ROLE OF MCP-1 IN RELAPSE OF ACUTE MYELOID LEUKEMIA AFTER

HEMATOPOIETIC STEM CELL TRANSPLANTATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to United States Provisional Patent Application No. 61/912,614, filed December 6, 2013, which is incorporated by reference in its entirety.

BACKGROUND AND SUMMARY

[002] This application relates to the correspondence between serum Monocyte Chemo-Attractant Protein- 1 (MCP-1) levels and Acute Myeloid Leukemia (AML) or Acute Myeloid Leukemia/Myelodysplastic Syndrome (AML/MDS) or the relapse of AML or AML/MDS. AML is the most common type of acute leukemia in adults. It is a malignancy that arises within the bone marrow, in which malignant leukemia cells proliferate along with a disruption of normal hematopoiesis or blood cell production. Patients with preexisting myelodisplasia (MDS patients) may be at increased risk of developing AML. While AML treatments have improved over the years, survival rates, particularly in elderly patients and AML/MDS patients, remain low, with a high risk of relapse.

[003] Current AML treatments may involve, for example, conditioning or induction chemotherapy, for example with DNA-damaging agents. While this initial chemotherapy may kill a majority of leukemia cells, further treatments may be needed to prevent relapse, such as hematopoietic stem cell transplantation (HCT or HSCT). However, relapse after HCT remains a leading cause of treatment failure, and as much as 30-40% of post-HCT patients may suffer a relapse. [004] Thus, there is a need in the art for a means of identifying patients who have received treatment for AML or AML/MDS but who are at high risk for relapse, for example, to provide further treatment or supportive care to those patients either to prevent relapse or reduce its intensity. For instance, it may be possible to modify the treatment of those patients at risk for relapse so that they receive further AML treatment before a relapse can be detected. There is also a need in the art for means of testing MDL patients for increased risk of developing AML.

[005] The present inventor has discovered that a higher than average level of the cytokine protein MCP-1 in an AML/MDS patient after HCT treatment correlates to an increased risk of relapse. (See, e.g., Figure 3.) In contrast, 41 other cytokine markers were tested but found not to correlate with relapse of AML/MDS in post-HCT patients. Accordingly, the present application encompasses methods of treating AML or

AML/MDS that incorporate information about the level of MCP-1 protein in a bodily fluid of the patient following previous AML treatment. Further, the present application also encompasses using of MCP-1 levels to detect risk of AML onset in MDS patients.

[006] Included herein is method of treating relapse of AML in a patient previously treated for AML, comprising obtaining a measurement of MCP-1 in a fluid sample from the patient, and providing further AML treatment to the patient if the patient is measured to have an MCP-1 level above that of an average MCP-1 level observed in a population of post-treatment AML patients, such as a population of patients who have received similar AML treatment. Also included is an ex vivo method of determining risk of relapse of AML in a patient previously treated for AML, comprising measuring MCP-1 level in a fluid sample from the patient or obtaining such a measurement, wherein an MCP-1 level above that of an average MCP-1 level observed in a population of post-treatment AML patients indicates that the patient is at risk for relapse. In alternative embodiments of these methods, the patient's MCP-1 level is compared to the average MCP-1 level of post-treatment AML patients who did not show relapse of AML or of AML/MDS within 3 months, 6 months, or 1 year after treatment.

[007] In some embodiments of the above methods, the patient suffers from AML/MDS, and in some embodiments, the above average MCP-1 level predicts relapse of MDS. Also included is an ex vivo method of determining whether an MDS patient is at risk of developing AML, comprising obtaining a measurement of MCP-1 in a fluid sample from the patient, wherein an MCP-1 level above that of an average MCP-1 level observed in a population of MDS patients indicates that the patient is at risk for developing AML.

[008] In some embodiments in which the patient has previously received AML treatment, the previous treatment comprises hematopoietic stem cell transplantation (HCT), such as allogenic HCT or autologous HCT, optionally combined with

chemotherapy. For example, some patients may have received a conditioning regimen of chemotherapy as well as HCT prior to measurement of their MCP-1 level and optional further treatment.

[009] The previous chemotherapy treatment may include treatment with one or more of cytarabine, daunorubicin, mitoxantrone, etoposide, idarubicin, fludarabine, busulfan, clofarabine, cyclophosphamide, topetecan, an azanucleoside such as 5- azacytidine or decitabine, sapacitabine, or vosaroxin. Two or more of these agents may be combined, such as a combination of busulfan and fludarabine (Bu-Flu treatment). In some embodiments, a patient may also have been previously treated with a tyrosine kinase inhibitor such as AC220.

[010] In some embodiments in which the patient has previously undergone HCT treatment, the MCP-1 level is measured post-transplant, such as approximately 20-40 days, approximately 25-35 days, approximately 27-33 days, or approximately 29-31 days post-transplant, or 30 days post-transplant. In some such embodiments, the average MCP-1 level against which the patient's MCP-1 level is compared is an average MCP-1 level in post-transplant patients, for example, an average MCP-1 level measured the same number of days after transplant that the patient's measurement was taken.

[011] In some embodiments, the further AML treatment given to patients showing higher than average MCP-1 levels comprises administering one or more of an inhibitor of MCP-1 , an azanucleoside such as 5-azacytidine (Vidaza®) or decitabine, lenalidomide (Revlimid®), a histone deacetylase inhibitor, or an angiotensin converting enzyme (ACE) inhibitor, or a combination of two or more of the above. In some embodiments, the further AML treatment comprises HCT, such as an allogenic or autologous HCT. For example, a patient who has already had HCT but who is at risk of relapse due to an above average MCP-1 level may be treated with a further HCT. In some embodiments, the further AML treatment comprises chemotherapy. In some embodiments, the patient may receive a combination of two or more of HCT or further HCT, chemotherapy, azanucleoside treatment (e.g. 5-azacytidine and/or decitabine), lenalidomide, histone deacetylase inhibitor treatment, and ACE inhibitor treatment.

[012] The MCP-1 level is determined ex vivo in a fluid sample of the patient, such as whole blood, plasma, or serum. In some embodiments, the MCP-1 level is measured by an assay comprising binding of an anti-MCP-1 antibody specific for MCP-1 protein in the fluid sample, such as an ELISA assay. In some embodiments, the MCP-1 level is measured at the messenger RNA level by reverse transcription and polymerase chain reaction (RT-PCT) amplification of messenger RNA in the sample using primers specific for the MCP-1 coding sequence.

[013] Additional objects of the invention will be set forth in part in the description that follows. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[014] Figure 1 shows the overall survival (OS) at 1 and 2 years and disease free survival at 1 and 2 years.

[015] Figure 2 shows cumulative incidence of treatment related mortality at 1 and 2 years and the cumulative incidence of relapse at 1 and 2 years respectively.

[016] Figure 3 is shows that of a subset of 30 patients where chemokine analysis was performed, only MCP-1 levels at day 30 post HCT were predictive of relapse out of the 42 biological markers tested.

DESCRIPTION OF EMBODIMENTS

[017] Reference is now made to the certain exemplary embodiments.

[018] This application encompasses methods of predicting and treating relapse of AML in patients who have previously received AML treatment. "Patients" herein refers to mammalian patients such as human patients, domestic mammals such as dogs and cats, and laboratory animals such as mice or rats. The term "treatment" as used herein covers any administration or application of a therapeutic or a medical procedure in a mammal, including a human, and includes inhibiting disease progression, partially inhibiting or slowing disease progression, preventing a relapse or partially inhibiting or slowing or reducing the intensity of a relapse, causing the disease to go into remission, and curing the disease.

[019] In some embodiments, the AML patients are AML/MDS patients.

AML/MDS is a form of AML that may arise from an antecedent myelodysplastic syndrome. MDS comprises a group of bone marrow disorders (myelodisplasias).

Among the types of MDS disorders are refractory anemia (low red blood cell count), refractory neutropenia (low white blood cell count), refractory thrombocytopenia (low platelet count), refractory anemia with ring sideroblasts (RARS), refractory anemia with excess blasts (RAEB-l and -2), refractory cytopenia with multilineage dysplasia (RCMD), myelodysplastic syndrome associated with isolated del (5q), as well as unclassified MDS syndromes. An antecedent MDS disorder may predispose a patient to developing AML. AML/MDS often occurs in elderly patients. AML/MDS patients have a particularly poor prognosis and a relatively high rate of relapse.

[020] Patients according to the inventive methods, in some embodiments, may have received previous treatment for AML, and thus the methods may help to predict relapse of AML or AML/MDS. "Relapse," as used herein, refers to a diagnosis of the return of AML or AML/MDS in the patient. Diagnosis of AML relapse or initial diagnosis of AML may be based upon detection of leukemia cells in the blood or bone marrow of the patient, for example in a bone marrow aspirate or biopsy, combined with cytogenetics, flow cytometry, and/ or visual inspection of cells. [021] Current AML treatments include chemotherapy regimens, often coupled with further treatment such as HCT. Chemotherapy may involve, for example, an initial conditioning or induction treatment intended to kill as many leukemia cells as possible. The chemotherapy may involve treatment with DNA damaging agents. Exemplary drugs for such treatments include one or more of cytarabine, daunorubicin, mitoxantrone, etoposide, idarubicin, fludarabine, busulfan, clofarabine, cyclophosphamide, topetecan, an azanucleoside such as 5-azacytidine or decitabine, sapacitabine, or vosaroxin, and combinations of two or more of the above agents such as busulfan and fludarabine (Bu- Flu treatment). Depending upon the health of the patient, the intensity of the conditioning chemotherapy may be varied. For example, reduced and full intensity conditioning (RIC, FIC) regimens employing intravenous busulfan plus fludarabine (Bu- Flu) have been used and both have resulted in improved treatment related mortality and comparable overall survival in patients undergoing allogeneic HCT for AML/MDS who are not candidates for more intense regimens. Bu-Flu based conditioning regimens, for example, have been observed to improve overall survival in patients with AML/MDS while not impacting relapse rate after allogeneic HCT.

[022] In some embodiments, the patient has previously received HCT before the patient's MCP-1 level is determined. In some embodiments, the HCT is "allogenic," meaning that the transplanted stem cells have been derived from a donor, such as a related donor (RD) or an unrelated donor that is otherwise a close match for the patient (a matched unrelated donor or MUD). HCT can also be "autologous," in which the patient's own stem cells are used. [023] In some embodiments, the MCP-1 measurement is obtained at a point in time post-HCT transplant, such as approximately 20-40 days, approximately 25-35 days, approximately 27-33 days, approximately 29-31 days, or approximately 30 days post- transplant. Obtaining the MCP-1 measurement at a particular point in time means that the fluid sample from the patient that is used for the MCP-1 measurement is taken at that point in time, i.e. at approximately 20-40 days, approximately 25-35 days, approximately 27-33 days, approximately 29-31 days, or approximately 30 days post-transplant.

[024] In some embodiments, to determine risk of relapse, the MCP-1 level measured for the individual patient is compared to an average MCP-1 level of a pool of similar patients. The terms "average" and "mean" are used interchangeably herein. For example, for a patient who has undergone prior AML treatment, the average may be the average MCP-1 level in patients who have undergone prior AML treatments or the average level in patients who have undergone a similar prior treatment. Thus, for patients who have received HCT, for example, the comparative average MCP-1 level may be that for a pool of AML patients who have also previously received HCT. For a patient whose sample post-HCT is taken within a certain number of days after HCT treatment, the comparative average MCP-1 level may be that of a pool of post-HCT patients whose MCP-1 levels were measured within the same number of days post HCT treatment. Thus, in some embodiments, where the patient's fluid sample for MCP-1 measurement is obtained approximately 20-40 days, approximately 25-35 days, approximately 27-33 days, approximately 29-31 days, or approximately 30 days post- transplant, the MCP-1 level in the patient's fluid sample is compared to that of an average of post-HCT patients whose fluid samples for MCP-1 measurement were taken within the same approximate date ranges as that of the patient. For an MDS patient tested to predict risk of developing AML, the average may be the average of a pool of MDS patients.

[025] In some embodiments, an MCP-1 level above that of the average MCP-1 level of a pool of similar patients may indicate risk of relapse or of developing AML. In some embodiments, the MCP-1 level indicating risk of relapse or of developing AML may be at least about 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, or 65% higher than of the average MCP-1 level of a pool of similar patients. In some embodiments, the MCP-1 level indicating risk of relapse or of developing AML may be higher than about 0.5 standard deviation, 1.0 standard deviation, 1.5 standard deviations, or 2 standard deviations above the average MCP-1 level of a pool of similar patients.

[026] In some embodiments, the pool or population of patients used for the comparative average MCP-1 level may be a historical pool, such that the average MCP-1 level against which the patient's MCP-1 level is compared is one that was determined in earlier AML clinical studies, for example.

[027] Alternatively, in some embodiments, the patient's MCP-1 level can be compared to the MCP-1 level in a pool of AML patients, such as patients who had received similar prior treatments, who did not show relapse after AML treatment, such as patients who did not show relapse after 3 months, 6 months ,or 1 year post-HCT transplant. In some such embodiments, the MCP-1 level indicating risk of relapse may be at least about 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, or 65% higher than of the average MCP-1 level of a pool of similar patients who did not relapse after a period of time such as 3 months, 6 months, or 1 year. In some embodiments, the MCP-1 level indicating risk of relapse may be higher than about 0.5 standard deviation, 1.0 standard deviation, 1.5 standard deviations, or 2 standard deviations above the average MCP-1 level of a pool of similar patients who did not relapse after a period of time such as 3 months, 6 months, or 1 year.

[028] For previously treated patients, prediction of relapse due to a high MCP-1 level may allow for the better tailoring of treatments seeking to prevent that relapse from occurring. For example, in some embodiments where a patient is receiving

immunosuppressive treatment following HCT, e.g. allogenic HCT, such treatments may be reduced. In some embodiments, the patient may be given a further chemotherapy treatment, such as with one or more of cytarabine, daunorubicin, mitoxantrone, etoposide, idarubicin, fludarabine, busulfan, clofarabine, cyclophosphamide, topetecan, an azanucleoside such as 5-azacytidine or decitabine, sapacitabine, or vosaroxin, such as a combination of busulfan and fludarabine (Bu-Flu treatment). In some embodiments, the patient may be given one or more further HCT treatments, such as a further allogenic HCT or an autologous HCT treatment, optionally in addition to further chemotherapy. In some embodiments, the patient may also be treated with an MCP-1 inhibitor. In some embodiments, the patient may be treated with lenalidomide and/ or a histone deacetylase inhibitor and/ or an antiotensin converting enzyme (ACE) inhibitor. In some

embodiments, the patient may be further treated with a combination of any of the above treatments.

[029] For MDS patients tested for MCP-1 levels as a means of predicting a first occurrentce of AML, treatment regimes may also be modified, for example, by providing an MCP-1 inhibitor such as azacytidine, or by testing for the presence of leukemia cells, such as in a bone marrow biopsy or aspirate at least once following the MCP-1 analysis.

[030] MCP-1 (also known as chemokine (C-C motif) ligand 2 or CCL2) binds to the CCR2 receptor protein in vivo. MCP-1 recruits monophages and mactocytes to sites of inflammation and may be overexpressed in sites of inflammation in diseases such as atherosclerosis, rheumatoid arthritis, and multiple sclerosis. A variety of chemokines including MCP-1 may have altered expression levels in certain tumor cell lines. The present inventor has found, in a study of 42 different chemokines or cytokines, however, that MCP-1 was the only one whose expression levels correlated with risk of relapse in post-HCT AML/MDS patients.

[031] The MCP-1 level may be tested in a variety of ways. A patient's bodily fluid, such as whole blood, plasma, or serum, may be used for the test. Thus, the test may be conducted ex vivo. MCP-1 may be detected in the sample at the protein level, for instance, by using an assay involving an MCP-1 -specific monoclonal antibody and detecting binding of the antibody to MCP-1 in the sample via a label. An exemplary specific antibody assay is an enzyme-linked immunoabsorbent assay (e.g. an ELISA assay). For instance, the sample may be imobilized on a plate or substrate and a solution containing a labeled MCP-1 -specific antibody may be added to it, and the sample may then be washed, leaving behind bound MCP-1 /antibody complexes. The label, for example a fluorescent or luminescent label, may be attached to the MCP-1 -specific antibody, or to a secondary or tertiary antibody that binds to the MCP-1 specific antibody, or the label may be activated through a chemical reaction that occurs upon binding of a secondary or tertiary antibody. [032] Alternatively, the MCP-1 -specific antibody may be immobilized on a substrate to which the fluid sample is applied. MCP-1 may then be detected by means of a label attached to the bound antibody, such as a luminescent or fluorescent label attached to a second MCP-1 -specific antibody.

[033] In other embodiments, MCP-1 level can be detected at the messenger RNA level using reverse transcription and polymerase chain reaction amplification using MCP-1 -specific primers.

[034] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

EXAMPLE

[035] In order to assess relapse following allogeneic HCT for AML/MDS, a retrospective analysis was performed to evaluate the outcomes of 55 consecutive patients with AML/MDS (49/ 6) who received intravenous Bu-Flu based conditioning. Blood samples were collected post HCT in a subset of those patients (30 patients). Serum values of 42 biological markers were measured at day 30 post HCT (2/ 30 patients were day 60 samples) using multiplex Luminex® assay. Patient characteristics are shown in Table 1.

Table 1

Patient Characteristics number (%)

Donor Type

Unrelated donor (MUD) 39 (71%)

Related donor (RD) 16 (29%)

Contitioning Regimen

Bu2Flu (FIC) 36 (65%)

Bu2Flu (RIC) 19 (35%)

Status at Transplant

CR1 36 (65%)

CR2 5 (9%)

PtF 14 (35%)

CIBMTR Risk

Low 39 (71%)

Intermediate 4 (7%)

PIF 14 (22%)

Cytogenic Bask

Low 33 (60%)

High 22 (40%)

>mplete remission; PIF— primary refractory

[036] With a median follow up of 18 months, the overall survival (OS) at 1 & 2 years was 73 ± 6% and 67 ± 7%, respectively, (Fig 1). Similarly, disease free survival at 1 and 2 years was 64 ± 7%. [037] As expected, there was low cumulative incidence of treatment related mortality of 8 ± 3% at 1 and 2 years while the cumulative incidence of relapse was 28.0 ± 3% and 31 ± 2% at 1 and 2 years respectively, (Fig 2).

[038] Cumulative incidence of grade II-IV acute graft versus host disease (GVHD) was 54% with grade III-IV of 25% at day 100. Cumulative incidence of chronic GVHD was 49, 54% at 1 and 2 years respectively.

[039] In a subset of patients where chemokine analysis was performed (30 patients), only MCP-1 levels at day 30 post HCT were predictive of relapse out of the 42 biological markers tested. The 7 out of 30 patients who relapsed in this subset (23%) had higher than mean level of MCP-1 at day 30 of 537, SD ±213 versus 324, SD ± 160, P=0.007 MCP-1 was predictive of leukemic relapse 82 days in advance on average prior to overt hematological relapse. Full chimerism (>95%) was detected at Day 30 in 5/7 patients who relapsed in the biological marker group. (See also Figure 3.)

[040] Although the embodiments have been described relative to various selected non-limiting examples, there are numerous variations and modifications that will be readily apparent to those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims hereto attached and supported by this specification, the embodiments may be practiced other than as specifically described.