyl of 1 to 8 carbon atoms, or CO-R ? -CH(R u ')NR 3 R 9 in which each of R 7 , R 8 , R 9 , and R 10 is as herein defined; and
R° is alkyl of 1 to 8 carbon atoms, benzo, eh!oro, or fluoro.
[00192] Specific examples f the compounds are of formula:
in which:
one of X and Y is C O and the other of and Y is C=0 or CH 2 ;
R° is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, chloro, or fluoro;
R ' is m-phenylene, p-phenylene or -(CnH2n)- in which n has a value of 0 to 4;
each of R 8 and R 9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8 and R 9 taken together are tetramethylene, pentamethylene, hexamethyiene, or - ( Ί ί . ,Π Ι ^ ' α Ι ! ,-· in which X 1 is -0-, -S- or -NH-; and
R 10 is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl,
[00193] Other specific immunomodulatory compounds are l -oxo-2-(2,6-dioxo-3-fluoropiperidin- 3yl) isoindoiines and l ,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in U.S. patent nos. 5,874,448 and 5,955,476, each of which is incorporated herein by reference. Representative compounds are of formula:
wherein:
Y is oxygen or H 2 and
226269-22850! / 12827-501-228
SDI-00022 107vl each of R ! , R y' , R \ and R 4 , independently of the others, is hydrogen, halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino,
[001 4] Other specific immunomodulatory compounds are the tetra substituted 2-(2,6- dioxopiperdin-3-yl)- 1 -oxoisoindolines described in U.S. patent no. 5,798,368, which is incorporated herein by reference. Representative compounds are of formula:
wherein each of R 1 , R 2 , R 3 , and R '* , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms.
[0Θ195] Other specific immunomodulatory compounds are 1-oxo and l,3-dioxo-2-(2,6- dioxopiperidin-3-yi) isoindolines disclosed in U.S. patent no. 6,403,613, which is incorporated herein by reference. Representative compounds are of formula:
in which
Y is oxygen or H 2 ,
a first of R 1 and R 2 is halo, alkyl, alkoxy, alkvlamino, dialkylamino, cyano, or carbamoyl, the second of R 1 and independently of the first, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl, and
R 3 is hydrogen, alkyl, or benzyl.
[00196J Specifi c examples of the compounds are of formula:
wherein
a first of R 1 and R 2 is halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl;
- 74 -
226269-228501 / 12827-501-228
SDI-60022 107vl the second of and R ' , independently of the first, is hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylamino in which each alky] is of from 1 to 4 carbon atoms, cyano, or carbamoyl; and R " ' is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl. Specific examples include, but are not limited to, l ~oxo-2-(2,6-dioxopiperidin-3-yl)-4~methylisoindoline,
[00197] Other representative compounds are of formula:
wherein:
a first of R 1 and R 2 is halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl;
the second of R 1 and ", independently of the first, is hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl; and R 3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl,
[00198] Other specific immunoniodulatory compounds disclosed herein are 1-oxo and 1,3- dioxoisoindo lines substituted in the 4- or 5 -position of the indoline ring described in U.S. Patent No. 6,380,239 and U.S. Patent No. 7,244,759, both of which are incorporated herein by reference. Representative compo nds are of formula:
in which the carbon atom designated C* constitutes a center of chirality (when n is not zero and R 1 is not the same as R"); one of X 1 and X y' is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X 1 or X'' is hydrogen; each of R 1 and R 2 independent of the other, is hydroxy or NH-Z; R 3 is hydrogen, alkyl of one to six carbons, halo, or haloalkyl; Z is hydrogen, aryl, alkyl of one to six carbons, formyl, or acyl of one to six carbons; and n has a value of 0, 1, or 2; provided that if X 1 is amino, and n is 1 or 2, then R 1 and R 2 are not both hydroxy; and the salts thereof.
[00199] Further representati ve compounds are of formula:
- 75 -
226269-228501 / 12827-501-228
SDI-60022 107vl
in which the carbon atom designated C* constitutes a center of chirality when n is not zero and R 1 is not R 2 ; one of X 1 and X 2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X' or X 2 is hydrogen; each of R 1 and R 2 independent of the other, is hydroxy or NH-Z; R 3 is alkyl of one to six carbons, haio, or hydrogen; Z is hydrogen, ary l or an alkyl or acy l of one to six carbons; and n has a value of 0, 1, or 2.
[00200] Specific examples include, but are not limited to, 2-(4-amino-l -oxo-l,3-dihydro- isoindo[-2-yl)-4-carbamoy[-butyric acid and 4-(4-amino- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl)-4- cabamoyl-butyric acid, which have the following structures, respectively, and pharmaceutically acceptable salts, solvates, prodrugs, and stereoisomers thereof:
in which the carbon atom designated C* constitutes a center of chirality when n is not zero and R 1 is not R 2 ; one of X ! and X 2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X'or X" is hydrogen; each of R 1 and R 2 independent of the other, is hydroxy or NH-Z; R ~ is alkyl of one to six carbons, halo, or hydrogen; Z is hydrogen, aryi, or an alkyl or acyl of one to six carbons; and n has a value of 0, 1, or 2; and the salts thereof,
[00202] Specific examples include, but are not limited to, 4-carbaraoyl-4-{4-[(furan-2-yl- methyl)-amino]-l,3-dioxo-l,3-dihydro-isomdol-2-yl} -butyric acid, 4-carbamoyl-2- {4-[(furan-2-yl- me†hy3)-ammo]-1 -diox«-1 -dihydro-isoindoi-2-yl} -butyric acid, 2-{4-[(furan-2-yl-meth l)- amino]- 1 ,3-dioxo- 1 ,3-dihydro-isoindoi-2-yl} -4-phenylcarbamoyl-butyric acid, and 2- {4-[(furan-2- yl-methyl)-amino]- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl} -pentanedioic acid, which have the
- 76 -
226269-228501 / 12827-501-228
SDI-60022 107vl following structures, respectively, and pharmaceutically acceptable salts, solvate, prodrugs, and stereoisomers thereof:
Other specific examples of the compounds are of formula:
wherein;
one of X 1 and X 2 is nitro, or NH-Z, and the other of X 1 or X" is hydrogen;
each of R 1 and R 2 , independent of the other, is hydroxy or NH-Z;
R 3 is alkyl of one to six carbons, halo, or hydrogen;
Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of one to six carbons; and n has a value of 0, 1 , or 2; and
if -COR" and -(CH 2 ) /2 COR 1 are different, the carbon atom designated C constitutes a center of chirality.
[00204] Other representative compounds are of formula:
wherein:
one of X 1 and X y' is alkyl of one to six carbons;
each of R 1 and R 2 , independent of the other, is hydroxy or NH-Z;
226269-228501 / 12827-501-228
SDI-60022 107vl R 3 is alky! of one to six carbons, halo, or hydrogen;
Z is hydrogen, phenyl, an acyl of one to six carbons, or an aikyi of one to six carbons; and n has a value of 0, 1 , or 2; and
if -COR " and -(CH?)«COR ! are different, the carbon atom designated C constitutes a center of chirality.
[00205] Still other specific immunomodulatory compounds are isoindoline- 1 -one and
isoindoline-1 ,3-dione substituted in the 2 -position with 2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S. patent no. 6,458,810, which is incorporated herein by reference. Representative compounds are of formula:
wherein;
the carbon atoms designated constitute centers of chirality;
X is ··( ·{())·· or -CH -:
R 5 is alky! of 1 to 8 carbon atoms or -NFIR 3 ;
R 2 is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen; and
R 3 is hydrogen,
alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms,
cycioalkyl of 3 to 18 carbon atoms,
phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of I to 4 carbon atoms,
benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms, or -COR 4 in which
R 4 is hydrogen,
alkyl of ! to 8 carbon atoms, unsubstituted or substituted with alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms,
cycioalkyl of 3 to 18 carbon atoms,
phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms, or
- 78 -
226269-228501 / 12827-501-228
SDI-60022 107vl benzyl, uns bstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms.
)6] Other specific compound s provided herein are of formula: and pharmaceutically acceptable salts, solvates, and stereoisomers thereof, wherein:
R 1 is : hydrogen; halo; -(Ο-ϊ 2 ) η 0Η; (C 1 -C 6 )alkyl, optionally substituted with one or more halo;
(C 1 -C 6 )alkoxy, optionally substituted with one or more halo; or
-(CH 2 ) n NHR a , wherein R a is:
hydrogen;
(Cs-C6)aikyi, optionally substituted with one or more halo;
~(CI¾) n ~(6 to 10 membered aryl);
··( ' {() )··{Π I ·■}„··('> to 10 membered aryl) or -C(0)-(CH 2 ) n -(6 to 10 membered
heteroaryl), wherein the aryl or heteroaryl is optionally substituted with one or more of: halo; -SCF ; (C-.-CcjalkyL itself optionally substituted with one or more halo; or (Cj-C,5)alkoxy, itself optionally substituted with one or more halo;
-C(0)-(C 1 -Cg)alkyl, wherein the alkyl is optionally substituted with one or more halo;
-C(0)-(CH 2 )n-(C 3 -C lo-cycloalkyl);
-C(0)-(CH2)„-NR b R c , wherein R° and R c are each independently:
hydrogen;
(Cj-Cejalkyl, optionally substituted with one or more halo;
(Ci-Ce koxy, optionally substituted with one or more halo; or
6 to 10 membered aryl, optionally substituted with one or more of:
halo; (C j -C6)alkyi, itself optionally substituted with one or more halo; or (C 1 -C6)alkoxy, itsel f optionally substituted with one or more halo;
-C(0)-(CH 2 )„-0-(C j -C 6 )alkyl; or
-C(0)-(Ci-I 2 )n-0-(C B2)n-i6 to 10 membered aryl);
R 2 is: hydrogen; -(CH 2 ) n OH; phenyl; -0-(CrC 6 )aikyl; or (Ci-C 6 )alkyl, optionally substituted with one or more halo;
- 79 -
226269-22850! / 12827-501-228
SDI-00022 107vl 3 is: hydrogen; or (C 1 -C 6 )alkyl, optionally substituted with one or more halo; and
n is 0, 1 , or 2.
[00207] Specific examples include, but are not limited to, 3-(5-ammo-2-metbyi-4-oxo-4H- quinazolin-3-yl)-piperidine-2,6-dione ( h has the following structure:
or an enantiomer or a mixture of enantiomers thereof; or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.
[00208] Compound A can be prepared according to the methods described in the Examples provided herein or as described in U.S. Pat. No. 7,635,700, the disclosure of which is incorporated herein by reference in its entirety. The compound can be also synthesized according to other methods apparent to those of skill in the art based upon the teaching herein, in certain
embodiments, Compound A is in a crystalline form described in U.S. Provisional Pat. App. No. 61/451 ,806, filed March 1 1 , 201 1 , which is incorporated herein by reference in its entirety, in some embodiments, the hydrochloride salt of Compound A is used in the methods provided herein.
Methods of treating, preventing and/or managing cancers and other diseases using Compound A are described in U.S. Provisional Pat. App. No. 61/451 ,995, filed March 1 1 , 201 1 , which is
incorporated herein by reference in its entirety.
[0Θ209] Other specific compounds provided herein are of formula:
or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, wherein:
X is ( 0 or CI ! .;
R 1 is -Y-R 3 ;
R 2 is H or (Ci-C 6 )aikyi;
Y is: 6 to 10 membered aryl, heteroaryl or heterocycle, each of which may be optionally
substituted with one or more halogen; or a bond;
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SDI-60022 107vl 3 is: -(CH 2 ) -aryl, -0-(CH 2 ) n -aryl or -(CH 2 ) n -0-aryl, wherem the aryi is optionally substituted with one or more: (Ci-Ccjaikyi, itself optionally
substituted with one or more halogen; (Ci-Cejalkoxy, itself
substituted with one or more halogen: oxo; amino; earboxyl; cyano;
hydroxy!; halogen; deuterium; 6 to 10 membered aryi or heteroaryi,
optionally substituted with one or more
(Ci-C 6 )alkyl, (Ct-C 6 )alkoxy or halogen; -CONH 2 ; or -COO-(Ci-C 6 )alkyl, wherem the alkyl may be optionally substituted with one or more halogen;
-(CH 2 ) n -heterocycle, -0-(CH 2 ) n -heterocycie or -(CH 2 ) n -0-heterocycie, wherein the
heterocycle is optionally substituted with one or more: (Ci-Ccjaikyi, itself optionally substituted with one or more halogen; (Ci-Cejalkoxy,
itself substituted with one or more halogen; oxo; amino; earboxyl;
cyano; hydroxyl; halogen; deuterium; 6 to 10 membered aryi or
heteroaryi, optionally substituted with one or more (¾- C 6 )alkoxy or halogen; -CONH 2 ; or -COG-iCi-Cejaikyi, wherem the
alkyl may be optionally substituted with one or more halogen; or
-(CH 2 )n-heteroaryl, -0-(CH 2 ) fl -heteroaryl or -(CH 2 )„-0-heteroaryl, wherein the
heteroaryi is optionally substituted with one or more: (C]-C 6 )alkyl, itself optionally substituted with one or more halogen; (C 1 -C 6 )alkoxy,
itself substituted with one or more halogen; oxo; amino; earboxyl;
cyano; hydroxyl; halogen; deuterium; 6 to 10 membered aryi or
heteroaryi, optionally substituted with one or more (Ci-Ceja!kyl, (C
C 6 )aikoxy or halogen; -CONH 2 ; or -COO-(C 1 -C 6 )alkyl, wherem the
alkyl may be optionally substituted with one or more halogen; and
n is 0, 1 , 2 or 3.
[0Θ210] Specific examples include, but are not limited to, 3-(4-((4-
(morphoiinomethy l)benzyi)oxy)- 1 -oxoisoindolm-2-y l)piperidine-2,6-dkme. In one embodiment, provided herein is the (S) stereoisomer of 3-(4-((4-(morpholinomethyl)benzyl)oxy)-l - oxoisomdolin-2-yl)piperidine~2,6~dione ("Compound B") e.g., for use in the methods described herein. Racemic 3-(4-((4-(morpholinomethyl)benzyI)oxy)- 1 -oxoisoindolin-2-yl)piperidine-2,6- dione and methods of preparing the same have been reported in U.S. Patent Publication No,
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SDI-60022 107vl 201 1/0196150, which is incorporated herein by reference in its entirety. Compound B has the following structure:
B
[00211] All of the compounds described can either be commercially purchased or prepared according to the methods described in the patents or patent publications disclosed herein. Further, optically pure compounds can be asymmetrically synthesized or resolved using known resolving agents or chiral columns as well as other standard synthetic organic chemistry techniques.
Additional information on immunomodulatory compounds, their preparation, and use can be found, for example, in U.S. Patent Application Publication Nos. US20060188475, US20060205787, and US20070049618, each of which is incorporated by reference herein in its entirety.
[00212] The immunomodulatory therapies may be small organic molecules having a molecular weight less than about 1 ,000 g/mol, and are not proteins, peptides, oligonucleotides,
oligosaccharides or other macromolecules.
[00213] It should be noted that if th ere is a discrepancy between a depicted structure and a name given that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it.
5o7 Combination Therapy
[00214] One or more additional therapies, such as additional active ingredients or agents, that can be used in combination with an immunomodulatory therapy, such as described in Section 5.6, supra. In a specific embodiment, one or more additional active ingredients or agents can be used in the methods and compositions provided herein with an immunomodulatory therapy. The one or
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SDI-60022 107vl more additional therapies can be administered prior to, concurrently with, or subsequent to the administration of an immunomodulatory therapy. Administration of an immunomodulatory therapy and an additional active agent to a patient can occur simultaneously or sequentially by the same or different routes of administration. The suitability of a particular route of administration employed for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without decomposing prior to entering the blood stream) and the cancer being treated. Preferred routes of administration for the additional active agents or ingredients of the invention are known to those of ordinary skill in the art. See, e.g., Physicians ' Desk Reference.
[0Θ215] In certain embodiments, an immunomodulatoiy therapy and an additional active agent are cyclically administered to a patient with a hematological cancer {e.g., DLBCL). Cycling therapy involves the administration of an active agent for a period of time, followed by a rest for a period of time, and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improves the efficacy of the treatment.
[00216] The additional active agents administered in combination with an immunomodulatory therapy can be large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules). In certain embodiments, the additional active agent is another immunomodulatory therapy. In other embodiments, the additional active agent is not an immunomodulatory therapy. Examples of large molecule active agents include, but are not limited to, hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies. In certain embodiments, large molecule active agents are biological molecules, such as naturally occurring or artificially made proteins. Proteins that are useful include proteins that stimulate the survival and/or proliferation of hematopoietic precursor cells and immunologically active poietic cells in vitro or in vivo. Others stimulate the di vision and differentiation of committed erythroid progenitors in cells in vitro or in vivo. Particular proteins include, but are not limited to: interieukins, such as IL-2 (including recombinant 1L-Ii ("rIL2") and canarypox I L-2), IL-10, IL-12, and IL-18; interferons, such as interferon alfa-2a, interferon alfa-2b, interferon alfa-nl, interferon alfa-ii3, interferon beta-I a, and interferon gamma-I b; GM-CF and GM-CSF; and EPO.
[00217] Particular proteins that can be used in the methods and compositions of the disclosure include, but are not limited to: filgrastim, which is sold in the United States under the trade name NEUPOGEN.RTM. (Amgen, Thousand Oaks, Calif); sargramostim, which is sold in the United States under the trade name LEUKINE, RTM. (Immunex, Seattle, Wash.); and recombinant EPO,
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SDI-60022 107vl which is sold in the United States under the trade name EPGEN.RTM, (Amgen, Thousand Oaks, Calif.),
[00218] Inhibitors of ActRII receptors or activin- Ac RII inhibitors may be used in the methods and compositions provided herein. ActRII receptors include ActRIIA inhibitors and ActRIIB inhibitors. Inhibitors of ActRJI receptors can be polypeptides comprising aetivm-binding domains of ActRII. In certain embodiments, the activin-binding domain comprising polypeptides are linked to an Fc portion of an antibody (i.e., a conjugate comprising an activin-binding domain comprising polypeptide of an ActRII receptor and an Fc portion of an antibody is generated). In certain embodiments, the activin-binding domain is linked to an Fc portion of an antibody via a linker, e.g. , a peptide linker. Examples of such non-antibody proteins selected for activin or ActRI IA binding and methods for design and selection of the same are found in WO/2002/088171,
WO/2006/055689, WO/2002/032925, WO/2005/037989, US 2003/0133939, and US
2005/0238646, each of which is incorporated herein by reference in its entirety.
[00219] Recombinant and mutated forms of GM-CSF can be prepared as described in U.S. Pat. Nos. 5,391 ,485; 5,393,870; and 5,229,496; the disclosure of each of which is incorporated herein by reference in its entirety. Recombinant and mutated forms of G-CSF can be prepared as described in U.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; the disclosure of each of which is incorporated herein by reference in its entirety.
[00220] This disclosure encompasses the use of native, naturally occurring, and recombinant proteins. The disclosure further encompasses mutants and derivatives (e.g., modified forms) of naturally occumng proteins that exhibit, in vivo, at least some of the pharmacological activity of the proteins upon which they are based. Examples of mutants include, but are not limited to, proteins that have one or more amino acid residues that differ from the corresponding residues in the naturally occurring forms of the proteins. Al so encompassed by the term "mutants" are proteins that lack carbohydrate moieties normally present in their naturally occurring forms (e.g. ,
nonglycosylated forms). Examples of derivatives include, but are not limited to, pegy laced derivatives and fusion proteins, such as proteins formed by fusing IgGl or IgG3 to the protein or active portion of the protein of interest. See, e.g., Penichet, M. L. and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).
[00221] Antibodies that can be used in combination with an immunomodulatory therapy include monoclonal and polyclonal antibodies. Examples of antibodies include, but are not limited to, trastuzumab (HERCEPTIN®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), pertuzumab
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SDI-60022 107vl (OM TARG™), tositumomab (BEXXAI ®), edrecolomab ( PANQREX®), panitumumab and G250. An immunomodulatory therapy provided herein can also be combined with or used in combination with anti-TNF-alpha antibodies.
[00222] Large molecule active agents may be administered in the form of anti-cancer vaccines. For example, vaccines that secrete, or cause the secretion of, cytokines such as IL-2, SCF, CXC14 (platelet factor 4), G-CSF, and GM-CSF can be used in the methods, pharmaceutical compositions, and kits of the disclosure. See, e.g., Emens, L. A,, et al., Curr. Opinion Moi. Ther, 3(l):77-84 (2001).
[00223] Additional active agents that are small molecules can also be used to alleviate adverse effects associated with the administration of an immunomodulatory therapy. However, like some l arge mol ecules, many are believed to be capable of providing a synergistic effect when
administered with (e.g., before, after or simultaneously) the immunomodulatory therapy. Examples of small molecule additional active agents include, but are not limited to, anti-cancer agents, antibiotics, immunosuppressive agents, and steroids.
[00224] Examples of anti-cancer agents include, but are not limited to: abraxane; ace-11;
acivicin; aclarubicin; acodazole hydrochloride; acronine; adozeiesin; aldesleukin; altretamine;
ambomycin; ametantrone acetate; amrubicin; amsacrine; anastrozole; anthramycin; asparaginase; aspeiiin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; caiusterone; caracemide; carbetimer; carbopiatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormapiatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitnicin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin
hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretimde; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; herceptin; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilrnofosine; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; lapatinib; letrozoie; leuprolide acetate; iiarozole hydrochloride; iometrexol sodium; lomustine; iosoxaiitrone hydrochloride; masoprocol;
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SDI-60022 107vl maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphafan; nienogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meruredepa;
mitindornide; mitocarcin; mitocromin; mitogillin; miiomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide;
pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porflmer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycm; puromycin hydrochloride; pyrazofurin; riboprine; romidepsin; safingol; safingol hydrochloride; semustine; simtrazene;
sparfosate sodium; sparsomvcin; spirogerma iimi hydrochloride; spiromustine; spiroplatin; stem cell treatments such as PDA-GO 1 ; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;
testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamirie; toremifene citrate;
trestoione acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin;
tubulozoie hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;
vinleurosine sulfate; vinoreibine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole;
zenip latin; zinostatin; and zorubicin hydrochloride.
[0Θ225] Other anti-cancer drugs include, but are not limited to: 20-epi-l,25
D3; 5-ethytiyturacii; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-T antagonists; aitretamine; ambamustine; amidox; amifostine; aminolevulinic acid;
amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1 ; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate;
apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustiiie; axinastatin 1; axinastatin 2; axinastatin 3;
azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzociilorins; benzoyistaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin B; betulinic acid; b-FGF inhibitor; bicaluiamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizeiesin; breflate; bropirimine; budotitane; buthionine sulfoximine;
ca!cipotriol; calphostin C; camptothecin derivatives; capeciiabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chiorins; cliloroquinoxaline
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SDI-60022 107vl sulfonamide; cicaprost; cis-porphyrin; ciadribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;
crisnatol; cryptophycin 8; cryptophycin A derivatives; curacm A; cyclopentanihraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxoL 9-;
dioxamycin; diplienyl spiroraustine; docetaxel; docosanol; dolasetron; doxirTuridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecoiomab;
eflornitliine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;
fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaiffiorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; gaiocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;
idarubicin; idoxifene; idramatitotie; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC®.), imiquimod; immunostimulant peptides; insulin- like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;
irsogladine; isobengazole; isohomohalicondrm B; itasetixni; jaspiakinolide; kalialalide F; lamellarin- N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin;
levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; iissoclinamide 7; lobapiatin; lombricine; lometrexol; lonidamine;
losoxantrone; loxoribine; lurtotecan; lutetium. texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metal foproteinase inhibitors; meiiogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor;
mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxaiitrone; mofarotene; molgramostim; Erbitux, human chorionic goriadotrophin; monophosphoryl lipid A-t-myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N- acetyidinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone +peritazocirie; napavin; naphterpin; nartograstim; nedapiatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitruliyn; oblimersen (GENASENSE®); O.sup.6-
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SDI-60022 107vl benzylguanine; octreotide; okicenone; oligonucleo tides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomyciii; paclitaxel;
paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peidesine; pentosan polysulfate sodium; pentostatm; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazmomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubiein; piritrexim; placetin A; placetiii B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone;
prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microaigal; protein tyrosine phosphatase mhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras mhibitors; ras-GAP mhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; R1I retinamide; rohitukine; romurtide; roquinimex; rubiginone Bl ; ruboxyl; safmgol; saintopin; SarC U; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction mhibitors; sizofuran; sobuzoxane; sodium borocaptate; sodium phenjdacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; spienopentiii; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; tenyposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; iitanocene bichloride; topsentin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; velaresol; veramine;
verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[00226] Specific additional active agents include, but are not limited to, oblimersen
(GENASENSE©), remicade, docetaxel, celecoxib, melphalan, dexamethasone (DECADRON®), steroids, gemcitabine, cisplatinimi, temozoiomide, etoposide, cyclophosphamide, temodar,
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SDI-60022 107vl carbo latin, procarbazine, giiadel, tamoxifen, topotecan, methotrexate, A ISA®., taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeioda, CPT-11, interferon alpha, pegylated interferon alpha (e.g. , PEG INTRON-A), capecitabme, eisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicm, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vmorelbine, zoledronic acid, palmitronate, biaxin, busulphan, prednisone, bisphosphonate, arsenic trioxide, vincristine, doxorubicin (DOXIL®), paclitaxel, ganciclovir, adriamycin, estramustine sodium phosphate) (EMCYT. degree,), suiiiidac, and etoposide.
5„8 Biological Samples
[00227 J In certain embodiments, the various methods provided herein use samples (e.g., biological samples) from subjects or individuals (e.g., patients). In a specific embodiment, the subject is a patient with a hematological cancer, such as multiple myeloma, leukemia or a lymphoma. The subject can be a mammal, for example, a human. The subject can be male or female, and can be an adult, child or infant. Samples can be analyzed at a time during an active phase of a disease or disorder, or when a disease or disorder is inactive. In a specific embodiment, a sample is obtained from a subject prior, concuiTently with and/or subsequent to administration of an immunomodulatory therapy. In certain embodiments, more than one sample from a subject can be obtained.
[00228 J In certain embodiments, the sample used in the methods provided herein comprises body fluids from a subject. Non-limiting examples of body fluids include blood (e.g., peripheral whole blood, peripheral blood), blood plasma, amniotic fluid, aqueous humor, bile, cerumen, eowper's fluid, pre-ejaculatory fluid, chyle, chyme, female ejaculate, interstitial fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubrication, vomit, water, feces, internal body fluids, including cerebrospinal fluid surrounding the brain and the spinal cord, synovial fluid surrounding bone joints, intracellular fluid is the fluid inside cells, and vitreous humour the fluids in the eyeball . In some embodiments, the sample is a blood sample. The blood sample can be obtained using conventional techniques as described in, e.g. Innis et al, editors, PGR. Protocols (Academic Press, 1990). White blood cells can be separated from blood samples using convention techniques or commercially available kits, e.g. RosetteSep™ kit (Stein Cell Technologies, Vancou ver, Canada). Sub-populations of white blood cells, e.g. mononuclear cells, B cells, T cells, monocytes, granulocytes or lymphocytes, can be further isolated using conventional
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SDI-60022 107vl techniques, e.g. magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescentiy activated cell sorting (FACS) (Becton Dickinson, San Jose, California),
[00229] In one embodiment, the blood sample is from about 0, 1 mL to about 10.0 ml., from about 0.2 mL to about 7 mL, from about 0.3 mL to about 5 mL, from about 0.4 mL to about 3.5 mL, or from about 0.5 mL to about 3 mL. In another embodiment, the blood sample is about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 mL,
[00230] In some embodiments, the sample used in the present methods comprises a biopsy (e.g., a tumor biopsy). The biopsy can be from any organ or tissue, for example, skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph node, hair, spleen, brain, breast, or other organs. Any biopsy technique known by those skilled in the art can be used for isolating a sample from a subject, for instance, open biopsy, close biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy.
[00231] In one embodiment, the sample used in the methods provided herein is obtained from the subject prior to the subject receiving a treatment for the hematological cancer. In another embodiment, the sample is obtained from the subject during the subject receiving a treatment for the hematological cancer. In another embodiment, the sample is obtained from the subject after the subject receiving a treatment for the hematological cancer. In various embodiments, the treatment comprises administering an immunomodulatory therapy (e.g., a compound provided in Section 5.6) to the subject.
[00232] In certain embodiments, the sample used in the methods provided herein comprises a plurality of cells. Such cells can include any type of cells, e.g., stem cells, blood cel ls (e.g., peripheral blood mononuclear cells), lymphocytes, B cells, T cells, monocytes, granulocytes, immune cells, or tumor or cancer cells. The tumor or cancer cells or a tumor tissue, such as a tumor biopsy or a tumor explains, T cells (T lymphocytes) include, for example, helper T cells (effector T cells or Th cells), cytotoxic T cells (CTLs), memory T cells, and regulatory T cells. In one embodiment, the cells used in the methods provided herein are CD 3 r T cells, e.g., as detected by flow cytometry. The number of T cells used in the methods can range from a single cell to about 10 9 cells. B cells (B lymphocytes) include, for example, plasma B cells, dendritic cells, memory B cells, Bl cells, B2 cells, marginal-zone B cells, and follicular B cells. B cells can express immunoglobulins (antibodies, B cell receptor).
[00233] Specific cell populations can be obtained using a combination of commercially available antibodies (e.g.. Quest Diagnostic (San Juan Capistraiio, Calif); Dako (Denmark)).
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SDI-60022 107vl [00234 J In some embodiments, the cancer is a hematological cancer. In one embodiment, the blood cancer is multiple myeloma. In another embodiment, the blood cancer is chronic
lymphocytic leukemia (CLL). In another embodiment, the blood cancer is DLBCL. In another embodiment, the blood cancer is myelodyspiastic syndrome, an acute leukemia, e.g., acute T cell leukemia, acute myelogenous leukemia (AML), acute promyelocytic leukemia, acute myeloblast! c leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia (Burkitt's lymphoma), or acute biphenotypic leukemia; a chronic leukemia, e.g., chronic myeloid lymphoma, chronic myelogenous leukemia (CML), chronic monocytic leukemia, Small lymphocytic lymphoma, or B-cell proiymphocytic leukemia; hair}' cell lymphoma; T-cell proiymphocytic leukemia; or a lymphoma, e.g, histiocytic lymphoma, lyniphoplasmacytie lymphoma (e.g., Waldenstrom macrogiobulinemia), splenic marginal zone lymphoma, plasma cell neoplasm (e.g., plasma cell myeloma, plasmacytoma, a monoclonal immunoglobulin deposition disease, or a heavy chain disease), extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), fol licular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B ceil lymphoma, intravascular large B ceil lymphoma, primary effusion lymphoma, T ceil large granular lymphocytic leukemia, aggressive N cell leukemia, adult T cell
ieukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sezary syndrome), a primary cutaneous CD30-positive T ceil lymphoproiiferative disorder (e.g., primary cutaneous anaplastic large cell lymphoma or lymphomatoid. papulosis), angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, a Hodgkin's lymphoma or a nodular lymphocyte-predominant Hodgkin's lymphoma.
[00235 J In certain embodiments, the sample used in the methods provided herein is from a diseased tissue, e.g., from an individual having a hematological cancer. In certain embodiments, the number of cells used in the methods provided herein can range from a single cel l to about 1 († cells. In some embodiments, the number of cells used in the methods provided herein is about 1 x 10 ' ', 5 x 10 4 , I x i0 5 , 5 x 10 5 , 1 x 1 if. 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , I x iO 8 , or 5 x ! 0\
[00236] The number and type of cells collected from a subject can be monitored, for example, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue specific or cell-marker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated cell
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SDI-60022 107vl sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling. These techniques can be used, too, to identify cells that are positive for one or more particular markers. Fluorescence activated cell sorting (FACS) is a well- known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151 :150-165). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture, in one embodiment, cell surface marker-specific antibodies or iigands are labeled with distinct fluorescent labels. Cells are processed through the cell sorter, allowing separation of cells based on their ability to bind to the antibodies used. FACS sorted particles may be directly deposited into individual wells of 96-well or 384-well plates to facilitate separation and cloning.
[00237] In certain embodiments, subsets of cells are used in the methods provided herein.
Methods to sort and isolate specific populations of cells are well-known in the art and can be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead based separation such as magnetic cell sorting, size-based separation (e.g., a sieve, an array of obstacles, or a filter), sorting in a microfiuidics device, antibody-based separation, sedimentation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc.
5.9 Methods for Detecting RNA Expression
[00238] Several methods of detecting or quantitating mRNA levels are known in the art.
Exemplary methods include but are not limited to northern blots, ribonuclease protection assays, PCR-based methods, and the like. The mRNA sequence can be used to prepare a probe that is at least partially complementary. The probe can then be used to detect the mRNA sequence in a sample, using any suitable assay, such as PCR-based methods, Northern blotting, a dipstick assay, and the like.
[00239] In other embodiments, a nucleic acid assay for testing for immunomodulator activity in a biological sample can be prepared. An assay typically contains a solid support and at least one nucleic acid contacting the support, where the nucleic acid corresponds to at least a portion of an mRNA encoded by a gene listed in Table 1 , 2, 3 or 4. The assay can also have a means for detecting the altered expression of the mRNA in the sample.
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SDI-60022 l07vl [00240 J The assay method can be varied depending on the type of mRNA information desired. Exemplary methods include but are not limited to Northern blots and PCR-based methods (e.g. , qRT-PCR). Methods such as qRT-PCR can also accurately quantitate the amount of the mRNA. in a sample.
[00241] Any suitable assay platform can be used to determine the presence of the mRNA in a sample. For example, an assay may be in the form of a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multi ell plate, or an optical fiber. An assay system may have a solid support on which a nucleic acid corresponding to the mRNA is attached. The solid support may comprise, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capil lary, a fi lm a plate, or a slide. The assay components can be prepared and packaged together as a kit for detecting an mRNA.
[00242] The nucleic acid can be labeled, if desired, to make a population of labeled mRNAs. In general, a sample can be labeled using methods that are well known in the art (e.g. , using DNA ligase, terminal transferase, or by labeling the R ' NA backbone, etc.; see, e.g., Ausubel, et ai, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, .Y.). In some embodiments, the sample is labeled with fluorescent label. Exemplary fluorescent dyes include but are not limited to xanthene dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6 carboxyfluorescein (FAM), 6 carboxy-2', 4 ',7', 4, 7-hexachloro fluorescein (HEX), 6 carboxy 4', 5 ' dichloro 2', 7' dimethoxyfktorescem (JOE or J), Ν,Ν,Ν',Ν' tetramethyl 6
carboxyrhodamine (TAMRA or T), 6 carboxy X rhodamine (ROX or R), 5 carboxyrhodamme 6G (R6G5 or G5), 6 carboxyrhodamme 6G (R6G6 or G6), and rhodamine 1 10; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; Alexa dyes, e.g. Alexa-fluor-555; coumarin, Diethylaminocoumarin, imibelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, Pyrene, Fluorescein Chiorotriazinyi, R110, Eosin, JOE, R6G,
Tetramethylrhodamine, Lissamine, ROX, Naptbofluorescein, and the like.
[00243] In some embodiments, the mRNA sequences comprise at least one mRNA selected from the mRNAs encoded by the genes listed in Table 3, or a f agment thereof. The nucleic acids may be present in specific, addressable locations on a solid support; each corresponding to at least a portion
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SDI-60022 l07vl of mRNA sequences that are differential!)' expressed upon treatment of an immunomodulatory compound in a cell or a patient.
[00244] A typical mRNA assay method can contain the steps of 1) obtaining surface-bound subject probes; 2) hybridization of a population of mRNAs to the surface-bound probes under conditions sufficient to provide for specific binding (3) post-hybridization washes to remove nucleic acids not bound in the hybridization; and (4) detection of the hybridized mRNAs. The reagents used in each of these steps and their conditions for use may vary depending on the particular application.
[0Θ245] Hybridization can be carried out under suitable hybridization conditions, which may vary in stringency as desired. Typical conditions are sufficient to produce probe/target complexes on a solid surface between complementary binding members, i.e., between surface-bound subject probes and complementar mRNAs in a sample. In certain embodiments, stringent hybridization conditions may be employed.
[00246] Hybridization is typically performed under stringent hybridization conditions. Standard hybridization techniques (e.g. under conditions sufficient to provide for specific binding of target mRNAs in the sample to the probes) are described in allioniemi et al. Science 258:818-821 (1992) and WO 93/18186. Several guides to general techniques are available, e.g., Trjssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993). For descriptions of techniques suitable for in situ hybridizations, see Gall et al. Meth. Enzymol., 21 :470- 480 (1981); and Angerer et al. in Genetic Engineering: Principles and Methods (Setiow and Holiaender, Eds.) Vol 7, pgs 43-65 (Plenum Press, New York 1985). Selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringenc of washing conditions, and the like will depend on experimental design, including source of sample, identity of capture agents, degree of com lementarity expected, etc., and may be determined as a matter of routine experimentation for those of ordinary skill in the art,
[00247] Those of ordinary skill will readily recognize that al ternati ve but comparable
hybridization and wash conditions can be utilized to provide conditions of similar stringency.
[00248] After the mRNA hybridization procedure, the surface bound polynucleotides are typically washed to remove unbound nucleic acids. Washing may be performed using any convenient washing protocol, where the washing conditions are typically stringent, as described above. The hybridization of the target mRNAs to the probes is then detected using standard techniques.
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SDI-60022 107vl | 00249| Other methods, such as PCR-based methods, can also be used to fol low the expression of the genes in Table 1 , 2, or 3. Examples of PCR methods can be found in the literature.
Examples of PCR assays can be found in U.S. Patent No. 6,927,024, which is incorporated by reference herein in its entirety. Examples of RT-PCR methods can be found in U.S. Patent No. 7, 122,799, which is incorporated by reference herein in its entirety. A method of fluorescent in situ PCR is described in U.S. Patent No. 7, 186,507, which is incorporated by reference herein in its entirety,
[00250] in some embodiments, Real-Time Reverse Transcription-PCR (qRT-PCR) can be used for both the detection and quantification of RNA targets (Bustin, et al , 2005, Clin. Sci. , 109:365- 379). Quantitative results obtained by qRT-PCR are generally more informative than qualitative data. Thus, in some embodiments, qRT-PCR-based assays can be useful to measure mRNA levels during cell-based assays. The qRT-PCR method is also useful to monitor patient therapy.
Examples of qRT-PCR-based methods can be found, for example, in U.S. Patent No. 7, 101 ,663, which is incorporated by reference herein in its entirety.
[00251 J In contrast to regular reverse transcriptase-PCR and analysis by agarose gels, real-time PCR gives quantitative results. An additional advantage of real-time PCR is the relative ease and convenience of use. Instruments for real-time PCR, such as the Applied Biosystems 7500, are available commercially, as are the reagents, such as TaqMan Sequence Detection chemistry. For example, TaqMan* Gene Expression Assays can be used, fol lowing the manufacturer's instructions. These kits are pre-formulated gene expression assays for rapid, reliable detection and quantification of human, mouse and rat mRNA transcripts. An exemplary PCR program, for example, is 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, then 60°C for 1 minute.
[00252] To determine the cycle number at which the fluorescence signal associated with a particular amplicon accumulation crosses the threshold (referred to as the CT), the data can be analyzed, for example, using a 7500 Real-Time PCR System Sequence Detection software vl .3 using the comparative CT relative quantification calculation method. Using this method, the output is expressed as a fold-change of expression levels. In some embodiments, the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set to be above the baseline but sufficiently low to be within the exponential growth region of an amplification curve.
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SDI-60022 107vl 5.10 Methods for Detecting Protein Expression
[0Θ253] Several protein detection and quantitation methods can be used to measure the level of proteins. Any suitable protein quantitation method can be used. In some embodiments, antibody- based methods are used. Exemplary methods that can be used include but are not limited to immunoblotting (western blot), enzyme-linked immunosorbent assay (ELIS A),
irnmunohistochemistry, flow cytometry, cytometric bead array, mass spectroscopy, and the like. Several types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.
5.11 Kits
[00254 J In one aspect, provided herein are pharmaceutical or assay kits comprising an immunomodulatory therapy or a pharmaceutical composition thereof, in one or more containers, and instructions for use. In certain embodiments, the kits useful for predicting the likelihood of an effective patient tumor response. In further embodiments, the immunomodulatory therapy, in a container, is accompanied by an apparatus or apparati necessary for administering the compound or composition thereof to a subject.
[0Θ255] In certain embodiments, a kit comprises an immunomodulatory therapy or
pharmaceutical composition thereof, in a container, and a reagent or reagents necessary for carrying out an assay(s) described herein, in one or more other containers. In certain embodiments, the kit comprises a solid support, and a means for detecting the RNA or protein expression of at least one biomarker (e.g., a differentially expressed gene identified in Table 1, 2, 3, or 4) in a biological sample. Such a kit may employ, for example, a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical fiber. The solid support of the kit can be, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capil lary, a film, a plate, or a slide.
[0Θ256] In a specific embodiment, the pharmaceutical or assay kit comprises, in a container, an immunomodulatory therapy' or a pharmaceutical composition thereof, and further comprises, in one or more containers, components for isolating RNA. In another specific embodiment, the pharmaceutical or assay kit comprises, in a container, an immunomodulatory therapy or a pharmaceutical composition, and further comprises, in one or more containers, components for conducting RT-PCR, RT-qPCR, deep sequencing or a microarray. In some embodiments, the kit comprises a solid support, nucleic acids contacting the support, where the nucleic acids are
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SDI-60022 107vl com lementary to at least 20, 50, 100, 200, 350, or more bases of mRNA, and a means for detecting the expression of the mRNA in a biological sample.
[00257] In another specific embodiment, the pharmaceutical or assay kit comprises, in a container, an immunomodulatory therapy or a pharmaceutical composition thereof, and further comprises, in one or more containers, components for isolating protein In another specific embodiment, the pharmaceutical or assay kit comprises, in a container, an immunomodulatory therapy or a pharmaceutical composition, and further comprises, in one or more containers, components for conducting flow cytometry or an ELISA.
[0Θ258] In another aspect, provided herein are kits for measuring biomarkers providing the materials necessary to measure the abundance of one or more of the gene products of the genes or a subset of genes (e.g., one, two, three, four, five or more genes) in Table 1 , 2, 3 or 4, or any combination thereof. Such kits may comprise materials and reagents required for measuring RNA or protein. In some embodiments, such kits include microarrays, wherein the microarray is comprised of oligonucleotides and/or DN A and/or RN A fragments which hybridize to one or more of the products of one or more of the genes or a subset of genes in Table 1 , 2, 3 or 4, or any combination thereof. In some embodiments, such kits may include primers for PCR of either the RNA product or the cD A copy of the RNA product of the genes or subset of genes, or both. In some embodiments, such kits may include primers for PCR as well as probes for Quantitative PCR. In some embodiments, such kits may include multiple primers and multiple probes wherein some of said probes have different fluorophores so as to permit multiplexing of multiple products of a gene product or multiple gene products. In some embodiments, such kits may further include materials and reagents for creating cDNA from RNA. In some embodiments, such kits may include antibodies specific for the protein products of a gene or subset of genes in Table 1, 2, 3, or 4, or any combination thereof. Such kits may additionally comprise materials and reagents for isolating RNA and/or proteins from a biological sample. In addition such kits may include materials and reagents for synthesizing cDN A from UNA isolated from a biological sample. In some embodiments, such kits may include, a computer program product embedded on computer readable media for predicting whether a patient is clinically sensitive to an immunomodulatory therapy. In some embodiments, the kits may include a computer program product embedded on a computer readable media along with instructions.
[0Θ259] In some embodiments, kits for measuring the expression of one or more nucleic acid sequences of a gene or a subset of genes in Table 1 , 2, 3 or 4 or a combination thereof. In a specific
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SDI-60022 107vl embodiment, such kits measure the expression of one or more nucleic acid sequences associated with a gene or a subset of genes in Table 1, 2, 3 or 4, or a combination thereof. In accordance with this embodiment, the kits may comprise materials and reagents that are necessary for measuring the expression of particular nucleic acid sequence products of genes or a subset of genes in Table 1, 2, 3 or 4, or a combination thereof. For example, a microarray or RT-PCR kit may be produced for a specific condition and contain only those reagents and materials necessary for measuring the levels of specific RNA transcript products of the genes or a subset of genes in Table 1, 2, 3 or 4, or a combination thereof to predict whether a hematological cancer in a patient is clinically sensitive to an immunomodulatory therapy. Alternatively, in some embodiments, the kits can comprise materials and reagents that are not l imited to those required to meas ure the expression of partic ular nucleic acid sequences of any particular gene in Table 1 , 2, 3, or 4, or a combination thereof. For example, in certain embodiments, the kits comprise materials and reagents necessary for measuring the levels of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the genes in Table 1 , 2, 3 or 4, in addition to reagents and materials necessary for measuring the levels of the expression of at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more genes other than those in Table 1, 2, 3 or 4. In other embodiments, the kits contain reagents and materials necessary for measuring the levels of expression of at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more of the genes in Table 1 , 2, 3 or 4, and 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or more genes that are genes not in Table 1 , 2, 3 or 4, or 1-10, 1-100, 1 -150, 1-200, 1-300, 1-400, 1 -500, 1-1000, 25-100, 25-200, 25-300, 25- 400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes that are genes not in Table 1, 2, 3 or 4.
[00260] For nucleic acid microarray kits, the kits generally comprise probes attached to a solid support surface. In one such embodiment, probes can be either oligonucleotides or longer length probes including probes ranging from 150 nucleotides in length to 800 nucleotides in length. The probes may be labeled with a detectable label. In a specific embodiment, the probes are specific for one or more of the gene products in Table 1, 2, 3 or 4. The microarray kits may comprise instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay. In a specific embodiment, the kits comprise instructions for
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SDI-60022 107vl predicting whether a hematological cancer in a patient is clinically sensitive to an
immunomodulatory therapy. The kits may also comprise hybridization reagents and/or reagents necessary for detecting a signal produced when a probe hybridizes to a target nucleic acid sequence. Generally, the materials and reagents for the microarray kits are in one or more containers. Each component of the kit is generally in its own a suitable container.
[00261] In certain embodiments, a nucleic acid microarray kit comprises materials and reagents necessary for measuring the levels of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the genes identified in Table 1 , 2, 3 or 4, or a combination thereof, in addition to reagents and materials necessary for measuring the levels of the expression of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more genes other than those in Tables 1 , 2, 3 or 4. In other embodiments, a nucleic acid microarray kit contains reagents and materials necessary for measuring the levels of expression of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more of the genes in Table 1, 2, 3 or 4, or any combination thereof, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or more genes that are not in Table 1, 2, 3 or 4, or 1-10, 1-100, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes that are not in Table 1 , 2, 3 or 4.
[00262] For Quantitative PGR, the kits generally comprise pre-selected primers specific for particular nucleic acid sequences. The Quantitative PGR kits may also comprise enzymes suitable for amplifying nucleic acids (e.g., polymerases such as Taq), and deoxynucleotides and buffers needed for the reaction mixture for amplification. The Quantitative PGR kits may also comprise probes specific for the nucleic acid sequences associated with or indicative of a condition. The probes may or may not be labeled with a fiuorophore. The probes may or may not be labeled with a quencher molecule. In some embodiments the Quantitative PGR kits also comprise components suitable for reverse-transcribing RNA including enzymes (e.g., reverse transcriptases such as AMV, MMLV and the like) and primers for reverse transcription along with deoxynucleotides and buffers needed for the reverse transcription reaction . Each component of the quantitative PGR kit is generally in its own suitable container. Thus, these kits generally comprise distinct containers suitable for each individual reagent, enzyme, primer and probe. Further, the quantitative PGR kits
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SDI-60022 107vl may comprise instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay. In a specific embodiment, the kits contain instructions for predicting whether a hematological cancer in a patient is clinically sensitive to an immunomodulatory therapy.
[00263] For antibody based kits, the kit can comprise, for example: (1) a first antibody (which may or may not be attached to a solid support) which binds to a peptide, polypeptide or protein of interest; and, optionally, (2) a second, different antibody which binds to either the peptide, polypeptide or protein, or the first antibody and is conjugated to a detectable label (e.g., a fluorescent label, radioactive isotope or enzyme). In a specific embodiment, the peptide, polypeptide or protein of interest is associated with or indicative of a condition (e.g., a disease). The antibody-based kits may also comprise beads for conducting an immunoprecipitation. Each component of the a ibody-based kits is generally in its own suitable container. Thus, these kits generally comprise distinct containers suitable for each antibody. Further, the antibody-based kits may comprise instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay. In a specific embodiment, the kits contain instructions for predicting whether a hematological cancer in a patient is clinically sensitive to an immunomodulatory therapy.
6. EXAMPLES
[00264] Certain embodiments of the invention are illustrated by the following non-limiting examples.
[00265] Gene expression differences between the baseline transcriptional profiles of refractory or relapsed diffuse large B-Cell lymphoma (DLBCL) patients who display response subsequent to lenalidomide treatment and those of patients who do not respond were investigated. A clinical trial was formed with four arms, each arm containing 25 patients. One arm contained patients classified as presenting germinal center B-cell-like DLBCL subtypes receiving lenalidomide, a second arm contained patients classified as presenting germinal center B-cell-like DLBCL subtypes receiving another therapy selected by the investigator, a third arm contained patients classified as presenting activated B-cell-like DLBCL subtypes receiving lenalidomide, and a fourth arm contained patients classified as presenting activated B-cell -like DLBCL subtypes receiving another therapy selected by the investigator. The patients in the four arms of the clinical trial received treatment until disease progression.
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SDI-60022 107vl [00266J For purposes of this exploratory analysis, a subset of the clinical data associ ated to each patient comprised the therapy arm, Revlimid (REV) or control drug (CON), and their corresponding response to the drug in both categorical variable {complete response (CR), partial response (PR), establish disease (SD), progression disease (PD) and Death} and continuous variables as progression free survival (PFS) and overall (OS), unit weeks. It also includes the predicted DLBCL sub-type (ABC/GCB) and other demographic data.
[0Θ267] Samples from patient biopsies taken prior to receiving therapy were hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip™ microarrays (ww w. affymetrix. com/) at the Molecular Characterization & Clinical Assay Development Laboratory, SAIC Frederick National Laboratory for Cancer Research, SAIC-Frederick, Frederick, MD. Biopsy samples were flash-frozen at screen (FF), archived having been formalin-fixed and paraffin embedded (FFPE archive), or FFPE treated at screen. All patients are associated with at least one gene expression profile obtained from one of the three sample types, some are associated with more than one profile. The results described in this Section 6 were obtained from analysis of only those profil es relating to FF samples and which passed QC.
[0Θ268] Microarray Data QC
[00269] Raw Affymetrix image (.eel) files were imported into the R statistical programming environment v3.G.O (r-project.org) using functionality of the Affy package of the related
Bioconductor sui te of open-source bioinformatics software ( bioconductor.org). Transcriptional profile QC was performed using the NUSE algorithm, implemented in the Bioconductor package arrayOuality Metrics (Kauffmann et ah, 2009), applied to a log2 transformation of raw signal.
[00270] The RMA (Irizarry et al., 2003) algorithm was applied to background-correct, quantile normalize and summarize profiles that passed QC. Annotation of probe-sets to genes was performed using the R packages annotate (Gentleman, 2013) and genefilter (Gentleman et al., 2013) selecting only one probeset per gene (Entrez Gene ID) and choosing the most variable across profiles according to inter-quartile range in cases wherein multiple probe-sets map to a single gene,
[00271] Clustering & Visualization
[00272] Data visualization via clustering and heatmap graphics was implemented in the R.
statistical programming environment. Either Euclidean distance and correlation (1 - Pearson correlation) were used to represent inter-profile dissimilarity and (1 -pearson correlation) to represent dissimilarity between genes across profiles. In both cases, hierarchical clustering using Ward's algorithm was applied. Comparative gene expression heatmaps were implemented using
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SDI-60022 107vl gplots (Warnes et al., 2013) and heatmap.plus (Day, 2012) packages from the Bioconductor suite, with colours generated using palettes from the RColorBrewer™ package (Neuwirth, 2011). Prior to clustering and visualization, individual gene expression across profiles was standardized to have zero mean and unit variance.
[0Θ273] Differesiiial Expression
[00274] The SAM algorithm (Tusher et al., 2001), as implemented in the R/Bioconductor package siggenes (Schwender, 2012), was applied to assess statistical significance of differential gene expression across discrete profile groups. Significance values obtained from multiple hypothesis tests were corrected for false-discovery by permutation as implemented in the SAM algorithm.
[00275] The Enrichr (Chen et al., 2013) tool (available at amp.pharm.mssm.edu/Enrichr/) was used to assess statistical over-representation of gene categories among genes deemed differentially regulated. The tool combines 35 gene set libraries sorted by categories including transcription, pathways, ontologies, diseases, etc. and totaling 31 ,026 gene-sets.
1 0276] Survival Analysis
[00277] The BioNet algorithm (Beisser et al., 2010), as implemented in the related Bioconductor package, was applied to the combined output of statistical tests for differential expression between refractory versus non-refractory best response groups and gene expression correlation with PFS and used the Human Interactome obtained from HI NT (Das and Yu, 2012). Optimal response-related sub-networks were visualized via the Cytoscape platform (Saito et al., 2012; Shannon et al., 2003; Smoot et al., 2011).
[00278] The Reactome FI package ( Wu and Stein, 2012) was implemented via Cytoscape and applied to Reactome annotation (Croft et al., 201 1) imported with the software. The "microarray data analysis" option was applied, with database version 2012, absolute value for Pearson correlation, and an inflation parameter of 5.0 for the Markov Cluster Algorithm. After network generation, Biological Process and Pathway enrichment and survival analyses were generated using associated functionality of the package. Survival analyses were calculated per module upon import of corresponding PFS and censor information.
[00279] Cox regression models and Kaplan-Meier curves were performed using the R package survival (Terry M. Therneau, 2013; Terry M. Therneau and Patricia M. Grambsch, 2000).
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SDI-60022 l07vl [00280] Decomposition
[0Θ281] Microarray gene expression profiles were decomposed using default functionality of the CellMix R/Bioconductor package (Gaujoux and Seoighe, 2013), which also provided collections of reference data used for decomposition. The decomposition method and reference collection of (Abbas et al., 2009) were applied to REV-DLC-001 profiles. Results were visualized and associated statistics calculated using native functionality of the R environment.
[00282] Results
[00283] Tables 3 and 4 provide lists of genes deemed significantly differentially regulated (empirical FDR 5%) pre-treatment, between patient groups determined as refractory and non- refractory further to Revlimid/lenalidomide therapy. Table 1 provides a list of genes deemed significantly upregulated (empirical FDR 5%) pre-treatment in patients non-refractory to further therapy relative to patients refractory to further Revlimid/lenalidomide therapy. Table 2 provides a list of genes deemed significantly downregulated (empirical FDR 5%) pre-treatment in patients non-refractory to further therapy relative to patients refractor}' to further Revlimid/lenalidomide therapy.
[0Θ284] Analysis was performed on the FF sample profiles from the lenalidomide-arm and estimated relative proportions of the reference immune cel l types were compared between profiles associated with binary lenalidomide response vs. non-response categories (see Figure 2). The distinction between lenalidomide response profiles and the non-responding profiles is characterized by lower estimated B-cell proportions and clear increases in estimated proportions of plasma cells, dendritic cells, and, in a smaller subset of responding patients, NK cells.
[0Θ285] It is of note that estimated proportions of monocytes and cytotoxic T-cells do not correlate strongly with Revlimid response, despite interpretations of the preceding analyses having suggested their presence. Rather, the patterns displayed appear more related to an earlier 'host response' (Monti et al., 2005) or immune infiltrate in combination with a corresponding reduction in particular B-cell populations - as in earlier interpretati on of the combined THRLBCL (Van Loo et al, 2010) / host response (Monti et al, 2005) gene signatures.
[00286] Figure 4 displays estimated proportion of BCR-ligated B-ceils (B algM) in baseline patient profiles plotted alongside corresponding progression free survivial (PFS) further to lenalidomide treatment. The association between low estimated B algM proportion and relatively high PFS is striking, in particular in the knowledge that one patient with both low B algM proportion and low PFS (< 3 weeks) died shortly after starting treatment.
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SDI-60022 107vl [00287 J The estimated proportion of plasma cells displays the opposite tendency. This observation lends relevance to the decomposition data, as the two cell types are directly linked (Caven et a!,, 2007) and strong up-regulation of plasma cell marker differentiation marker
PRDMl (BLIMP 1) is observed in profiles associated with response to treatment. This pattern persists to the extent that the sign of a simple subtracted difference between estimated proportions of BCR-ligated B-cells and plasma cells across Revlimid arm profile decompositions (see Figure 5 (lenalidomide-arm)) provides a statistically significant difference in PFS between two patient groups thus defined ( Wi!coxon rank sum, p = 0.04),
[0Θ288] The response-related decomposition values for resting NK-ceil proportions are interesting. Only four profiles are associated with non-zero estimated proportion of N -cells, but these profiles are associated with PFS of 27, 31.9, 32.4 & 85.3 weeks. Clearly, were these estimations to bear out in practice (they include two very small estimated proportions, 0.114, 0.158, 0.004 & 0.002 respectively), the presence of NK cells in the tumor sample may provide a good indicator for Revlimid response enrichment.
[00289 J Finally, relative estimated proportions of dendritic cells are higher and more stable than for NK cells, although they lack the binary 'on/off nature of BCR-ligated B-cell and plasma cell proportions in relation to best response categories. Figure 3 displays the relationship between estimated DC proportion (resting + activated) and PFS. Taken together with the partially response- discriminant populations above, the results suggest potential for, for example, a focused flow cytometry screen for future patients more likely to respond to Revlimid treatment.
References
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[00349] Table 1 , Table, 2, Table 3 and Table 4 are provided below.
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SDI-60022 107vl [00350 TABLE 1
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SD.l-600224J.07vi
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- 112 -
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SD.l-600224J.07v!
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SD.l-600224J.07vi
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SD.l-600224J.07vi
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SD.l-600224J.07vi
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SD.l-600224J.07v!
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SD.l-600224J.07vi
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SD.l-600224J.07v!
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SD.l-600224J.07v!
- 120-
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SD.l-600224J.07v!
- 121 -
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SD.l-600224J.07v!
- 122 -
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SD.l-600224J.07v!
- 123 -
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SD.l-600224J.07vi
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SD.l-600224J.07vi
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SD.l-600224J.07vi
- 126-
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SD.l-600224J.07v!
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SD.l-600224J.07vi
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SD.l-600224J.07vi
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SD.l-600224J.07v!
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SD.l-600224J.07v!
- 131 -
226269-228501 / 12827-501-228
SD.l-600224J.07v!
- 132 -
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SD.l-600224J.07vi
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SD.l-600224J.07vi
- 134 -
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SD.l-600224J.07vi
[00351 TABLE 2
135
226269-228501 / 12827-501 - 28
SD.l-600224J.07v!
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SD.l-600224J.07vi
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SD.l-600224J.07vi
[00352 TABLE 3
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SD.l-600224J.07v!
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SD.l-600224J.07vi
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SD.l-600224J.07v!
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SD.l-600224J.07vi
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SD.l-600224J.07vi
_ 144 ~
226269-228501 / 12827-501-228
SD -600224J.07vi
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SD.l-600224J.07vi
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SD.l-600224J.07v!
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226269-228501 / 12827-501-228
SD.l-600224J.07v!
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SD.l-600224J.07vl
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SD.l-600224J.07vi
- 151 -
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SD.l-600224J.07v!
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SD.l-600224J.07vi
226269-228501 / 12827-501-228 SD.l-600224J.07vi
protein phosphatase 2, regulatory subunit 3, delta [Source:HG
225066 at PPP2R2D ENSG00000175470 0.034190831 0.410023152 6.51512502 6.105101868 10 q26.3 Symbol;Acc:23732]
211977__at GPR107 ENSG00000148358 0.034190831 0.41506351 7.323330309 6.908266799 9 q34.ll G protein-coupled receptor 107 [Source: HGNC Symbol;Acc:178 family with sequence similarity 129, member A [Source:HGNC
217967_s_at FAM129A ENSG00000135842 0.034190831 1.631617618 9.361851904 7.730234286 1 q25.3 Symbol;Acc:16784]
207705 . _s . _at N!NL ENSG00000101004 0.034190831 0.458032479 7.469271682 7.011239203 20 pll.21 ninein-like [Source:HGNC Symbol;Acc:29163]
ubiquitin protein ligase E3 component n-recogriin 3 (putative)
230029 x at UBR3 ENSG00000144357 0.034212391 0.713232741 7.406813979 6.693581237 2 q31.1 [Source:HGNC Symbol;Acc:30467]
227236 TS PAN 2 ENSG00000134198 0.034240086 1.160525883 6.827257615 5.666731733 1 pl3.2 tetraspanin 2 [Source:HGNC Symbo!;Acc:20659j
tumor necrosis factor, alpha-induced protein 2 [Source: HGNC
202510_s_at " f NFAIP2 ENSG000O0185215 0.034240086 1.611311545 10.98695296 9.375641416 14 Q32.32 Symbol;Acc:11895]
202450 s at CTS ENSG00000143387 0.034333589 2.058768294 10.34330275 8.284534455 1 q21.3 cathepsin [Source:HGNC Symbol;Acc:2536]
22639S_at HOOK3 ENSG00000168172 0.034360811 0.907384172 9.099732802 8.192348631 8 pll.21 hook homolog 3 (Drosophi!a) [Source:HGNC Symboi;Acc:23576
219403 . _s . _at HPSE ENSG00000173083 0.034374363 1.273377491 7.073411349 5.800033858 4 q21.23 heparanase [Source:HGNC Symboi;Acc:5164]
calcium channel, voltage-dependent, alpha 2/deita subunit 1
227623 at CACNA2D1 ENSG000O0153956 0.034401348 1.509374159 5.523191348 4.013817189 7 q21.ll [Source:HGNC Symbol;Acc:1399]
poly (ADP -ribose) polymerase family, member 4 [Source:HGNC
202239_at PARP4 ENSG00000102699 0.034401348 0.920213526 9.568361079 8.648147553 13 ql2.12 Symbol;Acc:271]
dihydropyrimidine dehydrogenase [Source:HGNC
204646_at DPYD ENSG000001S8641 0,034480931 1.716538106 8.184742 6.468203894 p21.3 Symbol;Acc:3012j
209335 at DCN ENSG00000011465 0.034606801 2.0562205 8.106748111 6.05052761 12 q21.33 decorin [Source:HGNC Symbo!;Acc:2705]
202341_s_at TRIM2 ENSG00000109654 0.034606801 1.154998273 7.682678312 6.527680038 4 q31.3 tripartite motif containing 2 [Source:HGNC Symbol;Acc:15974]
203386_at J1D4 ENSG00000136111 0.034611968 1.59863556 9.927925002 8.329289442 13 q22.2 " f BCl domain family, member 4 [Source: HGNC Symbol; Acc: 191 procollagen C-endopeptidase enhancer [Source:HGNC
202465 at PCOICE ENSG00000106333 0.034687227 1.110793856 9.858341328 8.747547472 7 q22, l Syrrtbol;Acc:8738]
Rho guanine nucleotide exchange factor (GEF) 3 [SourceiHGNC
21850 l_at ARHGEF3 ENSG00000163947 0.034710054 1,2 1901006 9,645778775 8,433877769 pl4.3 Symbol;Acc:683]
203935_at ACVR1 ENSG00000115170 0,034710054 0.929748525 7.592939572 6.663 9 047 2 q24.1 activin A receptor, type 1 [SourceiHGNC Synibol;Acc: 171]
227726_at RNF166 ENSG00000158717 0.034778201 0.765255386 6,490241897 5.72498651 16 q24.3 ring finger protein 166 [SourceiHGNC Synibol;Acc:28856j family with sequence similarity 45, member A [Source:HGNC
225351_at FA 45A ENSG00000119979 0,034778201 0.712687022 7.985558444 7.272871422 10 q26.1 Syrrtbol;Acc:31793]
cA P responsive element modulator [Source:HGNC
209967 . __s_ . al CREM ENSG00000095794 0.034778201 1,498708212 8,008162888 6,509454676 10 pll.21 Symbol;Acc:2352i
217767 at C3 ENSG00000125730 0,034901896 1.610302964 11.06255643 9.452253469 19 pl3.3 complement component 3 [Source:HGNC Symbol;Acc:1318j leptin receptor overlapping transcript [Source: HGNC
202377_at LEPROT ENSGOOO0O213625 0.034901896 0,958758279 8,927718442 7,968960164 p31.3 Symbol;Acc:29477]
complement component 1, r subcomponent [Source:HGNC
212067__s C R ENSG00000159403 0.034901896 1.095883775 10.97729778 9.881414002 12 pl3.31 Symbol;Acc:1246]
206461 x at MT1H ENSG00000205358 0.034906504 0.937479848 10.94110484 10.00362499 16 ql3 metaiiothionein 1H [Source:HGNC Symbol;Acc:7400]
Rho-reiated BTB domain containing 1 [Source:l-IGNC
212651 .. at RHOBTB1 ENSG00000072422 0.034920435 1.278051787 5.30325947 4.025207683 10 q21.2 Symbol; Acc: 18738]
213077 .. _at YTHDC2 ENSG00000047188 0.034920435 1.082274718 7.160912963 6.078638245 c; q22.2 YTH domain containing 2 [Source:HGNC Symboi;Acc:24721]
238617 .. at KIF26B ENSG00000162849 0.034920435 1.816726723 7.548086863 5.73136014 1 q44 kinesin family member 26B [Source: HGNC Symbo!;Acc:25484]
201540__at FHL1 ENSG00000022267 0.034920435 2.090914464 8.481468308 6.390553844 X q26.3 four and a half LIM domains 1 [Source:HGNC Symbo!;Acc:3702]
227325_at PRR24 ENSG00000257704 0.034920435 0.590098253 7.823937835 7.233839582 19 Q13.32 proline rich 24 [Source:HG C Symbol;Acc:27406]
nudE nuclear distribution E homolog (A. nidulansj-like 1
203093 s at NDEL1 ENSG00000166579 0.034947401 0.741034838 8.576080766 7.835045928 17 pl3.1 [Source:HGNC Symbol;Acc:17620]
202006 .. at PTP 12 ENSG00000127947 0.035088557 1.018262804 9.540273742 8.522010938 7 qll.23 protein tyrosine phosphatase, non-receptor type 12 [Source:H
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[00354 J From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.
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