MULTIPARAMETER FLOW CYTOMETRIC CYTOTOXICITY

SYSTEMS, METHODS, COMPOSITIONS AND KITS FOR EVALUATING THE

SUSCEPTIBILITY OF CANCER CELLS TO A TREATMENT MODALITY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to co-pending U.S. Provisional Patent Application No. 60/702,438, filed July 25, 2005, which application is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Technical Field of the Invention

The present invention relates to the fields of oncology, cell biology, and flow cytometry. More specifically, provided herein are multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits for assessing, in a patient sample, whether one or more cancer cell(s) is susceptible to a chemotherapeutic or other cancer treatment modality.

Brief Description of the Related Art

Current practices in cancer treatment have been around since the 1940s. New drugs have been developed but the 'miracle' cure or prevention for cancer remains elusive to modern medicine. Given the anticipation for new diagnosis tools, new prevention methods, or even hopes of a cancer cure, there has been little emphasis on improving chemotherapy — the current highly toxic treatment of choice.

Once a patient presents with symptoms, a blood or biopsy sample of bone marrow, lymph node, or other tissue is sent to the pathology lab to determine whether or not cancerous cells are present. Current diagnosis technologies are very sensitive and can detect cancer cells even when present at very low levels. There are as many types of cancer as there are individuals. Since cancer cells multiply quickly, gene mutations or chromosomal abnormalities are frequently introduced during disease progression. Thus, cancers are typically comprised of multiple clonal cell populations. Cancer cells from one patient may differ in biology and, consequently, in response to a therapeutic regimen as compared to cancer cells from another patient afflicted with the same disease.

In the United States, blood cancers account for 7% of all cancers for a total of

100,000 new cases per year. B-cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in North America and Europe. The clinical course for CLL is quite variable. Montserrat, Hematol J. 5 Suppl. l:S2-9 (2004); Wendtner et al, Semin. Hematol. 41:224-233 (2004); and Leporrier, Hematol. J. 5 Suppl l:S10-19 (2004). Some patients have an indolent clinical course while for other patients treatment is indicated. For those patients requiring treatment, however, clinical responses are quite variable; many patients show no response or only partial response.

A diagnosis of CLL leads to three possible treatment options: (1) "watch and wait", (2) chemotherapy, and (3) bone marrow transplantation. Watch and wait is often chosen for non-aggressive types of cancer, largely because the available treatment regimens are more toxic than the disease itself. Chemotherapy is chosen for patients whose cancer is growing and poses a significant risk to a patient's short-term health. Bone marrow transplantation is commonly performed upon recurrence of disease following a course of chemotherapy, typically due to out-growth of residual drug resistant cancer cells.

A limitation in current cancer treatment regimens lies in the vast number of choices of chemotherapy drugs and the high incidence of resistance to these drugs. Currently there are over 50 drugs available for the treatment of blood cancers. Usually a patient is given 1 to 3 chemotherapy drugs for treatment. An oncologist prescribes the drugs that work based upon the previous responses of patients with similar cancers.

Currently the selection of appropriate drugs for the treatment of CLL remains empirical. The prediction of clinical response to a particular drug, or regimen for a given patient, remains a great challenge to clinicians. Although some biological factors, especially cytogenetic abnormalities, have been shown of prognostic value for survival, it is not clear whether these biological factors are directly involved in drug sensitivity or resistance.

If a patient's cancer cells are resistant to a prescribed chemotherapy, the patient will undergo an extremely toxic regimen yet realize little or no therapeutic benefit. If the drug(s) administered kills the majority of cancer cells, with residual disease unaffected and undetected, the cancer will likely recur. And, after an initial round of chemotherapy, a patient may be too compromised to undergo subsequent rounds with another therapeutic modality.

Fludarabine is a first line drug for B-cell chronic lymphocytic leukemia (CLL).

About 10 to 20% of patients, however, do not respond to Fludarbine therapy attributing to the poor prognosis for those patients. Assessment of Fludarbine sensitivity or resistance prior to therapy would be predictive of therapeutic outcome and would prevent patients from unnecessarily receiving expensive and ineffective chemotherapy. There remains an unmet need in the art for systems, methods, and associated compositions and kits for identifying patients within a patient population that are afflicted with a disease state comprising one or more cells that are susceptible to treatment with one or more candidate chemotherapy and/or other treatment modalities.

SUMMARY OF THE INVENTION

The present invention addresses these and other related needs by providing, inter alia, multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits that are suitable for assessing, prior to the initiation of treatment, whether a patient sample has one or more cancer cell(s) that is susceptible to one or more candidate chemotherapeutic or other cancer treatment modality. A tissue sample comprising one or more cancer cell(s) is collected from a patient and incubated in vitro with one or more treatment modality of interest. Cancer cells and non cancer cells within the tissue sample are identified based upon the presence of cell population specific surface markers (cell- surface markers; typically antigens) using a label such as a protein ligand, antibody, and/or antibody fragment that specifically binds to a cell population surface-marker within the tissue sample. Drug-induced cell death (cytotoxicity) in cancer cells as well as non-cancerous cells is subsequently detected and quantified without interference from healthy cells within the tissue sample. Cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is effective in a treatment regimen against those one or more cancer cells. In contrast, absence of cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is ineffective in a treatment regimen against those one or more cancer cell(s). Cytotoxicity of noncancerous cells within the same sample indicates an undesired toxic effect of the treatment regimen. Thus, within certain aspects, the present invention provides methods for assessing the suitability of a cytotoxic drug and/or treatment regimen comprising a cytotoxic drug for the treatment of a cancer patient. By such methods, the susceptibility to drug-induced toxicity of one or more normal cell in a patient sample is compared to the susceptibility to

drug-induced toxicity of one or more cancer cell in the same patient sample. Thus, by these methods, the susceptibility of the cancer cell(s) is compared to the susceptibility of the normal cell(s) and the relative susceptibility is calculated and a corresponding in vitro therapeutic index is determined. Such methods will find utility in assessing whether a given cytotoxic drug and/or treatment regimen is suitable for one or more individual cancer patient(s) wherein a high degree of susceptibility to a cytotoxic drug of one or more normal cell(s) as compared to one or more cancer cell(s) indicates that the cytotoxic drug is toxic the patient and consequently is not indicated for treatment of the patient whereas a low degree of susceptibility to a cytotoxic drug of one or more normal cell(s) as compared to one or more cancer cell(s) indicates that the cytotoxic drug is not toxic to the patient and consequently may be indicated for treatment of the patient without an expectation of in vivo toxicity.

The methods disclosed herein can be completed in two days or less, with early detection of cancer cell death, and are sensitive at a single cell level. Thus, the assay is suitable for determining the best chemotherapeutic or other treatment modality for individual cancer patients. The assay is suitable for all clinical samples including those with only a small amount of residual disease. The samples can be either freshly harvested from the patient or cryopreserved.

Patient tissue samples that may be subjected to the multiparameter flow cytometric cytotoxicity systems and methods disclosed herein include, but are not limited to, blood, bone marrow, and biopsy samples of solid tumors. Cancer cells within such patient samples that may be assayed for susceptibility to one or more chemotherapy or other treatment modality include, but are not limited to, cells associated with a wide variety of hematopoitic tancers including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous , leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (either T-cell, NK-cell, or B-cell lymphoma), and multiple myeloma. Other suitable patient samples include biopsy samples from solid tumors such as soft tissue sarcomas and cancers of the brain, esophagus, skin, uterine cervix, bone, lung, liver, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland, and prostate. It will be understood that other patient samples may also be tested by the systems and methods presented herein.

Candidate drugs or agents that may be tested for efficacy, individually or in combination, against one or more cancer cells from a patient sample include, without

limitation, one or more known chemotherapy drug(s) and other small molecules as well as one or more therapeutic biomolecule such as, for example, an antibody or fragment thereof and/or a protein ligand or soluble receptor.

Suitable chemotherapy drugs that may be tested for efficacy in the present systems and methods include, but are not limited to, Altretamine, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Estramustine, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Isotretinoin, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Prednisolone, Procarbazine, Streptozocin, Temozolomide, Teniposide, Thioguanine, Thiotepa, Thiotepa, Topotecan, Tretinoin, Vinblastine, Vincristine, and Vinorelbine. Further exemplified herein are systems, methods, compositions, and kits that employ the chemotherapy drugs Fludarabine, Chlorambucil, Cladribine, and Prednisolone. Suitable antibody therapeutics that may be tested for efficacy in the present systems and methods include, but are not limited to, Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab ozogamicin, Trastuzumab, and Rituximab.

Typically, labels that specifically bind to a cancer cell bind to a cell-surface marker such that cancer cells can be distinguished from admixed non-cancer cells. CeIl- surface markers include, for example, proteins, such as enzymes, hormones, oncofetal antigens, and blood-group antigens; and cell-surface displayed carbohydrates, including glycoproteins and glycolipids. Labels that may be suitably employed for specifically labeling one or more cancer cells include protein ligands, antibodies, and fragments thereof. Exemplary cell-surface protein markers include, but are not limited to, Alpha- fetal Protein (AFP), Human Chorionic Gonadotrophin (hCG), Calcitonin, CA27.29, CAl 5-3, CA19.9, CA125, CA72-4, Carcinoembryonic Antigen (CEA), CD22, CD3, CD33, CD13, CDw65, Lactoferrin (LF), CD68, CD15, CD15s, CD79a (MB-I), CD79b (B29), CD87 (uPA-R), and CDl 17 (c-kit). Exemplary cell-surface carbohydrate markers include, but are not limited to, N-linked and O-linked mucin-like glycoproteins; cell membrane-bound mucin-like carbohydrate structures such as Tn, sialyl-Tn, and T; and blood group-related carbohydrate structures such as Le(x), sialyl-Le(x), ABH, and Le(y). An exemplary cell-surface glycolipid is CA19.9.

In the case of cancers of the blood, including leukemias and lymphomas, one or more of the following cell-surface markers may be advantageously employed: (1) T-cell markers such as CDIa, CD2, CD3, CD4, CD5, CD7, CD8, TCR alpha-beta, and TCR gamma-delta; (2) B-cell markers such as CDlO, CD19 CD20, CD21, CD22, CD23, CD24, CD79b, CD 103, Kappa, Lambda, IgG, IgM, IgD, IgA, and FMC-7; and (3) Myeloid/Monocytic-cell markers such as CD13, CD14, CD15, CD33, CDl Ib, CDl 17, and myeloperoxidase. One or more other marker(s) may also be used such as, for example, CDl Ic, CD25, CD34, CD45, HLA-Dr, Tdt, CD16, CD30, CD38, CD41, CD42b, CD56, CD57, CD61, and glycophorin. In the specific case of B-CLL, exemplified herein, leukemic cells may be identified with a label comprising antibodies that specifically bind to CD5 and antibodies that specifically bind to CD19.

In order to achieve detection by the multiparameter flow cytometric cytotoxicity methods of the present invention, labels typically include one or more fluorescent dye such as, for example, allophycocyanin (APC), phycoerythrin (PE), and fluorescein- isothiocyanate (FITC). Labels may also include a tag such as biotin. Within related aspects, the label may be a first antibody that does not otherwise include a fluorescent dye or other tag. In such cases, a second antibody may be employed wherein the second antibody is capable of specifically binding to the first antibody. Within such aspects, the second antibody typically includes one or more fluorescent dye or other detectable tag.

A number of assay systems that are available in the art may be employed for measuring the cytotoxicity of a specifically labeled cancer cell treated with a candidate chemotherapeutic or other treatment modality. As exemplified herein, cytotoxicity may be measured by staining with annexin, anti-phosphatidylserine, and/or 7-AAD. Other assay systems may also be employed.

These and other aspects of the present invention will become apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE FIGURES

Figures IA- 1C present a gating strategy for measuring drug-induced cytotoxicity in B-CLL cells. Lymphocytes are gated based on forward and side scatter in region R

(Fig. IA). Viable cells, without binding of either 7-AAD or Annexin V, are gated in R2

(Fig. IB). The surviving B-CLL cells are those viable cells that express both CD5 and

CD 19 shown in the upper right quadrant (Fig. 1C).

Figure 2 presents the variability of in vitro drug sensitivity profiles of 43 samples of B-CLL. The drug concentrations for Fludarabine, Chlorambucil, Cladribine and Prednisolone are at 2.5 mg/ml, 2.5 mg/ml, 0.5 mg/ml, and 25 mg/ml, respectively. Leukemic survival indices are displayed as a mean ± SE of triplicate wells.

Figures 3A-3F present the correlation of in vitro drug sensitivity to Fludarabine, Chlorambucil, Cladribine, and Prednisolone. A good correlation is observed between Fludarabine and Chlorambucil (Fig. 3A), between Fludarabine and Cladribine (Fig. 3B), and between Chlorambucil and Cladribine (Fig. 3C). A poor correlation is seen between Prednisolone and Fludarabine (Fig. 3D), between Prednisolone and Chlorambucil (Fig. 3E), and between Prednisolone and Cladribine (Fig. 3F). Figures 4A-4B present differences of in vitro drug sensitivity among different cytogenetic groups. Abbreviations: del = deletion; +12 = trisomy 12; and -13q = 13q deletion. Leukemic survival indices are displayed in mean ± SE. Fludarabine in vitro resistance is significantly higher in cases with a p53 deletion (>10%) than other groups (p<0.05) (Fig. 4A). Prednisolone in vitro resistance is not associated with particular cytogenetic group of B-CLL (Fig. 4B).

Figures 5A-5B present in vitro drug sensitivity of B-CLL cells from patients at different clinical stages of disease. B-CLL cells from patients with early disease are more sensitive to Fludarabine than those from patients with advanced disease (Fig. 5A). No significant difference is seen in B-CLL cells from patients with early and advanced stage exposed to Prednisolone (Fig. 5B).

DETAILED DESCRIPTION OF THE INVENTION

As disclosed above, and as described in greater detail herein below, the present invention provides multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits for assessing a clinical response to one or more drug and/or agent either alone or in combination. By the present systems and methods, a patient sample comprising one or more cancer cells is collected from a patient and contacted in vitro with one or more treatment modality. Cancer cells are specifically identified in the patient sample by contacting the cell population with a label that specifically binds to one or more cancer cell(s) but does not specifically bind normal cells within the cell population. Efficacy of the one or more candidate drug or agent is assessed by assaying for drug-induced cancer cell death (i.e. cytotoxicity) in the specifically labeled cancer cell population. Cytotoxicity of one or more cancer cells within the cell population indicates

that the drug or agent is effective in a treatment regimen against those one or more cancer cells. In contrast, absence of cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is ineffective in a treatment regimen against those one or more cancer cells.

The multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits of the present invention are exemplified herein by their application to the evaluation of the efficacy of four commonly used chemotherapeutic agents (i.e. Fludarabine, Chlorambucil, Cladribine, and Prednisolone) on chronic lymphocytic leukemia (CLL) cells from 43 patients. Specific drug-induced cytotoxicity of leukemic cells was measured after subjecting a patient sample to treatment with Fludarabine, Chlorambucil, Cladribine, and/or Prednisolone. Leukemic cells were identified by staining with antibodies against CD5 and CD19. Cytotoxicity was assessed with Annexin V and 7- AAD. Leukemic cell survival indices were calculated by expressing viable leukemic cells in test culture as percent of viable leukemic cells in control cultures.

The data presented herein demonstrated a marked variability in in vitro drug sensitivity on leukemic cells after in vitro incubation with each of these four chemotherapeutic agents. Without wishing to be limited to any specific cytogenetic characteristics and/or mode of action for any of these chemotherapeutic agents, it is believed that in vitro resistance seen in B-CLL cells is correlative with deletions of p53 or ataxia telangiectasia-mutated gene (ATM), cytogenetic abnormalities that are associated with a poor in vivo therapeutic response to each of these agents.

The present methods can be advantageously completed in two days or less, with early detection of cell death, and are sensitive at a single cell level. The results achieved by these methods yield drug sensitivity profiles that will find utility in guiding the selection of a drug having efficacy against a given disease. The present methods also permit the identification of an appropriate drug for residual disease that exhibits resistance to an initial therapeutic regimen. Thus, within certain embodiments, the present invention permits a determination of a drug modality having efficacy as a second line of chemotherapy to kill the remaining cancerous cells.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of flow cytometry, cell biology, immunology, molecular biology, oncology, and protein chemistry within the skill of the art, many of

which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Shapiro, "Practical Flow Cytometry" (4 ^th Ed. Wiley-Liss, 2003); Longobardi Givan, "Flow Cytometry : First Principles" (2 ^nd Ed. Wiley-Liss, 2001); Watson, "Introduction to Flow Cytometry" (Cambridge University Press, 2004); Nguyen et al., "Flow Cytometry in Hematopathology: A Visual Approach to Data Analysis and Interpretation" (Humana Press, 2002); Hawley and Hawley, "Methods in Molecular Biology: Flow Cytometry Protocols" V. 263. (2 ^nd Ed., Humana Press, 2004); Radbruch, "Flow Cytometry and Cell Sorting" (2 ^nd Ed. Springer, 1999); Sambrook, et al., "Molecular Cloning: A Laboratory Manual" (2nd Edition, 1989); Maniatis et al., "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach, vol. I & II" (D. Glover, ed); "Animal Cell Culture" (R. Freshney, ed, 1986); Perbal, "A Practical Guide to Molecular Cloning" (1984); Ausubel et al, "Current protocols in Molecular Biology" (New York, John Wiley and Sons, 1987); Bonifacino et al, "Current Protocols in Cell Biology" (New York, John Wiley & Sons, 1999); Coligan et al, "Current Protocols in Immunology" (New York, John Wiley & Sons, 1999); and Harlow and Lane Antibodies: a Laboratory Manual Cold Spring Harbor Laboratory (1988).

All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The present invention will be better understood through the detailed description of the specific embodiments, each of which is described in detail herein below.

Patient Samples Comprising Cancer Cells

Patient samples that may be subjected to the multiparameter flow cytometric cytotoxicity methods disclosed herein include, but are not limited to, blood, bone marrow, and biopsy samples of solid tumors. Cancer cells within such patient samples that may be assayed for susceptibility to one or more chemotherapy or other treatment modality include, but are not limited to, cells associated with a wide variety of hematopoitic cancers including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (either T-cell, NK-cell, or B-cell lymphoma), and multiple myeloma. Other suitable patient samples include biopsy samples from solid tumors such as soft tissue sarcomas and cancers of the brain, eye, skin, liver, esophagus,

uterine cervix, bone, lung, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland, and prostate. (See, also, Table 2).

Methodologies for the isolation of blood, bone marrow, and solid tumor samples are well known in the art. Typically, blood is drawn from a vein (venipuncture) and bone marrow is aspirated from the posterior iliac crest. Specimens are typically collected in the presence of an anticoagulant such as, for example, ethylenediaminetetraacetic acid

(EDTA) or heparin. Red blood cells in blood or bone marrow samples are, generally, removed by a lysis procedure or by ficoll gradient centrifugation, by standard methods.

Mononuclear cells typically containing cancer cells are either used fresh or cryopreserved. An excisonal biopsy procedure is often performed to remove a cancer sample of an involved lymph node or other tissue. The cells are disaggregated, single cells are prepared, and they are either used fresh or cryopreserved.

Candidate Chemotherapeutic Modalities Candidate drugs or agents that may be tested for efficacy against one or more cancer cells from a patient sample include, without limitation, one or more known chemotherapy drugs and other small molecules as well as one or more therapeutic biomolecule such as, for example, an antibody or fragment thereof and/or a protein ligand or soluble receptor. Suitable chemotherapy drugs that may be tested for efficacy in the present systems and methods include, but are not limited to, Altretamine, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Estramustine, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Isotretinoin, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Prednisolone, Procarbazine, Streptozocin, Temozolomide, Teniposide, Thioguanine, Thiotepa, Thiotepa, Topotecan, Tretinoin, Vinblastine, Vincristine, and Vinorelbine. Further exemplified herein are systems, methods, compositions, and kits that employ the chemotherapy drugs Fludarabine, Chlorambucil, Cladribine, and Prednisolone.

Suitable antibody therapeutics that may be tested for efficacy in the present systems and methods include, but are not limited to, Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab ozogamicin, Trastuzumab, and Rituximab.

Each of the above mentioned treatment modalities has been shown to be effective in at least one type of cancer or other disease as summarized in Table 1.

Table 1

Cancers and Other Diseases that are Currently being Treated by Existing Candidate Treatment Modalities

Specific Labeling of Cancer Cells within a Patient Population

Typically, labels that specifically bind to a cancer cell bind to a cell-surface marker. Cell-surface markers include, for example, proteins, such as enzymes, hormones, oncofetal antigens, and blood-group antigens; and cell-surface displayed carbohydrates, including glycoproteins and glycolipids. Labels that may be suitably employed for specifically labeling one or more cancer cells include protein ligands, antibodies, and fragments thereof.

As used herein, the term "specifically bind" as used in the context of the binding of a label to a cell-surface marker refers to binding to the cell-surface marker at a detectable level (within, for example, an ELISA assay) and absence of detectable binding with unrelated polypeptides under similar conditions. Specific binding, as used in this context, generally refers to the non-covalent interactions of the type that occur between an antibody and an antigen for which the antibody is specific. The strength, or affinity of antibody-target antigen binding interactions can be expressed in terms of the dissociation constant (K _d ) of the interaction, wherein a smaller K _d represents a greater affinity. Specific binding properties of target-specific antibodies can be quantified using methods well known in the art. One such method entails measuring the rates of target-specific antibody/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (K _On ) and the "off rate constant" (K _Off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K _off /K _on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant Kd. See, generally, Davies et al, Annual Rev. Biochem. 59:439-473 (1990). By "specifically bind" herein is meant that the label binds to a cell-surface antigen, whether a protein, carbohydrate, glycoprotein, lipid, or glycolipid, with a dissociation constant in the range of at least 10 ^"6 -10 ^"9 M, more commonly at least 10 ^~7 - 10 ^{" 9} M.

Exemplary cell-surface cell-surface protein markers include, but are not limited to, Alpha-fetal Protein (AFP), Human Chorionic Gonadotrophin (hCG), Calcitonin, CA27.29, CA15-3, CA19.9, CA125, CA72-4, Carcinoembryonic Antigen (CEA), CD22, CD3, CD33, CD13, CDw65, Lactoferrin (LF), CD68, CD15, CD15s, CD79a (MB-I), CD79b (B29), CD87 (uPA-R), and CDl 17 (c-kit). Exemplary cell-surface carbohydrate markers include, but are not limited to, N-linked and O-linked mucin-like glycoproteins;

cell membrane-bound mucin-like carbohydrate structures such as Tn, sialyl-Tn, and T; and blood group-related carbohydrate structures such as Le(x), sialyl-Le(x), ABH, and Le(y). An exemplary cell-surface glycolipid is CAl 9.9.

In the case of cancers of the blood, including leukemias and lymphomas, one or more of the following cell-surface markers may be advantageously employed: (1) T-cell markers ~ CDIa, CD2, CD3, CD4, CD5, CD7, CDS, TCR alpha-beta, TCR gamma-delta; (2) B-cell markers - CDlO, CD19 CD20, CD21, CD22, CD23, CD24, CD79b, CD103, Total Ig, Kappa, Lambda, IgG, IgM, IgD, IgA, FMC-7; and (3) Myeloid/Monocytic-cell markers - CD13, CD14, CD15, CD33, CDlIb, CDl 17, myeloperoxidase. One or more other marker(s) may also be used such as, for example, CDl Ic, CD25, CD34, CD45, HLA-Dr, Tdt, CD16, CD30, CD38, CD41, CD42b, CD56, CD57, CD61, glycophorin. Table 2, below, further identifies specific cell-surface antigens and their hematopoietic disease associations. In the specific case of CLL, exemplified herein, leukemic cells may be identified with a label comprising antibodies that specifically bind to CD5 and antibodies that specifically bind to CD 19.

Table 2 Cell-surface Tumor Markers for Hematopoietic Malignancies

In order to achieve detection with the multiparameter flow cytometric cytotoxicity methods of the present invention, labels typically include one or more fluorescent dye such as, for example, allophycocyanin (APC), phycoerythrin (PE), and fiuorescein- isothiocyanate (FITC). Labels may also include a tag such as biotin. Within related aspects, the label may be a first antibody that does not otherwise include a fluorescent dye or other tag. In such cases, a second antibody may be employed wherein the second antibody is capable of specifically binding to the first antibody. Within such aspects, the second antibody typically includes one or more fluorescent dye or other detectable tag.

A wide variety of antibodies that specifically bind to the cell-surface antigens disclosed herein and that may be suitably employed in the systems and methods of the present invention are readily available from commercial sources. For example, Bioprocessing, Inc. (Scarborough, ME) provides antibodies to CaI 5-3, Ca27.29, breast tumor antigens, Cal25, ovarian tumor antigen, CaI 9-9, GI tumor antigen, CEA, carcinoembryonic antigen, AFP, alpha fetoprotein, Ca72.4, Tag-72, NSE, neuron specific enolase, Cyfra21-1, cytokeratin-19, PSA, prostate specific antigen using cell culture technologies; Diatec (Oslo, Norway) provides monoclonal antibodies including unconjugated antibodies and antibodies conjugated with FITC, PE, biotin, and APC against markers for leukemias and lymphomas; EMD Biosciences (San Diego, CA) and EXBIO Praha (Vestec, Czech Republic) provide monoclonal antibodies to human CD proteins; Euroclone (Pero, Italy) provides monoclonal antibodies to human CD antigens, cytokines, and apoptosis markers; ImmunoTools (Friesoythe, Germany) provides antibodies to cell markers for flow cytometric applications, anti-cytokine antibodies; Insight Biotechnology (Middlesex, UK) provides antibodies directed against a number of cytokines, CDs as well as secondary antibodies; Jackson ImmunoResearch Laboratories, Inc. (Bar Harbor, ME) provides a wide selection of secondary antibodies; Invitrogen/Molecular Probes (Eugene, OR) provides primary and secondary antibodies, including fluorescently tagged antibodies, for flow cytometry; QED Biosciences, Inc. (San Diego, CA) provides antibodies directed to antibodies for cancer markers and apoptosis; R&D Systems (Minneapolis, MN) provides antibodies against cytokine receptors; Vector Laboratories (Burlingame, CA) provides a wide range of primary and secondary antibodies including primary antibodies for flow cytometry and against various tumor markers, hnmunostaining procedures for labeling cell-surface markers are readily available in the art. For example, see Ormerod, "Flow Cytometry, A Practical Approach"

(3 ^rd Ed., Oxford University Press), which is incorporated herein by reference, describing the selection of fluorochromes, methodology for the preparation of single cell suspensions, and techniques for achieving the immunofluorescence of surface markers.

Methodology for" Assessing Cancer Cell Cytotoxicity The present invention provides multiparameter flow cytometry systems and methods to permit the rapid and simultaneous measurement of individual cells within the context of heterogenous cell populations. Typical flow cytometers comprise three major systems: fluidic, optical, and electronic. The fiuidics system pumps a suspension of cells through a nozzle, creating a stream with laminar flow properties. The optical system focuses one or more laser beams on the stream and collects scattered and fluoresced light from cells as they pass through the laser beam. The electronics measure and digitize these light emissions so that they can be analyzed on a desktop computer.

The following definitions are commonly used to describe flow cytometer parameters. As used herein, the terms "forward Scatter", "FSC", "FS" and "FALS" are used interchangeably to refer to the parameter that is a rough indicator of a cell's size. The terms "side scatter", "SSC", and "SS" are used interchangeably to refer to the parameter that is a rough indicator of cellular granularity, membrane complexity, and number of organelles. Taken together, the two scatter parameters provide a morphological fingerprint of the cells passing through the flow cytometer. Cell-surface markers, such as proteins, carbohydrates, and lipids (see, above) are detected with one or more label, typically an antibody, that has been conjugated to a fluorescent molecule such as, for example, FITC, PE, Texas Red, and APC. DNA content can be measured with propidium iodide (PI), 7-AAD, the Hoechst dyes, or DAPI. BrdU incorporation may be employed for measuring cell proliferation. Apoptosis can be measured in a number of ways. The TUNEL technique identifies DNA strand breaks. Annexin V labeling detects changes in the plasma membrane asymmetry. Nuclear condensation and DNA loss are demonstrated by hypodiploid peaks in DNA content experiments. Uptake of Hoechst 33258 also increases with apoptotic changes.

A number of other markers and assay systems have been described for assessing the following cellular functions: (1) mitochondrial function using Rhl23 or DiOC6; (2) oxidative burst using HE, DCFH-DA, or DHR; (3) reporter genes using GFP and LacZ, along with the substrate FDG; (4) glutathione/reductive reserve using MCB; (5) Ca++ flux using Indo-1; (6) enzyme activities using fluorescent substrates; (7) pH using

SNARF; (8) cell division and conjugation using fluoroscein, calcein, or PKH26; (9) total protein content using SRlOl or fluoroscein)

Assay systems that are available in the art may be employed for measuring the cytotoxicity of a specifically labeled cancer cell treated with a candidate chemotherapy modality and/or biomolecule. As exemplified herein, cytotoxicity may be measured by staining with annexin, anti-phosphatidylserine, and/or 7- AAD.

For example, Cell Technology, Inc. (Mountain View, CA) offers an assay system for measuring cell-mediated cytotoxicity/antigen-dependent cytotoxicity (CMC/ADCC).

By this methodology, a cell tracking dye CFSE is utilized to label a patient cell population. De Clerck et ah, J. Immunol. Meth. 172:115 (1994) and Bronner-Fraser, J. Cell Biol. 101:610 (1985). Cell death is measured by addition of 7AAD (live/dead).

Rabinovitch et al, J. Immunol. 136:2769 (1986) and Su, J. Immunol. 156:4198 (1996).

7AAD only enters membrane-compromised cells and binds to DNA. Flow cytometry is utilized to gate on the target cells and measure 7AAD negative vs. 7AAD postitve cells.

Percent cytotoxicity is calculated by the following equation: 7AAD positive (upper right quadrant) = Rl/ 7AAD Postitve (upper right quadrant) = Rl + 7AAD negative (lower right quadrant) = R2 xlOO

Assay systems for measuring apoptosis, such as those based on two-color flow cytometry, may also be employed in the systems and methods presented herein. After a candidate therapeutic modality is incubated with a patient sample comprising one or more cancer, the cancer cells are stained, as described above, with a target cell-specific label such as, for example, a PE-conjugated mAb. Annexin V-FITC, which binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell-surface, is subsequently added. Cancer cells are gated upon and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all cancer cells fall within discernible populations with respect to annexin V. This methodology permits the analysis of single cancer cells.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. These examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

EXAMPLES

Example 1

Fludarabine Resistance in Chronic Lymphocytic Leukemia (CLL) Detected by an In vitro Multiparameter Flow Cytometric Cytotoxicity Assay

This Example discloses an in vitro multiparameter flow cytometry-based drug sensitivity and resistance assay for routine analysis of clinical samples containing a heterogeneous population of both leukemic and normal hematopoietic cells.

43 clinical specimens of peripheral blood or bone marrow were isolated from patients with B-cell CLL diagnosed at the Oregon Health & Science University. Clinical characteristics of the corresponding patients are presented in Table 3.

Table 3 Clinical and Cytogenetic Features of B-cells from 43 Patients with CLL

Red cells were removed from each patient sample by Ficoll gradient centrifugation by standard methods. Flow cytometric analysis was performed with fresh samples for diagnostic classification. The remaining cells were cryopreserved in liquid nitrogen with RPMI 1640 culture medium (GIBCO, Grand Island, NY) containing 20% heat-inactivated fetal bovine serum (FBS) (GIBCO, Grand Island, NY) and 10% DMSO (Sigma, St. Louis, MO).

Cryopreserved cells were thawed in a water bath at 37 ⁰ C and transferred to RPMI 1640 tissue culture medium containing 2.5 mM MgCl ₂ and 200 units/ml DNAase I. After

incubation at room temperature for 10 minutes, cells were washed twice with phosphate buffered saline (PBS) and resuspended in RPMI 1640 supplemented with 20% FBS, 2 mM glutamine, 100 μg/ml streptomycin, and 100 μg/ml penicillin. Cell viability was determined by trypan blue dye exclusion. Cell concentration was adjusted to 1.5-3.0 x 10 ⁶ viable cells/ml. 150 μl of each cell suspension was added to each well of a 96-well microtiter plate containing 50 μl of either Fludarabine (Sigma, St. Louis, MO), Chlorambucil (Sigma, St. Louis, MO), Cladribine (Sigma, St. Louis, MO), or Prednisolone (Sigma, St. Louis, MO), at 4x their respective empirically derived cut-off concentrations (EDCC), which produced the largest scatter of B-cell CLL cell survival seen among 12 representative samples with test protocols. Each drug was tested in triplicate for each treatment group. The culture plates were incubated at 37 ⁰ C in humidified atmosphere containing 95% air and 5% CO ₂ for 48 hours. At the end of the incubation period, the culture plates were centrifuged at 1500 rpm for 5 minutes and the supernatants discarded.

The cells in each well were resuspended with 100 μl of RPMI 1640 supplemented with 10% FBS. 2.5 μl of CD19-APC (BD Biosciences, San Diego, CA) and 2 μl of CD5- PE (BD Biosciences, San Diego, CA) were added to each well. Plates were incubated on ice for 30 minutes in the dark. After one wash with 250 μl Ix binding buffer (BD Biosciences, San Diego, CA), 100 μl of Ix binding buffer was added. The cells in each well were resuspended and transferred to 120 x 75 mm tubes. 2 μl of Annexin V-FITC (BD Biosciences, San Diego, CA) and 1 μl of 1 mg/ml 7-

AAD (Sigma, St. Louis, MO) were added to each tube and incubated at room temperature in the dark for 10 minutes. 300 μl of Ix binding buffer was added to each tube.

Analysis was performed on a Becton Dickinson FACSCalibur flow cytometer. Results were analyzed using CellQuest Pro software. The specific strategy used is illustrated in Fig. 1. Cells that were CD5 positive, CD 19 positive, 7- AAD negative, and Annexin V negative were counted as surviving leukemic cells. A leukemic cell survival index (LCSI) was defined as the average number of viable leukemic cells in drug-treated cultures divided by the average number of viable leukemic cells in the untreated control cultures expressed as a percentage. Statistical analyses were performed using statistic software GraphPad InStat

(GraphPad Software, San Diego, CA). The difference of LCSI among different groups was determined by means of one-way analysis of variance or Student's /-test. Correlation

of drug effect between different drugs was measured by the Pearson correlation coefficient. Differences were considered to be statistically significant when p values were O.05.

Exposure to drugs for 48 hours yielded detectable cytotoxicity. The optimal drug concentrations that produced the greatest survival of B-CLL cells were 2.5, 2.5, 0.5, and 25 μg/ml for Fludarabine, Chlorambucil, Cladribine, and Prednisolone, respectively, when representative samples were tested. Although spontaneous apoptosis was significant and variable, it did not interfere with the measurement of drug induced cytotoxicity. Viable cells were separated from cells showing toxic effects including cells with early apoptosis (Annexin V positive but 7-ADD negative) and cells with complete loss of membrane integrity (Annexin V positive and 7-ADD positive). Marked variability was seen in the drug-induced cytotoxicity in leukemic cells of 43 cases of B-CLL tested. As shown in Fig. 2, LCSI after drug exposure in culture ranged from less than 10% to 189% among these samples.

A good correlation was observed among responses to Fludarabine, Chlorambucil, and Cladribine in a given B-CLL specimen. See, Figs. 4A-4C. Cross-resistance amongst these 3 drugs was not, however, always observed for individual samples. No correlation was observed for drug-induced cytotoxicity between Prednisolone and the other 3 drugs. See, Figs. 4D-4F. In a majority of the B-CLL samples, there was at least an additive effect of Prednisolone with Cladribine and Chlorambucil. No or very little additive drug effect was observed among a few samples with leukemic cells highly resistant to individual drugs. Occasionally, individual B-CLL samples showed resistance to all drugs and drug combinations tested.

The clinical features were retrospectively reviewed and correlated with the results of the assay for drug resistance. Of 29 patients with stage information available, there was a significant difference between the B-CLL cells from patients at early stages (0-II) and advanced stages (III/IV) (Fig. 5A). B-CLL cells from patients with advanced disease were less sensitive to Fludarabine, Chlorambucil, and Cladribine than were B-CLL cells from patients with early disease. No difference was observed, however, for Prednisolone. Fig. 6B. Of 25 patients with clinical follow-up information, 18 patients were treated with variable regimens and seven patients were on the "watch and wait" list because of indolent disease that does not meet the clinical criteria of treatment. B-CLL cells from patients who were not yet treated generally had greater sensitivity to Fludarabine,

Chlorambucil, and Cladribine as compared to patients who had more aggressive disease requiring treatment.

These data demonstrate that in vitro drug sensitivity assays are predictive of clinical response and prognosis and to monitor the development of drug resistance during treatment and to identify possible new therapeutic strategies in B-CLL patients. See, also, Bousanquet, Lancet 337:711-714 (1991); Larsson et al, Int. J. Cancer 50:177-185 (1992) ; Morabito et al, Br. J. Haematol. 102:528-531 (1998); Callea et al, Haematologica 84:863-864 (1999); Bousanquet et al., Br. J. Haematol. 106:71-77 (1999) Sturm et al., Cell Death Differ. 10:477-484 (2003); Bosanquet and Bell, Blood 87:1962- 1971 (1996); Bosanquet and Bell, Leuk. Res. 20:143-153 (1996); and Bosanquet et al., Br. J. Cancer 76:511-518 (1997).

The presently described assay system is relatively robust and could be adapted to routine clinical laboratory, superior to other in vitro assays such as the MTT assay (Twentyman et al, Br. J. Haematol. 71:19-24 (1989)), the DISC assay (Bosanquet, Lancet 337:711-714 (1991)), and FCMA (Larsson et al, Int. J. Cancer 50:177-185 (1992)), requiring a relatively homogeneous population of leukemic cells in clinical samples typically seen in advanced stage only.

A majority of clinical samples from CLL patients have leukemic cells admixed with a variable number of normal lymphocytes and other normal hematopoietic cells. CLL cells have morphological features indistinguishable from admixed normal lymphocytes that are almost always sensitive to chemotherapeutic drugs. Our protocol is suitable to all routine clinical samples regardless of the percentages of B-CLL cells in the samples.

Taking the advantage of multiparameter flow cytometry, cell population surface markers and drug-induced cytotoxicity are measured simultaneously. In addition, the present methods eliminate possible interference of spontaneous apoptosis that may overshadow the actual drug effect on the B-CLL cells seen in other assays. Therefore, the present assay system is suitable for both fresh and cryopreserved samples. This assay detects a remarkable variability in sensitivity to various drugs and their combinations in B-CLL cells, which is consistent with the reported results of other in vitro chemotherapy sensitivity assays. Morabito et al, Br. J. Haematol. 102:528-531 (1998); Callea et al, Haematologica 84:863-864 (1999) ; Bosanquet et al, Br. J. Haematol. 106:71-77 (1999); Bosanquet et al, Br. J. Haematol. 106:474-476 (1999).

The present data also demonstrate that cross-resistance among fludarabine, chlorambucil and cladribine was quite common. The difference of drug sensitivity among different cytogenetic groups of B-CLL provides additional validation for the reliability of in vitro drug effect as measured by our multiparameter flow cytometric cytotoxicity assay and supports the utility of personalization of therapy for B-CLL patients with different cytogenetic abnormalities and in vitro drug sensitivity profiles. The methodology disclosed herein permit the assessment of drug sensitivity and resistance in drug discovery, clinical trials, and routine clinically practice and offers a cost effective approach to help clinicians identify optimal therapeutic regimens for biologically heterogeneous diseases.

Example 2

In vitro Resistance to Fludarabine Correlates with Deletion of p53 or ATM This Example demonstrates that deletion of p53 and ATM genes is correlative of B-CLL resistance to Fludarabine. The prognostic significance of cytogenetic abnormalities has been well established for B-CLL over the last decade. Fenaux et al, Leukemia 6:246-250 (1992); el Rouby et al, Blood 82:3452-3459 (1993); Wattel et al, Blood 84:3148-3157 (1994); Dohner et al., Bloo d 85:1580-1589 (1995); Dohner et al., Leukemia 11 Suppl 2:S19-24 (1997); Shaw and Kronberger, Cancer Genet Cytogenet. 119:146-154 (2000); Oscier et al., Blood 100:1177 -1184 (2002); and Stilgenbauer and Dohner, Hematol. Oncol. Clin. North Am. 18:827-848 (2004).

Deletions of p53 or ATM are associated with an aggressive clinical course and poor survival. In addition, B-CLL patients with a p53 deletion have been found to be almost invariably resistant to therapy with Fludarabine, currently the first line drug. It is believed that such drug resistance, at least in part, results from a defect in activation of p53-dependent pro-apoptotic responses after DNA damage due to a loss of the p53 gene located on 17p. Rosenwald et al, Blood 104:1428-1434 (2004) and Stankovic et al, Blood 103:291-300 (2004).

Previous studies examining the association between p53 mutation and in vitro drug sensitivity to purine analogs or alkylating agents, have yielded inconsistent and sometimes conflicting results. Thomas et al, Oncogene 12:1055-1062 (1996); Johnston et al, Leuk. Lymphoma 26:435-449 (1997); Bromidge et al, Leukemia 12:1230-1235 (1998); Pettitt et al., Br. J. Haematol. 106:1049-1051 (1999); and Sturm et al, Cell Death

Differ. 10:477-484 (2003). Differences in in vitro drug sensitivity profiles among different cytogenetic subgroups of B-CLL has not been previously studied.

To correlate in vitro drug sensitivity/resistance results with cytogenetic abnormalities, each of the 43 cases described in Example 1 was further studied by interphase FISH in order to identify and characterize common cytogenetic abnormalities in B-CLL, including trisomy 12, deletion of p53, AMT, or 13q. Interphase FISH was performed with fresh slides made from the fixed cell pellets of untreated cells. Hybridization was performed using commercially available probes (Vysis, Downers Grove, IL) to detect genomic losses in 13q, ATM (I lq22.3), and p53 (17pl3.1), and to detect trisomy 12 (CEP12). 200 interphase nuclei were scored for hybridization signals for each probe. On the basis of the results in healthy controls, chromosomal losses and gains were interpreted as negative if they occurred in 5% or fewer of the nuclei.

The FISH results for 37 B-CLL cases are summarized in Table 2. 10 cases of B- CLL with p53 deletion were further divided into 2 subsets, based on the percentages of cells with p53 deletions as defined by FISH. As shown in Fig. 5A, 7 cases with high p53 deletion (i.e. > 10% of cells in the sample) were most resistant to Fludarabine-induced cytotoxicity, among these 5 different cytogenetic groups. Cases with ATM deletions had moderate resistance to Fludarabine. Cases with low p53 deletion (<10% of cells), were sensitive to Fludarabine similar to that of cases with normal cytogenetics, indicating that p53 deletion with in the 5-10% range is most likely false positive due to a variation of background.

No definitive dose effect was detected for p53 deletion. In case 1, four specimens collected at different times over a two-year period were compared. Similar drug resistance was demonstrated in B-CLL cells from peripheral blood, bone marrow, and lymph node. There were no significant changes in drug resistance (data not shown). The presence of additional cytogenetic abnormalities had no significant impact on drug resistance associated with p53 deletion.

Although there was cross-resistance between Fludarabine, Chlorambucil, and Cladribine, in general, in vitro sensitivities to Chlorambucil and Cladribine were less well predicted by the cytogenetic abnormalities of individual cases. Occasionally, leukemic cells with p53 deletions had moderate sensitivity to Chlorambucil and Cladribine. There was no association between cytogenetic abnormalities and in vitro sensitivity to prednisolone. Fig. 5B.

Table 4 lists individual correlations between interphase FISH cytogenetic abnormalities and clinical responses available in follow-up. The seven patients (cases 1 - 7) having the highest B-CLL cells survival indices to Fludarabine had p53 deletions and belonged to a high-risk group clinically. Three of these 7 patients (i.e. cases 4-6) were highly resistant to multiple chemotherapeutic drugs, including Fludarabine. The clinical in vivo effectiveness of Fludarabine could not be evaluated in the other patients because it had either not been used or was used in combination with other drugs. One specimen (i.e. case 7) from a patient with a p53 deletion who was clinically resistant to Fludarabine but responsive to Chlorambucil showed an identical response pattern in the in vitro assay.

Table 4 Summary of In vitro Response, Clinical Response, and Clinical Follow-up

Abbreviations: Del, deletion; Flu, fludarabine; ChI, chlorambucil; CIa, cladribine; Rit, rituxan; Pen, pentostatin; Pre, prednisolone; FCR, fludarabine, cyclophosphamide and rituxan; CHOP, cyclophosphamide, hydroxydaunomycin, vincristine and prednisone; BMT, bone marrow transplantation; CR, complete remission; PR, partial response; NR, no response; NA, not available. *Data show as detectable abnormalities and % of abnormal cells. ** data shown as leukemic cell survival index (%).

These data demonstrate that high resistance in vitro Fludarabine cytotoxicity in B- CLL cells correlates with p53 deletion. Moderate resistance was associated with ATM deletion, and no significant resistance to Fludarabine was associated with other cytogenetic abnormalities. We also found that leukemic cells from patients with p53 deletion were frequently resistant to Chlorambucil and Cladribine but only occasionally resistant to Prednisolone. These findings strongly support the concept that a loss of the function of the p53-dependent apoptotic signaling is a major contribution to drug resistance to DNA damaging agents in B-CLL patients. We therefore provide convincing evidence that B-CLL patients with p53 deletion would not benefit from Fludarabine therapy. The in vitro drug resistance seen in B-CLL cells with deletion of ATM is also likely related to the defect in activation of the p53 pathway which is partially dependent on ATM. Pettitt et al, Bloo d 98:814-822 (2001).

With the increasing therapeutic options available for B-CLL, an in vitro assay for drug sensitivity may have broad application in the clinical management. For cases with p53 or ATM deletions detected by FISH, this assay could be used to confirm the drug resistance to purine analogues or alkylating agents since occasional B-CLL with monoallelic loss or inactivation of the p53 gene may still have moderate Fludarabine sensitivity. In addition, the assay may be used for identification of drugs that are potentially more effective than Fludarabine since B-CLL cells with Fludarabine resistance in vitro are not always resistant to Chlorambucil or Cladribine. See, Case 7 in Table 2. An alternative effective therapy should be sought early for these patients given that they almost invariably have an aggressive course. In cases without p53 or ATM deletions, in vitro drug sensitivity are also variable, suggesting additional biological factors may affect the drug effect in B-CLL cells.