Stéphane de Botton

Prognostic factors for response and overall survival in 282 patients with higher-risk myelodysplastic syndromes treated with azacitidine

Prognostic factors for response and survival in higher-risk myelodysplastic syndrome patients treated with azacitidine (AZA) remain largely unknown. Two hundred eighty-two consecutive high or intermediate-2 risk myelodysplastic syndrome patients received AZA in a compassionate, patient-named program. Diagnosis was RA/RARS/RCMD in 4%, RAEB-1 in 20%, RAEB-2 in 54%, and RAEB-t (AML with 21%-30% marrow blasts) in 22%. Cytogenetic risk was good in 31%, intermediate in 17%, and poor in 47%. Patients received AZA for a median of 6 cycles (1-52). Previous low-dose cytosine arabinoside treatment (P = .009), bone marrow blasts > 15% (P = .004), and abnormal karyotype (P = .03) independently predicted lower response rates. Complex karyotype predicted shorter responses (P = .0003). Performance status ≥ 2, intermediate- and poor-risk cytogenetics, presence of circulating blasts, and red blood cell transfusion dependency ≥ 4 units/8 weeks (all P < 10(-4)) independently predicted poorer overall survival (OS). A prognostic score based on those factors discriminated 3 risk groups with median OS not reached, 15.0 and 6.1 months, respectively (P < 10(-4)). This prognostic score was validated in an independent set of patients receiving AZA in the AZA-001 trial (P = .003). Achievement of hematological improvement in patients who did not obtain complete or partial remission was associated with improved OS (P < 10(-4)). In conclusion, routine tests can identify subgroups of patients with distinct prognosis with AZA treatment.

Ecto-calreticulin in immunogenic chemotherapy

Summary: The conventional treatment of cancer relies upon radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Nonetheless, there are circumstances in which conventional anti‐cancer therapy can induce a modality of cellular demise that elicits innate and cognate immune responses, which in turn mediate part of the anti‐tumor effect. Although different chemotherapeutic agents may kill tumor cells through an apparently homogeneous apoptotic pathway, they differ in their capacity to stimulate immunogenic cell death. We discovered that the pre‐apoptotic translocation of intracellular calreticulin (endo‐CRT) to the plasma membrane surface (ecto‐CRT) is critical for the recognition and engulfment of dying tumor cells by dendritic cells. Thus, anthracyclines and γ‐irradiation that induce ecto‐CRT cause immunogenic cell death, while other pro‐apoptotic agents (such as mitomycin C and etoposide) induce neither ecto‐CRT nor immunogenic cell death. Depletion of CRT abolishes the immunogenicity of cell death elicited by anthracyclines, while exogenous supply of CRT or enforcement of CRT exposure by pharmacological agents that favor CRT translocation can enhance the immunogenicity of cell death. For optimal anti‐tumor vaccination and immunogenic chemotherapy, the same cells have to expose ecto‐CRT and to succumb to apoptosis; if these events affect different cells, no anti‐tumor immune response is elicited. These results may have far reaching implications for tumor immunology because (i) ecto‐CRT exposure by tumor cells allows for the prediction of therapeutic outcome and because (ii) the re‐establishment of ecto‐CRT may ameliorate the efficacy of chemotherapy.

Phase I studies of AVE9633, an anti-CD33 antibody-maytansinoid conjugate, in adult patients with relapsed/refractory acute myeloid leukemia

SummaryThe efficacy of anti-CD33 immunoconjugates had been previously demonstrated for gemtuzumab-ozogamicin. AVE9633 is an anti-CD33-maytansine conjugate created by ImmunoGen Inc. Phase I trials of AVE9633 were performed in patients with AML to evaluate tolerability, pharmacokinetics and pharmacodynamics. Three phase I studies of AVE9633 were performed in 54 patients with refractory/relapsed AML, evaluating drug infusion on day 1 of a 21-day cycle (Day 1 study), day 1 and 8 (Day 1/8 study) and day 1, 4 and 7 (Day 1/4/7 study) of a 28-day cycle. Toxicity was mainly allergic reaction during infusion (3 grade 3 bronchospasms). DLT was reached for the D1–D7 schedule at 150xa0mg/sqm (1 keratitis, 1 liver toxicity), and the MTD was set at 130xa0mg/sqm for this schedule. In the two other phases I, the DLT was not reached. In the Day 1/8 study, CD33 on peripheral blasts was saturated and down-modulated for doses of 75xa0mg/m2 × 2 or higher, which was correlated with WBC kinetics and plasma levels of AVE9633. Decrease of DM4/CD33 ratio on the blasts surface between day 1 and 8 was the rational for evaluating day 1/4/7 schedule. This induced relatively constant DM4/CD33 levels over the first 8xa0days, however no activity was noted. One CRp, one PR and biological activity in five other patients were observed in this study. The Day 1 and Day 1/4/7 studies were early discontinued because of drug inactivity at doses significantly higher than CD33 -saturating doses. No myelossuppression was observed at any trial of AVE9633. The pharmacokinetics/pharmacodynamics data obtained in these studies will provide very useful information for the design of the next generation of immunoconjugates.

Molecular determinants of immunogenic cell death: surface exposure of calreticulin makes the difference.

The treatment of cancer by chemotherapy causes tumour cell death, mostly by apoptosis. This tumour cell death may or may not elicit an immune response. At least in some cases, the efficacy of chemotherapy critically depends on the induction of immunogenic cell death that is a type of cell demise that stimulates the activation of an adaptative anti-tumour immune response, which in turn helps to eradicate residual cancer (stem) cells. Indeed, anthracyclins care more efficient in curing tumours in immunocompetent than in T cell-deficient mice. The molecular mechanism implicated in this anti-tumour T cell activation was recently discovered. Anthracyclins cause immunogenic cell death due to their specific capacity to stimulate the translocation of calreticulin to the cell surface. Calreticulin then acts as an “eat me” signal for dendritic cells, allowing them to phagocytose tumour cells and to prime tumour antigen-specific cytotoxic T cells. Importantly, non-immunogenic chemotherapy can be rendered immunogenic by adsorbing recombinant calreticulin to tumour cells or by enforcing the translocation of endogenous calreticulin to the cell surface by means of PP1/GADD34 inhibitors. This strategy could have major implications for the treatment of human cancer. Indeed, in vivo treatments with anthracyclins can cause the translocation of calreticulin to the surface of circulating tumour cells, in patients with acute myeloid leukaemia (AML). The challenge will be to determine whether the exposure of calreticulin translocation on the tumour cell surface is linked to chemotherapy-induced anti-tumour immune responses and therapeutic efficacy in human cancer.

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models

A CBFA2T3-GLIS2 fusion gene was identified in 31% of non–Down syndrome AMKL.

Treatment by Lenalidomide in lower risk myelodysplastic syndrome with 5q deletion--the GFM experience.

We treated 95 RBC transfusion dependent lower risk MDS with del 5q with Lenalidomide (10mg/day, 3 weeks/4 weeks). Median age was 70.4, median interval from diagnosis 29 months. IPSS was low in 31% and intermediate-1 in 69% patients. Del 5q was isolated, with 1 additional and >1 additional abnormality in 79%, 14%, and 6% patients, respectively. 62 (65%) patients achieved transfusion independence (TI). The only significant factor predicting TI was baseline platelet count >150 G/L and platelet decrease by at least 50% during the first weeks of treatment (p=0.001). Grade III-IV neutropenia and thrombocytopenia were seen in 74% and 37.9% of the cases, respectively, and 3 deaths were attributed to cytopenias. Eight (8%) patients developed deep venous thrombosis (DVT). Platelet decrease by less than 50% predicted a higher risk of DVT. Only 6 patients (6.3%) patients progressed to AML, but median follow-up time was short (18 months). We confirm the high rate of TI with Lenalidomide in lower risk MDS with del 5q. Very close patient monitoring for cytopenias and DVT is mandatory, especially during the first weeks of treatment.

Erlotinib antagonizes ABC transporters in acute myeloid leukemia.

Erlotinib was originally developed as an epidermal growth factor receptor (EGFR)-specific inhibitor for the treatment of solid malignancies, yet also exerts significant EGFR-independent antileukemic effects in vitro and in vivo. The molecular mechanisms underlying the clinical antileukemic activity of erlotinib as a standalone agent have not yet been precisely elucidated. Conversely, in preclinical settings, erlotinib has been shown to inhibit the constitutive activation of SRC kinases and mTOR, as well as to synergize with the DNA methyltransferase inhibitor azacytidine (a reference therapeutic for a subset of leukemia patients) by promoting its intracellular accumulation. Here, we show that both erlotinib and gefitinib (another EGFR inhibitor) inhibit transmembrane transporters of the ATP-binding cassette (ABC) family, including P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP), also in acute myeloid leukemia (AML) cells that do not overexpress these pumps. Thus, inhibition of drug efflux by erlotinib and gefitinib selectively exacerbated (in a synergistic or additive fashion) the cytotoxic response of KG-1 cells to chemotherapeutic agents that are normally extruded by ABC transporters (e.g., doxorubicin and etoposide). Erlotinib limited drug export via ABC transporters by multiple mechanisms, including the downregulation of surface-exposed pumps and the modulation of their ATPase activity. The effects of erlotinib on drug efflux and its chemosensitization profile persisted in patient-derived CD34+ cells, suggesting that erlotinib might be particularly efficient in antagonizing leukemic (stem cell) subpopulations, irrespective of whether they exhibit or not increased drug efflux via ABC transporters.

Abstract CT103: Clinical safety and activity in a phase I trial of AG-221, a first in class, potent inhibitor of the IDH2-mutant protein, in patients with IDH2 mutant positive advanced hematologic malignancies

Background: Cancer metabolism represents an emerging field of novel cancer target discovery. Somatic point mutations in the metabolic enzymes isocitrate dehydrogenase 1/2 (IDH1/2) confer a novel gain-of-function in cancer cells, which results in the accumulation and secretion of the onco-metabolite, R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α᠄KG dependent dioxygenases including histone and DNA demethylases, which regulate the epigenetic state of cells and result in altered cellular differentiation. IDH2 mutations have been identified in a spectrum of solid tumors and hematologic malignancies including chondrosarcoma, glioblastoma, acute myeloid leukemia (AML), and myelodyplastic syndromes (MDS). AG-221 is the first IDH mutant inhibitor in clinical trials; it is an oral, potent, reversible, and selective inhibitor of the mutated IDH2 protein. In a primary human AML xenograft model, AG-221 treatment reduced 2-HG levels and demonstrated a dose dependent survival benefit. Early results of the ongoing first in human phase I study of AG-221 in patients with advanced IDH2 mutant positive hematologic malignancies are reported here. Study Methods: This phase I study of oral AG-221 is designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD) including assessment of 2-HG levels, and clinical activity in patients with advanced hematologic malignancies. AG-221 is administered orally 2 times per day (BID) in continuous 28-day cycles. Sequential cohorts of up to 5 PK-evaluable patients are enrolled at higher dose levels, followed by multiple planned expansion cohorts. The eligible patient population includes those with relapsed or refractory AML, myelodysplastic syndromes (MDS,) and elderly untreated AML that harbor an IDH2 mutation. Blood and bone marrow is collected at multiple time points for determination of the PK and PD effects of AG-221. Response assessments via bone marrow examination are performed on Days 15, 29, 57, and every 56 days (2 cycles) thereafter. Study Status and Results: The study was activated in September 2013. As of February 26th 2014, a total of 19 patients have been enrolled; 18 with AML and 1 with MDS. All patients were IDH2-mutant positive by local testing. AG-221 doses administered included 30 mg BID (n=7), 50 mg BID (n=5), 75 mg BID (n=5), and 100 mg QD (n=2). Two patients were added to the 30 mg BID cohort to replace PK-unevaluable patients. Fourteen of 19 patients remain on study drug treatment. Therapy has been well tolerated; with no dose-limiting toxicities reported. The maximum tolerated dose has not been reached. Possible drug-related severe adverse events were reported in two patients: grade 2 hyperleukocytosis and grade 3 confusion. In the first cohort there were three deaths due to sepsis within 30 days of study drug termination. One of these was attributed as possibly related to study drug treatment. Preliminary analysis of PK at 30 and 50 mg doses demonstrated excellent oral AG-221 exposure and a mean plasma half-life of greater than 40 hours. Evaluation of the PD response demonstrated sustained reduction in 2-HG plasma levels of up to 97% following AG-221 dosing in cohort 1 and 2. Ten AML patients are evaluable for efficacy at this time: (N=5 at 30 mg BID, N=5 at 50 mg BID), 5 men and 5 women, with a median age (range) of 62.5 years (53-74). Eight patients had an R140Q mutation and two had a R172K mutation. The median number of prior regimens was 2 (1-4) including one patient who had relapsed after an allogeneic bone marrow transplant. Currently, 6 of 10 patients have had objective responses, including 2 complete remissions defined by the International Working Group Criteria (1 at 30mg BID and 1 at 50mg BID). The four other responses are ongoing and will be updated. Marked differentiation of myeloblasts into mature forms, consistent with preclinical models, was associated with responses. Only one patient experienced disease progression. One patient with a CR was removed from study to undergo allogeneic BMT. Five of the 6 responding patients remain on AG-221. Dose escalation continues along with exploration of a once daily dosing regimen. Expansion cohorts are being planned. Conclusions: AG-221, a novel, oral, potent IDH2 mutant inhibitor is well tolerated and shows promising initial clinical and pharmacodynamic activity in patients with relapsed and refractory IDH2 mutant hematologic malignancies, even in the lowest dose cohort. These data provide early validation of mutant IDH2 as a therapeutic target in hematologic malignancies. Additional safety and efficacy data from the ongoing study will be presented. Clinical Trial Information: NCT01915498 Citation Format: Eytan Stein, Martin Tallman, Daniel A. Pollyea, Ian W. Flinn, Amir T. Fathi, Richard M. Stone, Ross L. Levine, Samuel Agresta, David Schenkein, Hua Yang, Bin Fan, Kate Yen, Stephane De Botton. Clinical safety and activity in a phase I trial of AG-221, a first in class, potent inhibitor of the IDH2-mutant protein, in patients with IDH2 mutant positive advanced hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT103. doi:10.1158/1538-7445.AM2014-CT103

Tyrosine kinase inhibitors for the treatment of acute myeloid leukemia: Delineation of anti-leukemic mechanisms of action

Initially, tyrosine kinase inhibitors (TKIs) were developed as targeted therapies that would solely interfere with aberrant tyrosine kinase activation in malignant cells. Nevertheless, preclinical and clinical studies demonstrated that TKI also exhibit off-target effects, that is effects not mediated by the assumed mechanisms of action. We and others showed that the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib exert potent antineoplastic effects on EGFR-negative myeloblasts from patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Here, we undertook a side-by-side comparison of the anti-leukemic efficacy of four different TKI in MDS and AML. Besides the EGFR inhibitor erlotinib, which served as a point of reference, we employed the dual EGFR/HER2 TKI lapatinib, as well as the multikinase inhibitors dasatinib and sorafenib. All four drugs had anti-leukemic effects on cell line models of MDS/AML in vitro as well as on malignant blasts from MDS/AML patients ex vivo. We explored the biological phenomena underlying this anti-leukemic efficacy. Since it is established that a therapeutic benefit in MDS/AML can be conveyed by induction of cell cycle arrest, apoptosis and/or differentiation, we deciphered the individual contribution of these three phenomena to the anti-leukemic action of each of the four TKI. The concomitant assessment of the panel of TKI enables us thus to define (and quantify) their differential capacity to impact on the three biological phenomena, and provide further evidence that these mechanisms are not solely explained by on-target effects.

Erlotinib and gefitinib for the treatment of myelodysplastic syndrome and acute myeloid leukemia: a preclinical comparison.

Erlotinib and gefitinib, two inhibitors of the epidermal growth factor receptor (EGFR), can stimulate apoptosis and differentiation of myeloid cell lines that lack EGFR, unveiling a novel, therapeutically exploitable off-target effect of tyrosine kinase inhibitors. Here, we performed a side-by-side comparison of erlotinib and gefitinib effects on a broad spectrum of malignant myeloid cell lines, as well as on primary myeloblasts freshly purified from the bone marrow of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Both erlotinib and gefitinib induce apoptosis of a cell line (KG-1) that represents AML, and differentiation in another cell line (P39) derived from a patient with high-risk MDS. In this setting, erlotinib was more efficient than gefitinib. Erlotinib and gefitinib were equipotent in inducing apoptosis of primary CD34+ myeloblasts from MDS and AML patients, yet had no toxic effect on CD34+ progenitor cells from healthy donors. Although the response of individual MDS and AML patients in vitro was highly heterogeneous, the pro-apoptotic effects of erlotinib and gefitinib correlated significantly. These results suggest that erlotinib and gefitinib share a mechanistically related off-target effect that may be taken advantage of for the therapy of MDS and AML.