Bueso‐Ramos, Carlos E.

ABSTRACT Background Chronic myelomonocytic leukemia (CMML) is largely a disease of individuals older than 50 years; its occurrence in patients aged 50 years and younger is rare. The authors sought to characterize the clinicopathologic features and genetic profile of young‐onset (age 50 years and younger) CMML (y‐CMML) compared with older onset (older than 50 years) CMML (o‐CMML). Methods The authors conducted a multi‐institutional study of 32 patients with y‐CMML and 119 patients with o‐CMML, analyzing clinical, morphologic, cytogenetic, and molecular data. Results Patients who had y‐CMML frequently presented with splenomegaly (59% vs. 11%; p < .001) and hepatomegaly (25% vs. 2%; p < .001) compared with those who had o‐CMML. Although the overall incidence of cytogenetic abnormalities was similar, patients who had y‐CMML exhibited distinct mutational patterns. PTPN11 mutations were significantly more common in y‐CMML (13% vs. 3%; p = .034) than in o‐CMML, whereas TET2 (6% vs. 57%; p < .001) and SRSF2 (16% vs. 46%; p = .002) mutations were significantly less frequent. In addition, multi‐hit TET2 (6% vs. 34%; p = .002) and TET2‐SRSF2 (3% vs. 29%; p = .003) co‐mutations were rare in y‐CMML. However, the molecular differences did not affect the CMML‐specific risk‐stratification categories because the clinical outcomes, including overall survival and leukemia‐free survival, did not differ between the two groups. Conclusions CMML in patients aged 50 years and younger is rare and presents with unique clinical and mutational features, suggesting a distinct pathogenesis.

Abstract RAS pathway (RASp) mutations induce proliferative features, and promote transformation in chronic myelomonocytic leukemia (CMML). However, the unique clonal landscape and hierarchy of distinct RASp mutations remain unexplored. To characterize the landscape, architecture, and implications of unique RASp mutations in CMML, we evaluated a cohort of 814 patients with CMML. We identified 461 RASp mutations among 342 patients (42%). N/KRAS and CBL mutations were the most common, frequently involved the P-loop or RING domains, respectively, and frequently appeared as dominant events (63% and 65%, respectively). BRAF, NF1, and PTPN11 mutations spanned throughout the gene structure, and frequently appeared as subclonal events (75%, 64%, and 59%, respectively). CBL mutations frequently occurred in codominance with SRSF2 and multihit TET2, and were enriched for KIT mutations. PTPN11 mutations more frequently co-occurred with SETBP1 and DNMT3A mutations, and were infrequently codominant with TET2 or ASXL1. RASp mutations predicted for shorter overall survival (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.15-2.07; P = .0075) and leukemia-free survival (LFS; HR, 1.67; 95% CI, 1.26-2.20; P = .0011), influenced outcomes of myelodysplastic and TET2 mutant CMML, and cooperated with IDH2 and RUNX1 mutations to induce shorter LFS. These data set the bases for refined genomic classifications of CMML, and underscore the need to develop RAS-directed therapies for patients with CMML.

Abstract Objectives Myeloproliferative neoplasm, unclassifiable (MPN-U, revised to MPN, not otherwise specified in the fifth edition of the World Health Organization classification) is a heterogeneous category of primary marrow disorders with clinical, morphologic, and/or molecular features that preclude classification as a more specific MPN subtype due to stage at diagnosis, overlapping features between MPN subtypes, or the presence of coexisting disorders. Compared with other MPN subtypes, the contribution of the mutational landscape in MPN-U in conjunction with other clinical and morphologic biomarkers to prognosis has been less well investigated. Methods We performed a multicenter, retrospective study of MPN-U (94 cases) to better define the clinicopathologic features, genetic landscape, and clinical outcomes, including subgroups of early-stage, advanced-stage, and coexisting disorders. The Dynamic International Prognostic Scoring System (DIPSS) plus scoring system was applied to assess its relevance to MPN-U prognosis. Results Multivariate analysis demonstrated bone marrow blast count and DIPSS plus score as statistically significant in predicting overall survival. Univariate analysis identified additional potential poor prognostic markers, including abnormal karyotype and absence of JAK2 mutation. Secondary mutations were frequent in the subset analyzed by next-generation sequencing (26/37 cases, 70.3%) with a borderline association between high molecular risk mutations and overall survival. Conclusions This study, as one of the largest of MPN-U studies incorporating both clinicopathologic and molecular data, moves toward identification of biomarkers that better predict prognosis in this heterogeneous category.

Abstract The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

BackgroundCXCR4 mediates the retention and survival of acute myelogenous leukemia blasts in bone marrow and contributes to their resistance to chemotherapy. The authors evaluated a combination of the high‐affinity CXCR4 antagonist BL‐8040 with high‐dose cytarabine (HiDAC) chemotherapy in a phase 2a study of patients with relapsed and refractory AML.MethodsForty‐two patients received treatment with BL‐8040 monotherapy for 2 days followed by a combination of BL‐8040 with HiDAC for 5 days. Six escalating BL‐8040 dose levels were investigated (0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg/kg), and 1.5 mg/kg was selected as the dose for the expansion phase (n = 23).ResultsBL‐8040 in combination with HiDAC was safe and well tolerated at all dose levels. Clinical response was observed with BL‐8040 doses ≥1.0 mg/kg. The composite response rate (complete remissions plus complete remissions with incomplete hematologic recovery of platelets or neutrophils) was 29% (12 of 42) in all patients and 39% (9 of 23) in the 1.5‐mg/kg phase. The median overall survival was 8.4 months for all patients, 10.8 months in the 1.5‐mg/kg phase, and 21.8 months for responding patients in the 1.5‐mg/kg cohort. Two days of BL‐8040 monotherapy triggered the mobilization of blasts into peripheral blood, with significantly higher mean fold‐changes in responders versus nonresponders. This was accompanied by a decrease in bone marrow blasts.ConclusionsThe current results demonstrate the efficacy of CXCR4 targeting with BL‐8040 and support continued clinical development in acute myelogenous leukemia.

AbstractEnumeration of blasts and promonocytes is essential for World Health Organization (WHO) classification of myelomonocytic neoplasms. The accuracy of distinguishing blasts, promonocytes and monocytes, including normal vs abnormal monocytes, remains controversial. The objective of this analysis is to assess concordances between experienced hematopathologists in classifying cells as blasts, promonocytes, and monocytes according to WHO criteria. Each of 11 hematopathologists assessed glass slides from 20 patients [12 with chronic myelomonocytic leukemia (CMML) and 8 with acute myeloid leukemia (AML)] including blood and BM aspirate smears, and limited nonspecific esterase (NSE) stains. All cases were blindly reviewed. Fleiss’ extension of Cohen's kappa for multiple raters was used on these variables, separately for peripheral blood (PB) and bone marrow (BM). Spearman's rank correlation was used to assess correlations between each pair of hematopathologists for each measurement. For the classification based on the sum of blasts and promonocytes in the BM, Fleiss’ kappa was estimated as 0.744. For PB, categorizing patients according to the sum of blasts and promonocytes, Fleiss’ kappa was estimated as 0.949. Distinction of abnormal monocytes from normal monocytes in PB did not achieve a good concordance and showed strong evidence of differences between hematopathologists (P < .0001). The hematopathologists achieved a good concordance rate of 74% in CMML vs AML classification and a high k rate, confirming that criteria for defining the blasts equivalents (blasts plus promonocytes) could be applied consistently. Identification of monocyte subtypes (abnormal vs normal) was not concordant. Our results support the practice of combining blasts/promonocytes into a single category.

Abstract Background: Persistence of minimal residual disease (MRD) after induction, consolidation and salvage therapies is an independent poor prognostic marker in AML. The bone marrow (BM) microenvironment is a protective niche that promotes chemotherapy resistance of leukemia cells. This residual subset of chemotherapy-resistant cells present during remission eventually contributes to the leukemia relapse/persistence. Adhesion of AML cells to BM stroma is dependent on the CXCR4 receptor binding to its ligand CXCL12. High levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis will dislodge AML cells from the protective microenvironment and, when combined with chemotherapy, may improve the response to therapy. BL-8040 (BKT140) is a short peptide which inhibits the binding of CXCR4 to CXCL12, resulting in mobilization of leukemic blasts from the BM to the peripheral blood (PB). BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy and as a single agent induces differentiation and apoptosis of leukemia cells in preclinical models. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of relapsed/refractory AML patients was recently completed (NCT01838395). Here we report results from correlative studies done on samples from patients who participated in this trial. Objective: To assess BL-8040 single agent anti leukemia effects in relapsed/refractory AML. Method: Patients received a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5g/m2 for patients ≥60; 3g/m2 for patients <60) per day on days 3-7. Six dose levels of BL-8040 (0.5 - 2.0 mg/kg) were tested. PD parameters such as mobilization of leukemic blasts and induction of differentiation and apoptosis were assessed after monotherapy with BL-8040 using frequent PB sampling and BM aspirates at baseline and on day 3 prior to Ara-C administration. Results: 45 patients (including 3 receiving compassionate use BL-8040) with a median age of 61 yrs were treated. The majority of patients had poor risk disease with 24% secondary AML and 17% with prior allogeneic stem-cell transplantation. Most patients were heavily pretreated with 19% relapsing after a short first remission (CR1 ≤12 months), 17% had ≥2 relapses and 45% refractory to 1-2 inductions. The CR+CRi rate was 38% in subjects receiving BL-8040 dose ≥1.0 mg/kg (n=39). Ongoing follow-up (FU) of responding patients (expansion phase; N=19) shows median EFS of 9.3 months (range 4.3-12.8 months). At the time of this analysis 10/19 patients are alive with a FU range of 0.96 - 12.8 months. FACS analysis revealed that BL-8040 monotherapy triggered an average 4.3-fold increase of immature AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) in PB (i.e., mobilization). Response to treatment was associated with more robust mobilization of AML blasts following BL-8040 monotherapy (responders 9.5 vs non-responders 1.7 fold median). Preferential mobilization of AML blasts over normal cells (4.7 fold vs. 1.4 fold, respectively) was confirmed by FISH analysis in a subset of patients with informative cytogenetic abnormalities. Induction of granulocytic differentiation of immature leukemia progenitors (2.5 fold) in the BM following BL-8040 monotherapy was noted. Finally, induction of AML blasts apoptosis (40% increase) by BL-8040 alone was evident by FACS in day 3 (pre-araC) bone marrow biopsies. Conclusions: The data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 is safe and well tolerated and when given in combination with Ara-C improves the response rate achieved historically with Ara-C alone. In addition, treatment with BL-8040 as a single agent rapidly and efficiently induces mobilization, differentiation and cell death of AML blasts. This selective effect on chemotherapy-resistant cells may be translated into reduction of residual disease arguing for incorporation into front-line trials and such studies are ongoing. Disclosures Foran: Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; pfizer: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. DiPersio:Incyte Corporation: Research Funding. Peled:Biokine Therapeutics: Employment. Abraham:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Vainstein:BioLineRx Ltd.: Employment. Oberkovitz:BioLineRx Ltd.: Employment. Aharon:BioLineRx Ltd.: Employment. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Abstract Background: The bone marrow (BM) niche protects acute myeloid leukemia (AML) cells from chemotherapy. BM homing of AML cells is dependent on CXCR4 and its ligand CXCL12; high levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the leukemic blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a short synthetic peptide which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukemic blasts from the bone marrow to the peripheral blood. BL-8040 also inhibits CXCR4/CXCL12 mediated pro-survival ERK/Akt signaling and alters balance of pro/anti-apoptotic Bcl-2 proteins. BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy, and, in contrast to other CXCR4 inhibitors, as a single agent induces apoptosis of leukemia cells ex-vivo in clinical samples. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of adult relapsed/refractory AML patients is currently ongoing (NCT01838395). Objective: To assess the safety, efficacy and pharmacodynamics/pharmacokinetic parameters of BL-8040 in combination with Ara-C in first or second relapse and refractory AML patients. Method: The study includes a dose escalation phase (3+3 design) followed by an expansion phase. Each patient receives a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5 g/m2 for patients ≥60; 3 g/m2 for patients <60) on days 3-7. Five dose levels of BL-8040 (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) have been tested. Extensive pharmacodynamic parameters such as CXCR4 expression on the leukemic cells, receptor occupancy, mobilization of leukemic cells and induction of apoptosis are being assessed after monotherapy with BL-8040 using peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration. Clinical response to treatment is evaluated by BM biopsy on day 30 (-/+ 2). Results: Twenty two patients (median age, 61 y; range, 43-74 y) with relapsed/refractory AML were treated during the escalation phase. BL-8040 was escalated up to 1.5 mg/kg (recommended phase 2 dose) without reaching the MTD. Combination of BL-8040 with Ara-C was safe and well tolerated at all doses tested. Only one SAE (Sweet Syndrome) was reported as possibly related to study drug. The primary treatment related AEs were transient injection site and systemic reactions. The composite complete remission including complete remission (CR) and complete remission with incomplete blood count recovery (CRi) rate was 38% in subjects receiving BL-8040 doses of 1 mg/kg and higher (n=16). Two days of BL-8040 monotherapy triggered an average 40.2-fold (range 1.0-350.9) mobilization of immature AML progenitors (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Moreover, a significant apoptotic effect on the immature leukemia progenitors in the BM following 2 days of BL-8040 monotherapy was noted. Flow cytometric evidence of apoptosis by BL-8040 was corroborated by an increase in cleaved caspase 3 positive blasts in day 3 bone marrow biopsies. In addition, BL-8040 treatment resulted in a median decrease of 57.7% (range, -10.4% to -96.2%) in the number of BM Leukemia progenitor cells (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with the baseline biopsy. This decrease was accompanied with a 3.1 fold increase in granulocytes in the day 3 BM biopsy supporting differentiation effect. Finally, long receptor occupancy (up to 24 hours post dosing) was confirmed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed dose incremental BL-8040 exposure with a short plasma half-life. Conclusions: The current data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 has potent anti leukemic activity and in combination with Ara-C may improve the response typically achieved in this advanced AML population. Mechanistically, BL-8040 was able to induce apoptosis of leukemic progenitor cells, overcoming the stroma protection, inducing mobilization and differentiation. Disclosures Rowe: BioLineRx Ltd.: Consultancy; Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Novartis: Research Funding. Altman:Seattle Genetics: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; BMS: Consultancy. Peled:Biokine Ltd.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BiolineRX: Consultancy, Research Funding. Pereg:BioLineRx: Employment. Vainstein:BioLineRx: Employment. Aharon:BioLineRx: Employment. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Cortes:Teva: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

Abstract Abstract 2926 Background: RAS proteins are encoded by 3 proto-oncogenes (K-RAS, N-RAS, and H-RAS) and regulate the growth and differentiation of many cellular types, including the myeloid compartment. Codons 12, 13, and 61 (in N-RAS and K-RAS) have been reported to be common targets of mutations. These mutations result in constitutive activation of the RAS pathway by increasing intracellular RAS GTP levels, which result in activation of the RAS/MEK and the RAS/PI3K pathways. Its prognostic significance in MDS is yet to be clarified. Because the potential prognostic and therapeutic implications of RAS mutations in MDS, we decided to study the impact of RAS mutations in patients (pts) with MDS. Methods: Mutation analysis of KRAS and NRAS codons 12, 13, and 61 was performed by Pyrosequencing using biotinylated reverse primers. All pts referred to MD Anderson between 2000–2009 were reviewed. In addition to standard clinical characteristics, including karyotyping, mutational status of Flt3, c-Kit and JAK2 was also analyzed. Overall survival was calculated based on Kaplan-Meier. Results: Of the 1067 pts with MDS of age above 17 evaluated for RAS mutation, 43 pts (4%) were positive. Seventy nine percent of RAS mutations were of N-RAS type (codon12 (55%), codon 13 (12%), codon 61(12%)) with K-RAS 12 comprising the rest (21%). Median age was 68 years (range, 18–94) with 66% being males (703 pts). Their median white blood cell (WBC) was 3.3×109/L (range, 0.3–72.2), hemoglobin 9.8 (range, 3–17.5), and platelets 73×109/liter (range, 1–1040). Median bone marrow blast was 5% and 77 pts (7%) had leukemic transformation. More than half of the pts had RAEB or RAEB-t (44% and 9 % respectively) which translated into fewer patients in the low IPSS category (19%). The median age was 66 years (range 26–84) in the RAS mutated (muRAS) group compared to 68 in the wild type (wtRAS). WBC was higher in the muRAS group compared to wtRAS (6.8 ×109/L vs. 3.2 ×109/L, p<0.001 by Mann-Whitney median test (M-W-T)). Percent of bone marrow blasts was higher (9%) in muRAS group compared to 5% in wtRAS (p<0.001 by M-W-T). High (Int-2 and high) risk by IPSS was more frequent in muRAS 51% compared to 40% in wtRAS group (p=0.11). Leukemic transformation was 9% and 7% (p=0.61) in both muRAS and wtRAS groups respectively. RAS mutation was detected more frequently in the CMML subgroup (12/80 (15%) pts). There were no differences in terms of response to DNA methylating inhibitors (CR in 41% in muRAS versus 40% in wtRAS). ARA-C-based regimens resulted in better CR in wtRAS than muRAS (59% vs 25%); however, the size of muRAS group was too small (1 out 4 pts).There was a trend towards shorter overall survival (OS) in the muRAS group compared to wtRAS (395 vs. 500 days, p value=0.057). MuRAS RAEB (325 vs. 450 days, p= 0.13) and RAEB-t (125 vs. 375 days, p= 0.27) subgroups showed a trend towards worse OS compared to wtRAS group. There was a significant OS decrease in RCMD (150 vs. 700 day, p= 0.015). Surprisingly, muRAS CMML group showed a trend towards better OS compared to wtRAS (728 vs. 687 days, p=0.44). Additionally we analyzed patients for presence of Flt3 mutation. These were present in only 18 of the pts (4%) but none of them had also a RAS mutation. There was no c-Kit mutations found in this cohort of pts, compared to 9/37 (24%) pts tested positive for JAK2V617F. Conclusion: MuRAS occurs in 4% of pts with MDS. In the muRAS group, pts tended to have higher risk disease and risk of transformation to acute leukemia compared with wtRAS. OS tended to be worse in some subgroups in muRAS. Although the prognostic impact of RAS mutations in MDS appears to be limited, mutations could be used to develop targeted therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

AbstractObjectivesIn the 2008 World Health Organization classification, cases of acute myeloid leukemia (AML) and myelodysplastic syndrome that arise after chemotherapy or radiation therapy for a primary neoplasm are considered together as therapy-related myeloid neoplasms (TR-MNs). This concept, however, is not universally accepted since there are confounding variables in attributing myeloid neoplasms to earlier therapies.MethodsCases in session 6 of the 2013 Workshop of the Society for Hematopathology/European Association for Haematopathology illustrated myeloid neoplasms thought likely to be TR-MNs, and discussed the differences and biologic similarities with de novo myeloid neoplasms.ResultsWe reviewed data showing that diagnosis of TR-MN alters patient outcome only in specific subsets. The session also included examples of therapy-related AML with recurrent genetic abnormalities, such as t(15;17), inv(16), and t(8;21), and reports were highlighted showing that patients with these neoplasms have clinical outcomes similar to patients with their de novo counterparts.ConclusionsThe study of TR-MNs will likely provide insight into the pathogenesis of de novo myeloid disease and may explain why some patients with cancer develop TR-MN and evidently have a higher genetic susceptibility, whereas most patients treated with the same agents do not. These studies will also result in critical reappraisal of current concepts related to TR-MNs.