Chronic Myelomonocytic Leukemia Cohort Research Articles

ASXL1 Truncating Mutations Drive Leukemic Resistance to T Cell Attack

Restoration of graft-versus leukemia activity underlies the efficacy of donor lymphocyte infusion (DLI), a curative immunotherapy for leukemic relapse following allogeneic stem cell transplant (SCT). We found that while specific exhausted T cell subsets defined DLI response, no pattern of T cell dysfunction appeared in nonresponders (NRs) [PMID: 34758319]. We hypothesized that leukemic molecular pathways define DLI resistance (DLIr). We performed whole exome sequencing on bone marrow samples collected before and after DLI from 11 patients (pts) with relapsed chronic myeloid leukemia (CML) at Dana-Farber Cancer Institute. We used MuTect2 to discover somatic alterations using genomic DNA from recipient-derived mesenchymal stromal cells and donor peripheral blood cells as germline sources. The burden of nonsynonymous mutations and mutational spectra was similar between responders (Rs) and non-responders (NRs). Known non-Philadelphia (Ph) chromosome cancer drivers were found in 0/7 Rs and 4/4 NRs (RUNX1 (1/4), IKZF1 (2/4), and truncating exon 12 ASXL1 mutations (ASXL1 m) (3/4)). We profiled 6 more DLI-treated pts with relapsed CML from the MD Anderson Cancer Center with a targeted mutation panel that identified ASXL1 m in 4/5 NRs. Altogether, we found ASXL1 m in 78% (7/9) of NRs and 0% (0/10) of Rs (p=7e-4, two-sided Fisher's test), suggesting ASXL1 m as a driver of DLIr. To discover molecular pathways of DLIr, we interrogated unpublished non T-cell single cell (sc)RNA-seq data in our DLI cohort (8 R, 6 NR). Leukemic cells were identified for patients (n=8) with sex-mismatched SCT by male sex-defining genes, which correlated with Ph chromosomal positivity (Pearson correlation = 0.85). These cells were primarily confined to the myeloid population, so we performed a scRNA myeloid reclustering (138,152 cells) with Harmony batch correction and identified 5 populations enriched in R vs NR; 4 clusters in NR, and 1 monocyte cluster in R (p=0.039, 0.005, 0.038, 0.043, and 0.025, respectively). These NR clusters were enriched in progenitor cell identities, notably the LSC17 [PMID: 27926740] signature (p<2e-16). Interrogation of immune pathways in the LSC cluster showed downregulation of HLA-I and multiple antigen processing/presentation (APP) genes in NRs (p<2e-16), while HLA-II remained unchanged. Thus, ASXL1 m may mediate immune escape through suppression of HLA-I expression and antigen presentation. To directly test this hypothesis, we CRISPR corrected the endogenous ASXL1 m (Y591*) in a BCR-ABL-driven leukemic cell line (K562). This wildtype reversion (ASXL1 c) increased HLA-I expression at the RNA (RNA-seq; HLA-B/C, log2foldchange= -4.77, q=7e-42) and protein level (flow cytometry; 49.0% vs 1.62%, q=2e-4); here too, HLA-II expression remained unaffected by ASXL1 mutation status. Gene-set enrichment analysis also showed increased APP expression in ASXL1 c K562 (NES=-1.75, q=0.01). We then sought to identify whether correction of ASXL1m acted through known regulators of HLA-I. Treatment with IFNɣ or EPZ011989, an inhibitor of enhancer of zeste-homolog 2 (EZH2), resulted in greater HLA-I in K562 ASXL1 c (85.0% vs 50.2% and 89.5% vs 39.3%, respectively, q<0.001) suggesting that ASXL1 cacts orthogonally to IFNɣ or EZH2. ASXL1 c also mediated increased T-cell cytotoxicity towards K562 ASXL1 c vs ASXL1 m without (43% vs 16%, q=2e-4) and with IFNɣ pre-treatment (71% vs 34%, q=1e-6) at 10:1 target to effector ratios. Finally, to study other disease contexts, we examined published sc chromatin accessibility and scRNA-seq data from a chronic myelomonocytic leukemia cohort [PMID: 35301312] and an acute myeloid leukemia cohort [PMID:32649887], respectively. We found both chromatin and transcriptional states enriched in ASXL1m samples to have decreased accessibility and expression, respectively, of HLA-I and APP genes (p<2E-16). In summary, we integrated molecular analyses with immuno-functional studies to define a novel oncogenetic pathway of immune evasion. Specifically, we link DLIr to ASXL1 m that suppresses transcription of HLA-I and antigen presentation machinery in myeloid diseases; moreover, its correction restores anti-leukemic CD8+ T-cell cytotoxicity. Ongoing studies will examine additional clinical contexts and the epigenetic mechanisms by which ASXL1m regulates HLA-I expression.

A retrospective study of cladribine and low-dose cytarabine-based regimens for the treatment of chronic myelomonocytic leukemia and secondary acute myeloid leukemia.

Patients with higher risk chronic myelomonocytic leukemia (CMML) have limited therapeutic options beyond hydroxyurea and hypomethylating agents (HMAs). Regimens based on a backbone of cladribine (CLAD), low-dose cytarabine (LDAC), and an HMA are effective low-intensity therapies for acute myeloid leukemia (AML). The authors conducted a retrospective chart review to evaluate the efficacy of CLAD/LDAC/HMA in CMML and secondary acute myeloid leukemia (sAML) arising from CMML. Responses were evaluated according to the 2006 International Working Group criteria for CMML and the 2017 European LeukemiaNet criteria for AML. The overall survival (OS), leukemia-free survival (LFS), and duration of response were evaluated with the Kaplan-Meier method. Patients were stratified on the basis of prior HMA exposure. The authors identified 21 patients with CMML (eight with HMA-naive CMML and 13 with HMA-failure CMML) and 33 patients with sAML (11 with HMA-naive sAML and 22 with HMA-failure sAML) treated with CLAD/LDAC/HMA-based regimens. The CMML cohort was enriched for high-risk features (proliferative type, elevated blasts, and RAS/MAPK mutations). The overall response rate was 33% in CMML (50% in HMA-naive CMML and 23% in HMA-failure CMML) and 48% in sAML (82% in HMA-naive sAML and 32% in HMA-failure sAML). The median OS was 14.4, 8.8, 42.9, and 2.9months for HMA-naive CMML, HMA-failure CMML, HMA-naive sAML, and HMA-failure sAML, respectively. The median LFS was 14.4 and 3.9months for HMA-naive CMML and HMA-failure CMML, respectively. CLAD/LDAC/HMA-based regimens are effective in a subset of patients with higher risk CMML and sAML arising from CMML who have not previously experienced HMA failure. These findings must be confirmed in prospective studies.

Outcomes of Patients Treated with CPX-351 As First Line Therapy for AML Based on Their Antecedent History of Myeloid Malignancy

Outcomes of Patients Treated with CPX-351 As First Line Therapy for AML Based on Their Antecedent History of Myeloid Malignancy

Outcome with Hypomethylating Agent and Venetoclax Combination in Patients with Chronic Myelomonocytic Leukemia

Outcome with Hypomethylating Agent and Venetoclax Combination in Patients with Chronic Myelomonocytic Leukemia

Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelomonocytic Leukemia: Clinical and Molecular Genetic Prognostic Factors in a Nordic Population

Chronic myelomonocytic leukemia (CMML) is an aggressive disease in which survival after allogeneic hematopoietic stem cell transplantation (HCT) remains relatively poor. An assessment of prognostic factors is an important part of treatment decision making and has the potential to be greatly improved by the inclusion of molecular genetics. However, there is a significant knowledge gap in the interpretation of mutational patterns. This study aimed to describe outcomes of allogeneic HCT in patients with CMML in relation to clinical and molecular genetic risk factors. This retrospective study included 64 patients with CMML who underwent allogeneic HCT between 2008 and 2018, with a median follow-up of 5.4 years. Next-generation sequencing using targeted myeloid panels was carried out on saved material from 51 patients from the time of transplantation. Kaplan-Meier and Cox regression were used for analysis of overall survival (OS), and cumulative incidence with competing risks and Fine and Gray models were used for analysis of relapse and nonrelapse mortality (NRM). Mutations were detected in 48 patients (94%), indicating high levels of minimal residual disease (MRD) positivity at transplantation, even among those in complete remission (CR) (n=14), 86% of whom had detectable mutations. The most frequently mutated genes were ASXL1 (37%), TET2 (37%), RUNX1 (33%), SRSF2 (26%), and NRAS (20%). Risk stratification using the CMML-specific Prognostic Scoring System molecular score (CPSS-Mol) resulted in 45% of patients moving to a higher risk-group compared with risk stratification using the CPSS. High leucocyte count (≥13×109/L), transfusion requirement, and previous intensive chemotherapy were associated with higher incidence of relapse. Being in CR was not linked to better outcomes. Neither ASXL1 nor RUNX1 mutation was associated with a difference in OS, relapse, or NRM, despite being high risk in the nontransplantation setting. TET2 mutations were associated with a significantly higher 3-year OS (73% versus 40%; P=.039). Achieving MRD-negative CR was rare in this CMML cohort, which may explain why we did not observe better outcomes for those in CR. This merits further investigation. Our analyses suggest that the negative impact of ASXL1 and RUNX1 mutations can be overcome by allogeneic HCT; however, risk stratification is complex in CMML and requires larger cohorts and multivariate models, presenting an ongoing challenge in this rare disease.

Promising Results of a Phase 1/2 Clinical Trial of Ruxolitinib in Patients with Chronic Myelomonocytic Leukemia

▪Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by GM-CSF hypersensitivity, marrow dysplasia, blood monocytosis, and a propensity for acute myeloid leukemia transformation. The overall survival is estimated at 34 months with no drug therapies known to improve survival. We previously reported results of a phase 1 study demonstrating that ruxolitinib, a JAK1/2 inhibitor, is safe and has clinical activity in CMML at doses up to 20mg BID. To confirm efficacy we performed a phase 2 clinical trial and here report the combined results of the phase 1 and 2 clinical studies. Methods: All CMML WHO subtypes were included without regard to previous therapy. Key exclusion criteria included an ANC < 0.25x103 c/dL and a platelet count < 35x103c/dL. The use of GM-CSF analogs was prohibited. The Kaplan-Meier method was used to estimate survival and log-rank test to determine statistical significance. Somatic mutations were identified at baseline using a 49 gene targeted next generation sequencing panel (RainDance Technologies) and a custom 46-plex Luminex assay was used to profile baseline cytokine levels (R&D). The study was registered at clinicaltrials.gov NCT01776723 Results: Between April 2015 and October 2016, 29 patients were enrolled in the phase 2 study resulting in a combined 49 CMML patients treated with ruxolitinib. In phase 2, 56 percent of patients had the proliferative subtype (MPN-CMML) defined by WBC >13x109/L and 28% had higher-risk disease by the Global MD Anderson Scoring System. Six patients (20%) received a prior hypomethylating agent and 85% of patients were WHO subtype CMML-1. Therapy-emergent toxicities were similar to those reported in phase 1. The mean duration of therapy was 219 days (range, 6-675 days). Three hematologic responses, including bi- or trilineage responses (2 HI-E, 2 HI-P, 1 HI-N), one marrow CR, and one partial bone marrow response per 2006 IWG criteria were observed. Six of 13 patients (46%) with splenomegaly had a 50% or greater reduction in spleen size by physical exam. When combining IWG and spleen responses, the overall response rate was 46% (n=11 of 24), including clinical benefit by MDS/MPN IWG response criteria. Using the Myeloproliferative Symptom Assessment Form (MPN-SAF) administered electronically daily, a reduction in total symptom score was noted. The median OS (mOS) from start of treatment was 28 months (95% CI 18-38), and, for CMML-MDS and CMML-MPN, was 32 months and 28 months respectively, (p=0.3). To evaluate whether ruxolitinib may have disease-modifying properties in CMML, analysis of mOS from time of diagnosis was performed comparing all ruxolitinib treated patients (n=49) to an historical dataset with over 1800 CMML patients (Padron et al 2015). The mOS at the time of diagnosis in CMML ruxolitinib-treated CMML patients was 69 months compared to 31 months in the historical CMML cohort (p=0.03). Adjusting for age, Global MD Anderson model, CMML WHO subtype, CMML-MPN, and the interval from diagnosis, treatment with ruxolitinib was associated with improved OS (HR 0.42, p <0.005). Although no mutation or class of mutations was associated with response, peripheral blood plasma levels of CXCL9 (p=0.02) and decreased levels of CCL5 (p=0.018) and RAGE (p=0.016) were statistically associated with higher likelihood of response. Conclusions: Ruxolitinib has promising activity in CMML patients with particular benefit in those with proliferative symptoms and may have disease-modifying activity. The MPN-SAF may be a useful tool to measure symptom burden in the context of ruxolitinib therapy for CMML. Overexpression of cytokines and soluble innate immune receptors is associated with a higher likelihood of response to ruxolitinib therapy. Further clinical study is warranted to validate the biomarkers of response and disease modifying capacity of ruxolitinib in CMML. DisclosuresPadron:Incyte: Honoraria, Research Funding. Lancet:Bio-Path Holdings: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy; Jazz Pharmaceuticals: Consultancy; BioSight: Consultancy; Erytech: Consultancy; Janssen: Consultancy; Pfizer: Other: Institutional research funding, Research Funding; Novartis: Consultancy. Mesa:Promedico: Research Funding; Ariad: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy; Gilead Sciences, Inc.: Research Funding; Celgene Corporation: Research Funding; Galena Biopharma, Inc.: Consultancy; Incyte Corporation: Research Funding; CTI BioPharma Corp.: Research Funding. Roboz:AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy; Cellectis: Research Funding. Steensma:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Equity Ownership; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy, Research Funding; H3 Biosciences: Consultancy; Onconova: Consultancy; Pfizer: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria.

A Phase II Trial of Eltrombopag in Chronic Myelomonocytic Leukemia (CMML) with Thrombocytopenia

Context: Thrombocytopenia is a frequent complication in CMML associated with worse prognosis (Itzykson JCO 2013, Patnaik Leukemia 2013). Thrombopoietin (TPO) protects HSCs from DNA damage (de Laval Cell Stem Cell 2013). The TPO analog eltrombopag (ELT) has shown efficacy in MDS (Oliva Lancet Hematol 2017). The limited data on ELT in CMML is restricted to a small cohort (n=7) with high risk features, where 4 pts had disease progression during ELT (Ramadan Clin Lymphoma Myeloma Leuk 2016). We report interim results of a multicenter phase 2 trial investigating the efficacy, toxicity and biomarkers of ELT in 25 CMML with platelets (PLT) < 50 x109/L (NCT02323178).Methods: Key inclusion criteria were: WHO 2008 defined HMA-naive CMML, PLT < 50 x109/L, marrow blasts ≤ 5%, IPSS low/int-1 in MD-CMML, and in MP-CMML no or 1 severity criteria (Hb < 10 g/dL, ANC > 16 x109/L, abnormal karyotype, extramedullary disease) ,spleen size < 16 cm. ELT was started at 100 mg/d with escalating doses to 300 mg/d. Platelet Response (HI-P) was evaluated at 12 weeks, according to IWG 2006 criteria, and responders could continue treatment. Gene mutations were analyzed by NGS at inclusion. DNA repair was explored by counting gH2AX foci in CD34+ cells obtained at inclusion, evaluation and study exit.Results: We report on the first 19 evaluable pts (median age 79y, M/F 15/4), all CMML-1 by WHO 2008, including 14 MD-CMML and 5 MP-CMML, with a low/int-1/int-2 CPSS (Such Blood 2013) in 61%, 33% and 6% of pts respectively (resp). Median PLT count was 29 x109/L (IQR 19 - 43.5) and 10 (53%) pts had PLT transfusion dependency (PLT-TD). TET2, RUNX1, ASXL1, SRSF2, PHF6, and RAS pathway mutations were present in 17, 10, 6, 7, 4 and 7 of 18 pts sequenced, resp.The median maximal dose of ELT received was 200 mg/d (IQR: 150 - 300 mg/d). Clinical grade ≥3 toxicities, all cardiovascular or pulmonary, were noted in 5 pts in the first 12 weeks, and biological grade ≥3 toxicities in 6 pts, without grade ≥3 ALT/AST elevation. Six (32%) pts went off study before week 12 because of lack of efficacy (n=2), presence of in circulating blasts (n=1), progression to CMML-2 (n=1), pancreatic cancer (n=1) and patient decision (n=1). All were considered non-responders. At 12 weeks, 12 (63%, 95%CI: 38 - 84) pts had achieved HI-P, including 3/5 MP-CMML and 9/14 MD-CMML (p=1). Responses were obtained in 7/10 pts with baseline PLT-TD and 5/9 pts without PLT-TD (p=0.65). The mutational profile of responders was comparable to that of non-responders except that none of the 4 PHF6mut pts responded (p=0.01). Responders at 12 weeks were receiving a median of 125 mg/d of ELT, compared to 225 mg/d in non-responders.Responders received ELT for a median of 21 weeks (IQR 15 - 38). With a median follow-up of 18 months (95%CI: 16.8 - NR), 11 of the responders discontinued ELT because of loss of efficacy (n=2), patient decision (n=1), death from unrelated causes (n=2), liver toxicity (n=2), renal failure (n=1) and disease progression (n=3). Median response duration was 8 months (95%CI: 5 - NR), with one pt still on treatment with a response lasting more than 14 months. The 12-month cumulative incidence of AML was 19% (95%CI: 4 - 41%) and 12-month progression-free and overall survival (OS) were 41% (95%CI: 23 - 73) and 65% (95%CI: 45-92%). By comparison, in the 21 CMML pts with PLT < 50 x109/L and ≤5% BM blasts from our previous CMML cohort (Itzykson JCO 2013), none of whom received ELT, the 12-month estimates for cumulative incidence of AML and OS were 10% (95%CI 0 - 23%) and 71% (95%CI 47 - 86%), resp. An updated analysis including all 25 pts will be presented.At protocol screening, a higher number of gH2AX foci were seen in CMML bone marrow CD34+ cells (n=14) compared to age-matched controls (n=5, p<0.01), reflecting higher levels of baseline DNA damage. However, in the 5 pts for whom serial bone marrow samples were obtained during ELT, gH2AX foci were reduced in CD34+ cells from all 5 pts, suggesting that in vivo exposure to ELT protected CMML stem/progenitor cells from DNA damage.Conclusion: Our interim results suggest that ELT is manageable in CMML pts with no excess of marrow blasts and thrombocytopenia and results in a high response rate, with no obvious increase in disease progression, though prolonged responses seem infrequent. ELT could potentially protect CMML stem/progenitor cells from DNA damage. Larger pt numbers and longer follow-up are however required to determine precisely the role of ELT in thrombocytopenic CMML pts. DisclosuresItzykson:Janssen: Research Funding; Novartis: Research Funding. Fenaux:Astex: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

Transcriptome of DNA Repair Genes in CMML: Rationale for Targeting ERCC1 and CDKN1A As a Synthetic Lethality Approach

Background: Chronic myelomonocytic leukemia (CMML) is characterized by features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). Despite the main role of DNA repair pathways in cancer, in myelodysplastic disorders, somatic mutations are not found in these pathways. The novel discovery of non-classical leukaemogenesis mediated by splicing defects, profound influence of epigenetic anomalies, deeper knowledge of the DNA repair network, and development of unbiased high-throughput sequencing approaches, prompted us to revisit whether these processes might be disrupted by non-mutational mechanisms in CMML and related myeloid disorders. Study aims: i) to screen the transcriptome of DNA repair genes in a CMML discovery cohort; ii) to validate their misregulation in an independent large CMML cohort; and iii) determining how those candidates behave through the spectrum of myeloid malignancies. Methods: RNA-seq was performed in the discovery cohort: 27 CMML bone marrow samples (BM) at diagnosis and 9 healthy BM samples, and in 7 patients treated with Azacitidine in BM collected before and after 4 cycles. RT-qPCR was used to validate 9 DEGs through the myeloid spectrum and in a CMML validation cohort: 74 additional CMML, 70 MDS, 66 AML , and 25 CML patients. Candidates were chosen based on clinical considerations: i) druggable oncogenes highly overexpressed ii) oncogenes infra-expressed with inhibitory molecules already being tested in myeloid neoplasms. Global pattern of DNA repair gene expression was compared with MDS and AML extracted from MILE study data. The DEGs methylation status was studied using available public data and we confirmed by pyrosequencing the methylation status of our main candidates. The CMML validation cohort was mutationally characterized by targeted sequencing of 18 CMML recurrently mutated genes. Alternative splice sites, skipped exons, mutually exclusive exons and retained introns were quantified using rMATS.Results: Of 27 CMML patients and 9 healthy donors, the expression of 30 genes was significantly different between the two groups (8 genes up-regulated and 22 down-regulated). Defects on genes predominantly unique to a single strand breaks repair pathway included: NEIL1 and OGG1 in base excision repair, XPA and MSS19 in nucleotide excision repair and RPA4 in mismatch repair. XRCC4 and MSH4 overexpression and PRKDC down-regulation were found as significant changes among the genes associated exclusively to double strand breaks repair. We next extracted the DNA repair transcriptional components from MILE study: 206 MDS, 47 ckAML and 73 healthy donors. MDS misregulation was characterized by upregulated genes while ckAML showed a global disruption, mostly downregulation of Homologous Recombination genes. Some genes showed opposite sense of misregulation depending on the myeloid entity: TDP1 was upregulated in CMML cases and downregulated in ckAML, and viceversa for BAP1 . CDK1 and EXO1 were upregulated in MDS cases but downregulated in ckAML cases. Statistically significant and direction-concordant dysregulation of CDKN1A, ERCC1, XRCC4, NEIL1, MSH4, PARP1 and BAP1 were confirmed in our independent CMML validation cohort. The methylation study showed a global demethylated status of our misregulated DNA repair genes in CMML. Either responder s (n=3) or non-responders (n=2) to azacitidine failed to achieve a substantial expression change in their DNA Repair DEGs. PARP1 infraexpresion was associated with TET2 mut cases. Lower CDKN1A and BAP1 expression was related to chromatin regulator mutations. SRSF2 mutations induced widespread aberrant splicing events in DNA repair genes in CMMLConclusions: We have identified by means of an unbiased high-throughput approach, and validated in an independent cohort, a subset of DNA repair genes consistently misregulated in CMML. The genes identified warrant further study as potential novel therapeutic targets, both directly and through modulation of associated compensatory pathways. CDKN1A and ERCC1 in particular emerge as realistic candidates for a synthetic lethality approach. Our findings suggest that the promise of PARP1 inhibition appears less pertinent to CMML than for other myeloid diseases. These differences might also partially explain the different genomic and phenotypic manifestations of different myeloid neoplasms, and shed insights into their distinctive biology. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Eltrombopag in Chronic Myelomonocytic Leukemia (CMML) with Severe Thrombocytopenia: Final Results of a Multicenter Phase II Study

Eltrombopag in Chronic Myelomonocytic Leukemia (CMML) with Severe Thrombocytopenia: Final Results of a Multicenter Phase II Study

Clinical Outcomes and Influence of Mutation Clonal Dominance in Oligomonocytic and Classical Chronic Myelomonocytic Leukemia

Clinical Outcomes and Influence of Mutation Clonal Dominance in Oligomonocytic and Classical Chronic Myelomonocytic Leukemia

Bone Marrow Mastocytosis Is Independently Associated with Inferior Survival in Chronic Myelomonocytic Leukemia

Introduction: Systemic mastocytosis with associated hematologic neoplasm (SM-AHN) is defined by the presence of a concomitant hematologic malignancy with chronic myelomonocytic leukemia (CMML) being a particularly common partner. The overall survival of patients with SM-AHN is inferior to those with SM alone, even when matched for relevant prognostic covariates. However, the prognostic impact of mastocytosis in patients with CMML is unknown. Methods: CMML patients with concomitant mastocytosis were identified from the Moffitt Cancer Center CMML database. We assessed baseline demographic, clinical, and molecular findings and used Kaplan-Meier method to estimate overall survival (OS). The log-rank test was used to compare Kaplan-Meier curves, We then compared the SM-CMML cohort to a well-established institutional database of CMML patients. Baseline demographic and clinical variables were analyzed using GraphPad Prism and SPSS was used for Cox Regression Analysis. Results: Between 5/2004 and 5/2019 22 of 645 CMML patients (3.4%) were identified to have concomitant mastocytosis. The median follow-up for the 22 patients with SM-CMML was 51 months. Nine (41%) patients were diagnosed with de novo CMML prior to SM. In these cases, secondary SM-CMML occurred at a median time of 7 months after CMML diagnosis. Ten patients (45%) were diagnosed with CMML and SM concurrently and 3 (14%) were diagnosed with SM prior to CMML. Among 17 patients tested for KIT mutations, 12 were found to harbor a mutation. The remaining five patients did not undergo high-sensitivity KIT testing on a bone marrow aspirate. Eleven patients had extended gene sequencing performed with the most common additional mutations involving TET2 (45%), SRSF2 (55%), ASXL1 (27%), RAS (27%), DNMT3A (27%), and RUNX1 (27%). The median overall survival (OS) was estimated to be 38.6 months. Next, we compared this cohort of SM-CMML patients to a large, established database of CMML patients (excluding those with concomitant SM). Age at diagnosis, baseline white blood cell count, hemoglobin, and platelet count were well matched between the two groups. Applying the Mayo CMML Prognostic Model to the cohort of SM-CMML patients demonstrated that 32%, 41%, and 27% were low, intermediate and high risk, respectively. In the CMML cohort, 13%, 35%, and 51% were low, intermediate and high, risk respectively, suggesting the SM-CMML was more common in the lower-risk group (p=0.025). The median OS was similar between the two cohorts (median OS 31.3 vs 38.6 months, p = 0.43). However, multivariate analysis including Mayo Prognostic Scoring System, age &gt; 65, and SM component revealed all three variables to be independently associated with survival (HR 1.8, p &lt; 0.001; HR 1.7, p = 0.047; and HR 1.5, p 0.003, respectively). Assessing the impact of mastocytosis in low, intermediate, and high-risk groups separately, the inferior prognostic impact of mastocytosis was most prominent in high-risk patients (OS 19.6 mo vs. 5.4 mo; p = 0.049). Survival outcomes between SM-CMML and CMML were not statistically different in intermediate and low-risk groups (p = 0.47 and p = 0.19, respectively). Among 16 deaths in the SM-CMML cohort, cause of death was able to be assessed in 13 patients. Four (31%) patients died after transformation to acute myeloid leukemia (AML). These patients were either intermediate- or high-risk by Mayo Prognostic Model. Nine patients (69%) died due to multisystem organ failure due to progressive systemic mastocytosis without development of acute leukemia. Among these, 4 (44%) were low-risk, 3 (33%) were intermediate-risk, and 2 (22%) were high-risk. Conclusions: SM-CMML typically presented with lower-risk disease when graded by the Mayo CMML Prognostic Model. Compared head-to-head, OS was similar between SM-CMML and CMML; however multivariate analysis revealed the SM component to be a significant adverse prognostic factor. The presence of bone marrow mastocytosis is associated with inferior survival in high-risk CMML cases. Cause of death among SM-CMML patients was attributable to both progressive mastocytosis and transformation to AML. AML transformation was limited to intermediate- and high-risk group while progressive mastocytosis was seen across the risk spectrum. Future studies are warranted to determine if SM therapy can mitigate this outcome. Figure 1 Disclosures Kuykendall: Abbvie: Honoraria; Celgene: Honoraria; Incyte: Honoraria, Speakers Bureau; Janssen: Consultancy. Talati:Celgene: Honoraria; Agios: Honoraria; Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria. Komrokji:DSI: Consultancy; Incyte: Consultancy; Agios: Consultancy; JAZZ: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; pfizer: Consultancy; celgene: Consultancy.

Loss of Asxl1 Cooperates with Oncogenic Nras to Drive CMML Progression

Chronic myelomonocytic leukemia (CMML) belongs to the group of "mixed myelodysplastic/myeloproliferative neoplasm". There is currently no effective chemotherapy to treat CMML. Approximately 30% of CMML cases evolve to acute myeloid leukemia (AML) soon after their initial diagnosis, contributing to the poor prognosis of CMML patients. Ras pathway genes are frequently mutated in CMML patients (NRAS:11%; CBL:10%; KRAS:8%). Our whole exome sequencing of CMML patient samples and targeted sequencing of CMML patient-derived myeloid colonies showed that oncogenic NRAS could serve as an initiating or progression mutation. Consistent with human genetics, all the oncogenic Nras mice we and others characterized (NrasG12D/+, NrasG12D/G12D, and NrasQ61R/+), develop myeloproliferative (MP)-CMML like phenotypes.However, oncogenic NRAS alone is insufficient to drive CMML progression or transformation to AML. Additional sex combs-like 1 (ASXL1) is a human homolog of fly Asx. Mutations in ASXL1are predominantly nonsense and frequently identified in all myeloid malignancies (e.g. 40% in CMML). ASXL1mutations predict inferior overall survival in univariable analysis in multiple large CMML cohort studies and are significantly associated with NRASmutations in CMML patients. Therefore, we hypothesize that loss of Asxl1 cooperates with oncogenic Nrasto drive CMML progression. To test our hypothesis, we used Vav-Cre to drive oncogenic Nras expression and/or Asxl1 deletion in hematopoietic system. We refer these mice as NrasG12D/+, Asxl1-/-and NrasG12D/+;Asxl1-/-(NA) mice. Consistent with the literature, Asxl1-/-mice are normal at 6-week old. NrasG12D/+ mice showed mild CMML-like phenotypes, including enlarged spleen, increased white blood cell and monocyte counts, and expanded LSKs (Lin-Sca-1+c-Kit+) and myeloid progenitors (MPs). Compared to NrasG12D/+ mice, NA mice showed more sever phenotypes that were associated with hyperactivated ERK signaling in MPs at both basal level and upon GM-CSF stimulation. Our new NrasG12D/+mice developed a fully penetrant MP-CMML with the median survival of ~430 days. Loss of Asxl1 significantly shortened the median survival of NrasG12D/+ mice to 220 days. Half of NA mice displayed CMML transformation to AML. The transformed AML was serially transplantable to recipients. To investigate the mechanism(s) underlying NA-driven CMML/AML, we performed RNA-Seq analysis in Lin- c-Kit+BM cells from age-matched control, Asxl1-/-, NrasG12D/+, and moribund NA mice with CMML or AML phenotypes. We found 844 differentially expressed genes (DEG) in AML cells compared to control cells (FDR&lt;0.05 and fold change&gt;2). Among these genes, 341 are common between NA and published Nf1+/-;Asxl1+/-AML cells, including AP-1 complex genes (Jun, Junb, Jund and Fosb) that are crucial for malignant propagation in several AML subtypes. The remaining 503 genes are unique in NA AML, including Flt3, one of the mostly mutated genes in AML. These data suggest that our NA AML model demonstrates some distinct mechanisms from Nf1+/-;Asxl1+/-model. In-depth analysis of RNA-seq data is still ongoing. We also examined multiple histone marks using Western blot in BM cells of 6-week old mice. Our data showed that levels of H3K4me3, H3K4me1, and H3K27me3 were downregulated in Asxl1-/-cells, whereas H3K27Ac level was comparable to that of control cells. In contrast, H3K27Ac level was upregulated in NrasG12D/+and NA cells, while levels of H3K4me3, H3K4me1, and H3K27me3 were indistinguishable from those of control cells. BRD4 is a member of the BET family of bromodomain-containing proteins that binds to acetylated histones to activate gene transcription. Thus, we hypothesized that Ras/MEK/ERK signaling and BRD/BET are potential targets to inhibit AML progression. To test this hypothesis, we treated the AML cells and BMT recipients using trametinib (a MEK inhibitor approved by FDA to treat melanoma) and GSK525762 (a pan BET inhibitor under clinical development). We demonstrated thatcombined treatment significantly inhibited the growth of leukemia cells in vitro and prolonged the survival of recipients in vivo. It was more effective than single agent alone. Overall, we established a novel CMML/AML mouse model, which represents a significant group of CMML patients with poor prognosis. Our study provides a strong rational to treat these patients with combined MEK and BET inhibition. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Clinical Relevance of Somatic Mutations in Chronic Myelomonocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Clinical Relevance of Somatic Mutations in Chronic Myelomonocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Comprehensive mutation profiling and mRNA expression analysis in atypical chronic myeloid leukemia in comparison with chronic myelomonocytic leukemia.

Atypical chronic myeloid leukemia (aCML) and chronic myelomonocytic leukemia (CMML) represent two histologically and clinically overlapping myelodysplastic/myeloproliferative neoplasms. Also the mutational landscapes of both entities show congruencies. We analyzed and compared an aCML cohort (n = 26) and a CMML cohort (n = 59) by next‐generation sequencing of 25 genes and by an nCounter approach for differential expression in 107 genes. Significant differences were found with regard to the mutation frequency of TET2, SETBP1, and CSF3R. Blast content of the bone marrow revealed an inverse correlation with the mutation status of SETBP1 in aCML and TET2 in CMML, respectively. By linear discriminant analysis, a mutation‐based machine learning algorithm was generated which placed 19/26 aCML cases (73%) and 54/59 (92%) CMML cases into the correct category. After multiple correction, differential mRNA expression could be detected between both cohorts in a subset of genes (FLT3, CSF3R, and SETBP1 showed the strongest correlation). However, due to high variances in the mRNA expression, the potential utility for the clinic is limited. We conclude that a medium‐sized NGS panel provides a valuable assistance for the correct classification of aCML and CMML.

Time-to-Treatment in Chronic Myelomonocytic Leukemia - a Novel Prediction Model

IntroductionFor chronic myelomonocytic leukemia (CMML) several scores exist which prognosticate overall survival (OS) based on different clinical and genetic parameters. The time-to-treatment (TTT) among CMML patients is highly variable, and a predictive model to specifically estimate TTT in CMML has not been described so far. The aims of this single-center retrospective study were (a) to test and validate established myelodysplastic syndrome (MDS)-specific and CMML-specific prognostic scores in our patient cohort, (b) to evaluate which baseline factors were relevant to the time point of treatment initiation with either hydroxyurea or azacitidine, and (c) to propose a prediction model for TTT in CMML.MethodsThis retrospective analysis was based on the data of 55 unselected, consecutive CMML patients diagnosed and/or treated at our tertiary center between 2004 and 2015. We applied the following published prognostic models to our CMML cohort, using both OS and TTT as endpoints: the MD Anderson Prognostic Score (MDAPS), the modified MDAPS (MDAPS M1), the CMML-specific Prognostic Scoring System (CPSS), the Mayo Prognostic Model, the Düsseldorf Score, the International Prognostic Scoring System (IPSS), and the Revised International Prognostic Scoring System (IPSS-R).ResultsAccording to the CMML-specific MDAPS, 27% of our patients were classified as "higher-risk" (23% intermediate-2, 4% high-risk) (Figure 1). At the time of data analysis, 38% and 24% of patients had received azacitidine and hydroxyurea as first-line treatment. A total of 40 (73%) patients had died at the time point of data analysis. The median time of follow-up was 24.8 months (range 1.7-74.8 months).All applied MDS-specific (Düsseldorf Score, IPSS, IPSS-R) and CMML-specific (MDAPS, MDAPS M1, CPSS, Mayo Prognostic Model) prediction scores were able to significantly discriminate patient cohorts with different OS probabilities. The following variables were associated with a shorter TTT in the univariate analysis: the presence of immature myeloid cells in the peripheral blood, white blood cell count ≥14.5 G/L, platelet count <55 G/L, absolute neutrophil count ≥6 G/L, absolute lymphocyte count ≥2.3 G/L, absolute monocyte count ≥2.8 G/L, serum lactate dehydrogenase ≥223 G/L, peripheral blood blasts >0%, bone marrow blast percentage ≥7.5%, red blood cell transfusion-dependence, palpable spleen and/or symptomatic splenomegaly, and the presence of B-symptoms at the time of initial diagnosis. In multivariate analysis, the following factors remained independently associated with TTT: lactate dehydrogenase (HR 5.428; p = 0.008), bone marrow blast count (HR 4.570; p = 0.001), and platelet count (HR 2.660; p = 0.027).These three clinical parameters were included in the TTT prediction model and CMML patients were stratified into three subgroups: low-risk, intermediate-risk and high-risk. Median TTT was not reached for low-risk patients, 16.5 months for intermediate-risk patients, and almost immediate treatment initiation (0.6 months) was observed in the high-risk group (Figure 2).ConclusionsWe validated seven existing MDS-specific and CMML-specific prognostic scores in 55 CMML patients treated at the center in Salzburg. We were able to demonstrate that lactate dehydrogenase, bone marrow blast percentage and platelet count at initial diagnosis were the most relevant parameters for predicting time to treatment initiation in our CMML cohort. Based on these three parameters, we propose the first TTT prediction score for treatment-naïve CMML patients. Clinical implications of this score include the identification of CMML patients for early investigational trials, as well as the tailoring of individual follow-up intervals. [Display omitted] [Display omitted] DisclosuresHuemer:Roche: Other: Travel funding; Merck: Other: Travel funding. Egle:Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: travel support; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel support. Greil:Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Eisai: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Merck: Honoraria; Janssen-Cilag: Honoraria; Genentech: Honoraria, Research Funding; Novartis: Honoraria; AstraZeneca: Honoraria; Roche: Honoraria, Research Funding; Sanofi Aventis: Honoraria; GSK: Research Funding; Ratiopharm: Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria. Pleyer:Celgene: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan Pharmaceuticals: Honoraria.

DNTM3A Mutations and Prognosis in Chronic Myelomonocytic Leukemia

▪Background: DNMT3A mutations result in epigenetic dysregulation and impart a negative prognostic impact in acute myeloid leukemia and myelodysplastic syndromes. In chronic myelomonocytic leukemia (CMML), DNMT3A mutations are seen in 2-5% of patients. In a large Groupe Français des Myélodysplasies (GFM) study (n=312), DNMT3A mutations were seen in 2% and were not included in further survival analyses (Itzykson JCO 2013). In a prior Mayo Clinic study (n=175), DNMT3A mutations were seen in 5% (n=9) and on univariate, but not multivariate analysis (Patnaik Blood C J 2016), were associated with shortened over-all survival (OS). We carried out this study on a larger CMML cohort (n=261), with more (n=15) informative cases to assess the impact of DNMT3A mutations.Methods: 261 patients with World Health Organization (WHO)-defined CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. The 2016 WHO diagnostic criteria were used.Results: Among the 261 study patients, 65% were males and median age was 70 years (range, 28-91). 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations (LT) were documented. Mutational frequencies ≥4% were encountered in; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%.i) DNTM3A mutated CMML: phenotypic and molecular correlatesDNMT3A mutations were seen in 15 (6%) patients; 64% male with a median age of 64 years. DNMT3A amino acid substitutions included; R882H 50%, R882C 29%, R910P 7%, R598* 7% and R320* 7%. The median variant allele frequency burden was 45%. Concurrent gene mutations were detected in; TET2 43%, ASXL1 21%, SF3B1 21%, U2AF1 14%, RUNX1 14%, SETBP1 14%, NRAS 14%, SRSF2 7%, JAK2 7% and Tp53 7%. There was no difference between DNMT3A mutated and wild-type patients in terms of age and gender distribution, hemoglobin level, leukocyte, monocyte (AMC), and platelet counts, peripheral blood (PB) or BM blast content. Concurrent gene mutations were equally distributed with the exception for a higher prevalence of SF3B1 (p=0.003) and a lower prevalence of SRSF2 (p=0.004) mutations in DNMT3A mutated CMML. Four (29%) patients underwent leukemic transformation.ii) Impact on OS and leukemia-free survival (LFS):Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was shorter in DNMT3A mutated (median 8 months) versus wild-type (median 27 months) patients (p=0.0007; HR 2.9, 95% CI 1.5-5.7; Figure 1A). Other variables of significance, in univariate analysis, included lower hemoglobin (p=0.002), higher leukocyte count (p=0.0009), higher AMC (p=0.0012), PB blast % (p=0.001), circulating immature myeloid cells (IMC, p=0.01), BM blast % (p=0.045), abnormal karyotype (p=0.02), and ASXL1 (p=0.01) mutations. Survival was also adversely affected by the presence of either (n=133) or both (n=3) ASXL1/DNMT3A mutations (0=0.007, Figure 1B). In multivariable analysis (MVA) excluding ASXL1 and DNMT3A mutations, hemoglobin (p=0.03), IMC (p=0.013) and AMC (p=0.02) retained significance. When ASXL1 mutations were added to the MVA, ASXL1 (p=0.01) mutations, AMC (p=0.012) and IMC (p=0.03) retained significance. Similarly, when only DNMT3A mutations were added to the MVA, DNMT3A (p=0.003) mutations, IMC (p=0.01) and AMC (p=0.02) retained significance. When both DNMT3A and ASXL1 mutations were added to the MVA, only DNMT3A (p<0.0001) and ASXL1 (p=0.004) mutations remained significant. DNMT3A mutations predicted shortened OS, independent of the ASXL1 inclusive GFM model (p<0.0001) and Mayo Molecular Model (p=0.002). DNMT3A mutations (p=0.0018), along with low hemoglobin levels (p=0.003) independently predicted for a shorter LFS.Conclusions:DNMT3A mutations are seen in ~5% of patients with CMML and impart a negative prognostic impact on both OS and LFS. This finding warrants inclusion of DNMT3A mutations in molecularly integrated CMML prognostic models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of ASXL1 Mutations in MDS and CMML

▪Background Molecular profiling through next-generation sequencing (NGS) in myeloid diseases has greatly improved prognostic discrimination. In myelodysplastic syndromes (MDS), mutations of TP53, EZH2, ETV6, RUNX1 and ASXL1 were shown in a multivariate model to predict for inferior survival although ASXL1 mutations were least predictive (HR 1.38, P =.049; Bejar et al., 2011). In contrast, mutation analysis of two large chronic myelomonocytic leukemia (CMML) cohorts both identified ASXL1 mutation as the only mutation to be independently predictive of inferior survival in multivariable analysis when including only nonsense and frameshift mutations (Itzykson et al., 2013; Patnaik et al., 2014). Therefore, our goal was to investigate the relative impact of ASXL1 mutations in MDS and CMML.Patients and Methods NGS profiled MDS and CMML cases were retrospectively identified from the Moffitt Cancer Center MDS database. We genotyped ASXL1 and up to 20 additional genes in our cohort. The lower limit of detection was set at 5% mutant allele reads and the minimum depth of coverage was 500X. Clinical variables and outcomes of MDS patients were characterized at the time of sample procurement. Fisher's exact and t-tests were used for comparative analyses. Kaplan-Meier estimates were used to estimate overall survival and analyzed from the date of mutation identification. Multivariate Cox regression models were created to adjust for clinical characteristics.Results From May 2013 to October 2014, 248 patients with NGS for somatic mutations were identified in our database. The baseline characteristics of the MDS and CMML cohorts stratified by ASXL1 mutation status are shown in Table 1. In this cohort, 89% of patients (n=220) had at least one pathogenic mutation. ASXL1 frameshift or nonsense mutations occurred in 22% of patients (n=54) with increased frequency in the CMML cohort (50% versus 18%, P =0.0002). An additional 7% of patients (n=17) had a missense mutation of ASXL1 and were predominantly restricted to the MDS subgroup (16/17). The most common type of ASXL1 mutation were frameshift mutations in the MDS and CMML cohorts, 51% and 69% respectively, with the c.1934dupG; p.G646Wfs*12 variant observed in 33% of CMML patients (n=5) and 21% of MDS patients (n=8). ASXL1 mutant patients had similar survival to wild-type patients with median OS of 15.3 versus 13.7 months, respectively (P = 0.80). Exclusion of missense mutations had no effect on the prognostic impact of ASXL1 mutation (P =0.78). In the MDS cohort, there was again no difference in OS (median OS 14.3 versus 13.3 months; P =0.47) and type of ASXL1 mutation had no effect (P = 0.85). In the CMML cohort (n=30), median OS was 8.8 months in ASXL1 mutant patients and not reached in wild-type patients (HR 3.0, 95% CI 0.87 to 10.37, P =0.08). In comparison to wild-type patients, class of ASXL1 mutation was predictive of survival in the CMML cohort (P =0.008). CMML patients with frameshift mutations of ASXL1 had shorter OS in comparison to nonsense mutations although this did not reach significance (P =0.21). In multivariable analysis incorporating age and IPSS, ASXL1 mutation status was predictive for inferior survival among CMML patients (HR 2.5, 95% CI 1.08 to 5.85; P =0.03).Conclusion In MDS patients, neither ASXL1 mutation status nor exclusion of missense mutations was predictive for inferior survival, while ASXL1 mutant CMML patients had inferior OS, particularly with frameshift mutations. Together, these results highlight biological differences between MDS and CMML and support collaborative efforts in mutational profiling of these patients to improve prognostication. Table 1Baseline Characteristics by ASXL1 Mutation Status (Frameshift and Nonsense)MDS CohortCMML CohortASXL1 MTASXL1 WTASXL1 MTASXL1 WTn=39n=179n=15n=15Median age72 (54-91)71 (36-100)73 (48-88)72 (57-94)Male29 (74%)109 (61%)13 (87%)9 (60%)Female10 (26%)70 (39%)2 (13%)6 (40%)Median Hemoglobin99.410.111.1Median Platelets64827976Median ANC1.321.395.394.23Median Monocyte count0.230.241.912.46Median BM Blast %4445IPSSlow5 (15%)39 (25%)5 (33%)6 (40%)intermediate 117 (50%)50 (33%)7 (47%)6 (40%)intermediate 23 (9%)36 (24%)2 (13%)2 (13%)high9 (26%)28 (18%)1 (7%)1 (7%) DisclosuresVaupel:Genoptix: Employment; Novartis: Employment, Equity Ownership. Lancet:Celgene: Consultancy, Research Funding; Amgen: Consultancy; Pfizer: Consultancy; Kalo-Bios: Consultancy; Boehringer-Ingelheim: Consultancy; Seattle Genetics: Consultancy. Hall:Novartis: Employment, Equity Ownership; Genoptix: Employment. List:Celgene Corporation: Honoraria, Research Funding. Komrokji:Incyte: Consultancy; Celgene: Consultancy, Research Funding; Pharmacylics: Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Chronic Myelomonocytic Leukemia (CMML) Can be Categorized By Ancestral Mutational Events

CMML heterogeneous with a spectrum of dysplastic or proliferative clinical features. Except for rare balanced translocations (involving PDGFbR or PDGFaR), cytogenetic defects are overlapping with MDS and MPN. Recently, due to the introduction of NGS, many somatic mutations have been discovered in myeloid neoplasms. Analysis of mutational spectrum in CMML may facilitate understanding of the pathogenesis of this disease. However, with the recognition of the complexity of clonal architecture and intra-tumor heterogeneity, not only a share presence of individual mutations/their combinations, but also the clonal hierarchy may be of diagnostic importance. Ancestral events may correspond to clinically distinguished CMML subtypes or allow for a new sub-categorization reflective of common pathogenetic features. Here we studied somatic mutational spectra of 306 patients, including 150 CMML cases (97 CMML-1, 23 CMML-2, and 30 post CMML sAML) with 55% dysplastic (MD-CMML) and 44% proliferative form (MP-CMML), abnormal cytogenetics were found in 45% cases. In 10 cases serial analysis was performed. Comparisons were also performed with JMML (N=92) and M4/M5 AML (N=64). We performed analyses of WES in 108 paired cases with deep targeted DNA NGS in the remaining patients.Within CMML cohort, the most frequently mutated somatic genes were TET2 (43%), SRSF2 (31%), ASXL1 (23%), RUNX1 (17%), CBL (13.3%), U2AF1 (12%) followed by EZH2, SETBP1, DNMT3A, and K/NRAS. Cross-sectional analysis demonstrated distinct variations in the distribution of individual mutations between subtypes. Similarly, global analysis of mutational frequencies showed only a few differences between clinical subtypes, e.g., SRSF2 mutations were found in 47% of MP-CMML vs. 24% in MD-CMML. JMML was showed a clearly distinct mutational pattern from CMML with PTPN11, NF1, K/NRAS and JAK3 mutations more commonly encountered in JMML. While ASXL1, EZH2 and TET2 (common in CMML) were not detected in JMML (p<0.001). CBL and SETBP1 were equally distributed in both diseases. When sAML from CMML was compared to Non-CBF AML M4/5, TET2 and ASXL1 mutations were more commonly found in sAML than in M4/M5 (40% vs. 6%; 30% vs. 0%, P<.0001). NPM1FLT3 and DNMT3A mutations were significantly more common in M4/M5.Study of clonal architecture can allow for identification of ancestral vs. secondary events by serial or cross-sectional analyzes of variant allelic frequencies. Totally, 81% of TET2 mutations and 90% of SRSF2 mutations were ancestral, while 75% of U2AF1 and 78% of K/NRAS were secondary. Overall, 110 ancestral mutations were identified in 25 driver genes. CMML with founder clones included TET2 in 25%, SRSF2/ZRSR2 19%, ASXL1 12%, EZH2 5%, BCOR/BCORL1 8%, RUNX1 9%, SETBP1 5%, DNMT3A 5%, CBL/RAS 10% while all the others were present in 10% of CMML cases (of note is that 13% of them were co-dominant founder mutation). To examine whether this sub-classification corresponds to clinical phenotypes, they were correlated with clinical variables. For instance, ancestral mutations of TET2, ASXL1 and RUNX1 were commonly identified in both MD-CMML and MP-CMML while ancestral SETBP1, JAK2, SRSF2 and NRAS mutations were present only in MP-CMML, suggesting that initial genetic events of these 4 genes determine proliferative characteristics. In contrast, ancestral mutational events of SF3B1, DNMT3A, STAG2 and CBL were observed exclusively in MD-CMML. Secondary events further contribute to the clinical heterogeneity. For example, CBL secondary mutations were found in MP-CMML cases, while NRAS secondary mutations were observed in MD-CMML mainly in combination with ancestral STAG2 mutations. When we analyzed the impact of ancestral events on survival, EZH2 and U2AF1 mutant CMML conveyed worse prognosis (HR 3.7 and HR 2.2), but secondary EZH2 and U2AF1 mutations did not affect the outcome.In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations. DisclosuresNo relevant conflicts of interest to declare.

Importance of Genetics in the Clinical Management of Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by dysplasia, peripheral monocytosis, ineffective hematopoiesis, and an increased risk of transformation to acute myeloid leukemia. Because the clinical phenotype of CMML displays features of both myelodysplastic syndromes (MDSs) and myeloproliferative neoplasms (MPNs), the classification of CMML has historically been a dynamic process. Before 2001, CMML was classified as a subtype of MDSs, as initially reported by the French-American-British Group, because of its characteristic dysplasia, cytopenias, and acute myeloid leukemia transformation rate. However, as it became increasingly clear that CMML is clinically distinct from MDS, CMML was placed in a provisional category within the World Health Organization (WHO) classification of hematologic neoplasms known as MDS/MPN overlap diseases. This classification schema was formalized by WHO in 2008 and defines CMML as having a persistent peripheral blood monocytosis greater than 1 10/L, dysplasia in at least one hematopoietic lineage, and less than 20% myeloblasts and promonocytes in the peripheral blood and bone marrow. Although it is clinically heterogeneous, the overall survival in CMML is poor. It is the most aggressive chronic myeloid malignancy, with 3-year survival on the order of 20%. Because of its ontologic connection to MDSs, most therapeutic interventions and prognostic tools for CMML have been derived from studies with end points that were more focused on MDSs. However, next-generation sequencing technology has allowed for the comprehensive and cost-effective annotation of genetic mutations across the spectrum of hematologic malignancies and has uncovered that CMML seems to be a unique disease entity, with mutations in a divergent array of cellular processes that include epigenetic modification, alternative splicing, and receptor kinase signaling. Although recurrent mutations in CMML, such as those in ASXL1 and SRSF2, are not disease-specific, it is the high frequency of these mutations that gives CMML its unique genomic identity. This CMML genomic fingerprint now gives CMML researchers a platform to construct genomically based, CMML-specific therapeutics and prognostic tools. In the article by Itzykson et al that accompanies this editorial, this genomic fingerprint is leveraged to create a robust CMML-specific prognostic tool using the combined presence of recurrent mutations and clinical parameters. Prognostic models for CMML have traditionally incorporated predictors of survival that are biased toward patients with MDSs. At least seven prognostic scoring systems derived from unique cohorts have been published to date that either exclusively included patients with CMML or included patients with CMML in a larger MDS cohort (Table 1). However, most of these models have either never been formally externally validated in the context of CMML or were externally validated before the use of hypomethylating agents. Furthermore, the International Prognostic Scoring System (IPSS) and revised IPSS (IPSS-R) specifically excluded patients with CMML who had a WBC greater than 12 10/L, making their application to CMML as a whole problematic. Significant uncertainty exists, given that limited data are available to guide clinicians when they are attempting to choose the highest performing and most applicable tool in CMML. Only two articles have addressed the comparative power of various prognostic tools within a single CMML cohort: one was published before the widespread use of hypomethylating agents, and the other was a preliminary analysis that compared the IPSS, Global MD Anderson Scoring System, Dusseldorf Scoring System, Spanish Score, and MD Anderson Prognostic Score for CMML in the era of hypomethylating agents. More study is needed to determine the comparative prognostic power of existing tools, but the prognostic model proposed by Itzykson et al is particularly robust, given that it included a training set of over 300 patients, was internally validated using a two-step bootstrapping process, and was externally validated using an independent CMML cohort. After retrospectively collecting clinical data and interrogating up to 18 genes that are recurrently mutated in CMML, the authors found that age older than 65 years, WBC of greater than 15 10/L, platelet count of less than 100 10/L, and ASXL1 mutation were each independently prognostic of poorer survival in a large hypomethylating agent–naive and smaller hypomethylating agent– treated group. Using hazard ratio estimations for weighting of variables, a novel prognostic score was created that was able to improve on the predictive outcome of the MD Anderson Prognostic Score for CMML and the Bournemouth CMML prognostic tools. This proposed tool represents an advance in the prognostication of CMML because it demonstrates that the incorporation of genetic mutations with clinical variables can result in a model with improved prognostic power compared with models that incorporate clinical variables alone. However, for this model to be adopted in clinical practice as the gold standard for CMML prognostication, detailed comparisons with other widely used prognostication tools in CMML such as the Global JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 19 JULY 1 2013

A Comparison of Prognostic Models for Chronic Myelomonocytic Leukemia (CMML) in the Era of Hypomethylating Agents

AbstractAbstract 1695 Introduction:CMML is a genetically and clinically heterogeneous malignancy characterized by peripheral monocytosis, cytopenias, and a propensity for AML transformation. Several prognostic models attempt to stratify patients into subcategories that are predictive for overall survival (OS), six models of which are specific to CMML. However, these models have either never been externally validated in the context of CMML or were externally validated prior to the use of hypomethylating agents. We externally validate and perform a detailed statistical comparison between the International Prognostic Scoring System (IPSS), MD Anderson Scoring System (MDASC), MD Anderson Prognostic Score (MDAPS), Dusseldorf Score (DS), and Spanish Scoring Systems (SS) in a large, single institution cohort. Methods:Data were collected retrospectively from the Moffitt Cancer Center (MCC) CMML database and charts were reviewed of patients that satisfied the WHO criteria for the diagnosis of CMML. The primary objective of the study was to validate the above prognostic models calculated at the time of initial presentation to MCC. All prognostic models were calculated as previously published. All analyses were conducted using SPSS version 15.0 (SPSS Inc, Chicago, IL). The Kaplan–Meier (KM) method was used to estimate median overall survival and the log rank test was used to compare KM survival estimates between two groups. Results:Between January 2000 and February 2012, 123 patients were captured by the MCC CMML database. The median age at diagnosis was 69 (30–90) years and the majority of patients were male (69%). By the WHO classification, the majority of patients had CMML-1 (84% vs. 16%) and most patients were subcategorized as MPN-CMML (59%) versus MDS-CMML (39%) by the FAB CMML criteria. The median overall survival of the entire cohort was 30 months and the rate of AML transformation was 44% (54). Twenty-two patients (18%) were treated with decitabine and 66 (54%) patients were treated with 5-azacitidine. Risk group stratification according to specific prognostic model is summarized in Table 1. The IPSS, MDASC, DS, and SS all predicted OS (p<0.05) while the MDASP could not be validated (p=0.924). When only patients who were treated with 5-azacitadine were considered, the MDASC, DS, and SS continued to predict OS (p<0.05) while the IPSS (p=0.15) and MDASP (p=0.239) did not. Previous reports have demonstrated that the MDASC provides further discrimination to refine stratification by the IPSS in Myelodysplastic Syndromes (MDS). Except for the low-risk DS patients, we grouped patients in our CMML cohort into lower and higher risk disease with each prognostic score and attempted to further stratify patients by the MDASC using KM and the log rank test. The MDASC was able to further risk stratify patients in each group for all prognostic models except those in the higher risk groups by the SS (p=0.07) and DS (P=0.45). When a similar statistical analysis was applied to each prognostic scoring system, only the MDASC was consistently able to further stratify the majority of risk groups as described in Table 2. The Dusseldorf scoring system was able to further stratify all lower risk groups regardless of model but was not able to do so in higher risk disease. Conclusions:This represents the first external validation of existing CMML prognostic models in the era of hypomethylating agent therapy. Except for the MDASP, we were able to validate the prognostic value all models tested. The MDASC represents the most robust model as it consistently refined the stratification of other models tested and remained predictive of OS in 5-azacitidine treated patients. Multi-institution collaboration is needed to construct a robust CMML specific prognostic model. Comparison to the IPSS-R is in progress.Table 1MDASCMDASPDSIPSSSSRISK GROUPPercentPercentPercentPercentPercentlow2524132257int-I32416753-int-II2530-20-high18520543Table 2Initial Staging<– Refining Models–>MDASCMDASCDSIPSSSSLower Riskp<0.05p=0.41p<0.05Higher Riskp=0.24p=0.22p=0.79DSLower Riskp<0.05p=0.39p=0.14Higher Riskp=0.46p=0.99p=0.20IPSSLower Riskp<0.05p<0.05p<0.05Higher Riskp<0.05p=0.17p=0.73SSLower Riskp<0.05p<0.05p<0.05Higher Riskp=0.07p=0.10p=0.58 Disclosures:No relevant conflicts of interest to declare.