Chronic Myelomonocytic Leukemia Cases Research Articles

Outcomes of intermediate or high-risk CMML patients treated with hypomethylating agents combined with venetoclax: A single center experience.

Chronic myelomonocytic leukemia (CMML) treatment remains a pressing clinical challenge. We conducted a retrospective analysis on 52 CMML cases, exploring the effectiveness of combining venetoclax (Vene) with hypomethylating agents (HMAs). The study's findings show promise: the HMAs plus Vene group (n = 13, 53.8%) demonstrated superior overall response rates compared to the HMA monotherapy (mono) group (n = 19, 31.6%) and HMA plus arsenic trioxide group (n = 9, 22.2%) by the second cycle, and notably higher response rates (53.8% vs. 15.7%, p = 0.04) compared to the HMA mono group after four cycles. Over a median follow-up of 14.7 months, the HMAs plus Vene group exhibited significantly lower cumulative mortality (23.1%) compared to the other two groups (p = 0.003 and p = 0.008, respectively). Furthermore, this group displayed extended overall survival compared to the others. The study also delved into the molecular mechanisms, revealing significant BCL2 mRNA overexpression in patients with CMML. These findings suggest the potential for HMAs combined with Vene therapy in CMML but emphasize the necessity for further prospective studies to determine its precise role in managing CMML.

Cellular Interactions within Clonal Dendritic Cell Aggregates Drive Immune Tolerance in Chronic Myelomonocytic Leukemia Bone Marrow Microenvironment

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

CSF3R-mutant chronic myelomonocytic leukemia is a distinct clinically subset with abysmal prognosis: a case report and systematic review of the literature

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) chacaterized by persistent peripheral blood monocytosis, hypercellular bone marrow and dysplasia at least in one myeloid lineage. CMML shares much of its molecular landscape with other myeloid neoplasms, while differs from others such as chronic neutrophilic leukemia (CNL), given the high frequency of CSF3R mutations in the latter. In this article, we report a case of CSF3R-mutated CMML and dissect this rare entity by reviewing the medical literature, with the intent to understand how this rare mutation shapes CMML's clinical and morphological phenotype. CSF3R-mutated CMML emerges as a rare entity meeting the ICC/WHO diagnostic criteria for CMML and simultaneously showing clinical-pathological and molecular traits of CNL and atypical chronic myeloid leukemia, rising an important and difficult diagnostic and therapeutical issue.

Clinical Characteristics and Survival Analysis of Patients with Chronic Myelomonocytic Leukemia

To investigate the clinical characteristics, prognostic factors and efficacy of hypomethylating agent (HMA) in patients with chronic myelomonocytic leukemia (CMML). The clinical data of 37 newly diagnosed patients with CMML was analyzed retrospectively, and their clinical characteristics and the efficacy of HMA were summarized. Kaplan-Meier and Log-rank test were used for univariate survival analysis, and Cox proportional hazards regression model was used for multivariate analysis. The median age at diagnosis was 67 years old. Their common manifestations included fatigue, bleeding, abnormal blood routine and fever. Most patients had splenomegaly. According to FAB classification, there were 6 cases of myelodysplastic CMML and 31 cases of myeloproliferative CMML, while according to WHO classification, 8 patients belonged to CMML-0, 9 patients to CMML-1 and 20 patients to CMML-2. At the time of diagnosis, the median white blood cell count was 32.84×109/L, median hemoglobin (Hb) was 101 g/L, median platelet count was 65×109/L, median absolute monocyte count was 9.53×109//L, median absolute neutrophil count (ANC) was 11.29×109//L and median lactate dehydrogenase (LDH) was 374 U/L. Cytogenetic abnormalities were found in 4 cases among the 31 patients who underwent karyotype analysis or fluorescence in situ hybridization detection. There were 12 patients who had analyzable results and gene mutations were identified in 11 cases, including ASXL1, NRAS, TET2, SRSF2 and RUNX1. Among the 6 patients who were treated with HMA and could be evaluated for efficacy, 2 patients achieved complete remission, 1 patient achieved partial remission and 2 patients achieved clinical benefit. Compared with the non-HMA treatment group, overall survival (OS) time was not significantly prolonged in the HMA treatment group. Univariate analysis showed that Hb<100 g/L, ANC≥12×109/L, LDH≥250 U/L and peripheral blood (PB) blasts ≥5% were significantly associated with poor OS, while WHO classification CMML-2, Hb<100 g/L, ANC≥12×109/L, LDH≥250 U/L and PB blasts≥5% were significantly associated with poor leukemia-free survival (LFS) (P<0.05). Multivariate analysis showed that ANC≥12×109/L and PB blasts≥5% were significantly associated with poor OS and LFS (P<0.05). CMML has high heterogeneity in clinical characteristics, genetic changes, prognosis and treatment response. HMA can not significantly improve the survival of CMML patients. ANC≥12×109/L and PB blasts≥5% are independent prognostic factors of OS and LFS in patients with CMML.

Chronic myelomonocytic leukemia in a 72-year-old male from Nepal: A case report.

Herein, the authors report a case of CMML in a 72-year-old male who presented with fever and abdominal pain for 2 days with a history of easy fatigability. Examination revealed pallor and palpable supraclavicular nodes. Investigations showed leukocytosis with a monocyte count of 22% of white blood cell count, 17% blast cells in bone marrow aspiration, increased blast/promonocytes, and positive markers in immunophenotyping. The patient is planned for injection of azacitidine, 7 days cycle for a total of six cycles. CMML is classified as overlapping myelodysplastic/myeloproliferative neoplasms. It can be diagnosed based on a peripheral blood smear, bone marrow aspiration and biopsy, chromosomal analysis, and genetic tests. The commonly used treatment options are hypomethylating agents like azacitidine and decitabine, allogeneic hematopoietic stem cell transplant, and cytoreductive agents like hydroxyurea. Despite various treatment options, the treatment is still unsatisfactory, demanding standard management strategies.

Clinical Characterization and Outcomes of Patients with NPM1-Mutated Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background: Nucleophosmin (NPM1) gene mutations are rarely detected in patients with non-acute myeloid neoplasms (MN), such as myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Retrospective case series suggested that they are typically associated with an aggressive disease course and progress into acute myeloid leukemia (AML) in a short period of time, representing a distinct clinicopathological entity. However, the long-term outcomes and prognostic factors of these MNs in the era of modern therapies remain not well defined given their rarity. Methods: In the current study, we retrospectively reviewed clinical features, treatments, and outcomes of patients with NPM1-mutated MDS or CMML seen at Moffitt Cancer Center between 2009 and 2022. In comparison, AML patients with confirmed prior histories of MDS or CMML (sAML) that had NPM1 mutations detected at their AML diagnoses were used as the control group. Results: We identified 22 patients with NPM1-mutated MNs (median age 66 years at diagnosis and 54.5% male), including 16 cases of MDS (68.8% excess blasts) and 6 cases of CMML (50% CMML-2). Most patients had a normal karyotype (72.7%), and the median variant allele frequency of NPM1 mutation was 30.7%. Compared to NPM1-mutated sAML (n = 31), patients with NPM1-mutated MNs had lower bone marrow blast counts as expected and a trend for less IDH1/2 and FLT3-ITD/TKD mutations (Table). The majority of NPM1-mutated MNs (77.3%) were treated with upfront hypomethylating agents, and treatment response was unsatisfactory, with only one complete response. Four patients ultimately underwent allogeneic stem cell transplant. In contrast, most NPM1-mutated sAML (71.0%) received intensive chemotherapy as the frontline therapy, which resulted in a higher rate of complete response (81.8%), and 12 patients (38.7%) subsequently underwent allogeneic stem cell transplant. The overall survival was significantly inferior in NPM1-mutated MNs compared to sAML (median 22.1 vs. 41 months, p = 0.041; p = 0.023 if data censored at the time of transplant), which remained significant after adjusted by age (hazard ratio 2.15, 95% confidence interval 1.07-4.34, p = 0.032). During follow-up, 14 patients (63.6%) with NPM1-mutated MNs transformed into AML with a median time to transformation of 21.1 months. Among them, nine patients were able to receive intensive chemotherapy as induction for AML, and four achieved complete responses. In patients with mutational data available at both MN and subsequent AML phases, NPM1 mutations remained detectable in 75% of cases, and gain of FLT3-ITD was commonly seen (41.7%) at the AML phase. In terms of prognostic factors, concurrent baseline DNMT3A mutations conferred a trend of inferior outcomes (median 16.0 vs. 30.7 months; p = 0.116), and patients who received transplant showed improved outcomes (median 59.7 vs. 18.1 months; p = 0.029). Conclusion: Our findings suggest that NPM1-mutated MNs are an aggressive clinicopathological entity and support the recent WHO proposal to reclassify these MNs into AML irrespective of the blast counts, as they may benefit from more intensive treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P693: DIAGNOSTIC ROLE OF CD26+ LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA

Background: Diagnosis of CML consists of persistent leucocytosis with the demonstration of the Philadelphia (Ph) chromosome abnormality, the t(9;22)(q34;q11), by routine cytogenetics, fluorescence in situ hybridization (FISH) or by molecular studies such as RT-PCR.These conventional methods suffer from a longer turn around time, higher costs and requirement of labs with adequate expertise. CD26 is a highly specific marker expressed in CML stem cells, which is not present on normal hematopoietic stem cells or on Leukemia stem cells (LSCs) of other myeloid neoplasms. A flow-cytometry based assessment of CD26+LSCs may prove to be an optimal biomarker for the diagnosis and monitoring of CML patients. Aims: 1)Assessment of CD 26 expression in suspected cases of CML-chronic phase 2)Correlation of CD 26+ stem cells with clinico-pathological parameters at baseline and its kinetics on tyrosine kinase inhibitor (TKI) treatment on further follow-up at 12 months. Methods: Suspected patients of CML-CP were included in the study. Peripheral blood were utilized for flowcytometric assessment of CD26+ LSCs. The results were correlated with conventional diagnostic tests of CML. Patients with myeloid neoplasms other than CML who proved negative for BCR-ABL1 by RT-PCR and normal HSCs donors treated with granulocyte-colony stimulating factors (G-CSF), were included as negative controls. All samples were processed using standard stain -lyse-wash method within 24 hr of collection. To reach a sensitivity of 10−5, acquisition of at least 1.0 × 106 cells was performed on FACS Canto II flow cytometer data a FACS DIVA 8.0.3 software (BD, Biosciences).CD26 expression was evaluated by a sequential gating strategy. After exclusion of doublets and debris CD45 dim to intermediate/CD34+/CD38- population was gated sequentially and then the expression of CD 26+ was studied on this population. All the samples were simultaneously assessed for BCR-ABL1 transcript by standard RT-PCR analysis. Patients on treatment were also followed up at 12 months to detect levels of CD26+ LSCs and correlated with BCR -ABL1 levels by qPCR. Results: A total of 86 patients of suspected cases of CML were included in the study from June 2020 to Dec 2021.The On flowcytometry analysis, CD 26 expression was present in 75 cases. All the cases with positive CD26+ LSCs were found to be positive on RT-PCR for BCR-ABL transcript. Eleven cases which were scored negative for CD26+ LSCs were also found to be negative for any BCR-ABL transcript. These negative cases included 8 cases of primary myelofibrosis, 2 cases of chronic myelomonocytic leukemia and 1 case of Polycythaemia Vera. The median percentage of CD26+ LSCs was 39.8% (Range-0.5-100). There was no significant correlation of the CD26 levels with age (p value=0.96), sex (p value=0.7), percentage of blasts (p value = 0.88), total leucocyte counts (p value=0.32), basophils (p value = 0.19), Sokal score (p value = 0.99) and ELTS score (p value = 0.99). Follow-up data was available for 7 patients and at 12 months all of these patients had achieved major molecular remission on TKIs. Two patients scored negative for CD26+LSCs while five patients showed significant fall in the level of CD 26+ LSCs however it was still detectable by flowcytometry (p value= 0.04). Summary/Conclusion: Flowcytometry assessment of CD26 + LSCs is a rapid and cheap diagnostic tool for diagnosis of CML with high diagnostic efficacy. Falling levels on TKIs warrants further research to prove it as a surrogate for monitoring response by conventional methods.

Monocytosis and its association with clonal hematopoiesis in community-dwelling individuals

AbstractMonocytosis may occur in numerous inflammatory conditions but is also the defining feature of chronic myelomonocytic leukemia (CMML). Clonal somatic mutations detectable in CMML may occur with aging in otherwise healthy individuals, so-called “clonal hematopoiesis” (CH). We investigated whether the combination of CH and monocytosis would represent an early developmental stage of CMML. We studied community-dwelling individuals with monocytosis (≥1 × 109/L and ≥10% of leukocytes) in the population-based Lifelines cohort (n = 144 676 adults). The prevalence and spectrum of CH were evaluated for individuals ≥60 years with monocytosis (n = 167 [0.8%]), and control subjects 1:3 matched for age and sex (n = 501). Diagnoses of hematological malignancies were retrieved by linkage to the Netherlands Cancer Registry (NCR). Monocyte counts and the prevalence of monocytosis increased with advancing age. Older individuals with monocytosis more frequently carried CH (50.9% vs 35.5%; P < .001). Monocytosis is associated with enrichment of multiple gene mutations (P = .006) and spliceosome mutations (P = .007) but not isolated mutated DNMT3A, TET2, or ASXL1. Persistent monocytosis over 4 years was observed in 30/102 evaluable individuals and associated with a higher prevalence of CH (63%). Myeloid malignancies, including 1 case of CMML, developed in 4 individuals with monocytosis who all carried CH. In conclusion, monocytosis and CH both occur at an older age and do not necessarily reflect clonal monocytic proliferation. In a fraction of older subjects with monocytosis, CH might constitute early clonal dominance in developing malignant myelomonocytic disease. Mutational spectra deviating from age-related CH require attention.

Prognostic Factors and Survival Outcomes in Blast Transformed Chronic Myelomonocytic Leukemia: A Mayo Clinic-MD Anderson Cancer Center Study of 171 Cases

Background:Blast transformed (BT) chronic myelomonocytic leukemia (CMML); defined by ≥20% blood or bone marrow (BM) blasts, occurs in 15-30% of patients and is a major cause of mortality. Reported risk factors include high risk karyotype, circulating blast count, circulating immature myeloid cells (IMC), CMML morphological subtype, and the presence of ASXL1 mutations. In the current study, we reviewed consecutive cases of BT CMML from the Mayo Clinic, MN, and the MD Anderson Cancer Center, Texas, USA, with the intent to examine i) prognostic factors, ii) survival trends, and iii) treatment outcomes.Methods:Diagnoses of CMML and BT-CMML were according to the 2016 WHO criteria. Treatment response in BT-CMML patients was assessed according to standard AML response criteria. Statistical analyses considered clinical and laboratory data collected at the time of BT. Survival was calculated from the date of BT to the date of death or last contact.Results:171 cases of BT CMML were identified from the Mayo Clinic (n=84) and MDACC (n=87). The median age at BT was 69 years and 64% were male. Eight one (49%) had received prior hypomethylating agent (HMA) therapy (Decitabine 50, 5-azacitidine 31), with 30% having achieved a complete remission (CR) prior to BT. CMML patients that received prior HMA therapy were more likely to have thrombocytopenia (p=0.03), higher LDH levels (p=0.007), lower frequency of TET2 mutations (p=0.04) and higher risk stratification by the Mayo prognostic model (p=0.009). The two institutional cohorts were comparable with regards to variables, both at CMML diagnosis and BT.Patient characteristics at time of BT: The median time from CMML diagnosis to BT was 12 months (range, 1-36). At BT, 16 (10%) presented with extramedullary disease, 81 (63%) with an abnormal karyotype, 41 (34%) with cytogenetic clonal evolution, with the following mutational distribution; TET2 50%, ASXL1 39%, NRAS 39%, FLT3 ITD 20%, NPM1 6%, RUNX1 6%, and 5% each for IDH1/2, EZH2 and JAK2 . Molecular clonal evolution was seen in 4 (22%) of 18 evaluable cases.Survival trends: After a median follow-up of 4.4 months (range 0-122), 141 (82%) deaths were recorded. Median overall survival (OS) was 6 months with 1- and 3-year survival rates of 25% and 9%, respectively; survival trends were similar (p=0.4) for patients diagnosed prior to and after the year 2006 (figure 1A).Treatment and response rates: Specific treatment for BT-CMML was documented in 157 cases and included induction chemotherapy (n=61), induction chemotherapy followed by stem cell transplant (HCT, n=18), HCT alone (n=3), HMA (n=18), novel therapeutic agents (n=18), and best supportive care (BSC, n=39). Treatment response was assessable in 113, including 76 treated with induction chemotherapy, 18 with HMA and 16 with novel therapeutics; with respective CR rates of 24%, 0% and 41%; with an additional 24%, 16% and 16% of patients achieving CR with incomplete blood count recovery (CRi), with a survival similar to those with CR (figure 1B). HCT was reported in 25 patients, 17 myeloablative and 8 reduced intensity, with a median OS of 7 months.Predictors of survival in BT-CMML: In univariate analysis of variables recorded at time of BT, risk factors adversely impacting survival included older age, lower hemoglobin, PB blast %, adverse cytogenetics at BT, prior exposure to HMA, not receiving induction chemotherapy, not achieving CR or CRi and not receiving HCT; all of these risk factors remained significant when analysis was adjusted for age. In multivariable analysis, lack of prior HMA exposure (p=0.001, HR 5.1; 95% CI 1.9-14.1) and the achievement of CR/CRi (p=0.02, HR 1.4; 95% CI 1.2-3.4) remained significant while HCT lost its significance (p=0.09). Gene mutations, including FLT3 ITD, cytogenetic and molecular clonal evolution at the time of BT did not impact survival. The 3-year survival rates for BT patients receiving induction chemotherapy, induction chemotherapy followed by HCT, HMA, novel agents and BSC were 8%, 20%, 12%, 3% and 0% respectively (figure 1C).Conclusions: Survival in patients with BT- CMML is dismal (≈6 months) and is adversely impacted by prior exposure to HMA and by the failure to achieve CR/CRi. After BT, the use of HMA therapy is largely ineffective (0% CR), with intensive therapies such as induction chemotherapy with or without HCT, failing to achieve durable remissions. [Display omitted] DisclosuresJabbour:Amgen: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Takeda Oncology: Research Funding. Kantarjian:Amgen: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: 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.

IRF8 is a Reliable Monoblast Marker for Acute Monocytic Leukemias.

Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the aspirate smears. While blasts of most nonmonocytic acute leukemias express CD34, available immunohistochemical antibodies to monocytic blasts also mark normal background mature monocytes. We searched for a potential biomarker candidate by surveying specific gene expression profiles of monocyte progenitors. Our investigations led us to IRF8, which is a lineage-specific transcription factor critical for the production of monocytic and dendritic cell progenitors. In this study, we tested and validated a monoclonal antibody to IRF8 as a novel immunohistochemical stain for trephine core biopsies of human bone marrow. We assessed the expression of IRF8 in 90 cases of AMoL, including posttherapy staging bone marrows, 23 cases of chronic myelomonocytic leukemia, 26 cases of other acute myeloid leukemia subtypes, and 18 normal control marrows. In AMoL, there was high correlation of IRF8-positive cells to aspirate blast count (R=0.95). Comparison of IRF8 staining to aspirate blast percentage in chronic myelomonocytic leukemia also showed good correlation (R=0.86). In contrast, IRF8-positive cells did not correlate with blast count in other subtypes of acute myeloid leukemia (R=0.56) and staining was <5% in all normal control marrows, even those with reactive monocytosis. We found that IRF8 was also weakly reactive in B cells and hematogones, with the latter accounting for rare cases of discrepancies. When IRF8 was used to categorize cases as AMoL, positive for residual leukemia or negative, the sensitivity was 98%, specificity was 82%, positive predictive value was 86%, and negative predictive value was 98%. These results demonstrate that IRF8 may serve as a clinically useful immunostain to diagnose and track AMoLs on bone marrow core biopsies. This can be particularly impactful in the setting of poor aspiration and focal blast increase. In the era of new targeted therapies that have been reported to induce monocytic outgrowths of leukemia, a marker for malignant monoblasts may prove even more critical.

Mutations in chronic myelomonocytic leukemia and their prognostic relevance.

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy that overlaps with myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) and tends to transform into acute myeloid leukemia (AML). Among cases of CMML, > 90% have gene mutations, primarily involving TET2 (~ 60%), ASXL1 (~ 40%), SRSF2 (~ 50%), and the RAS pathways (~ 30%). These gene mutations are associated with both the clinical phenotypes and the prognosis of CMML, special CMML variants and pre-phases of CMML. Cytogenetic abnormalities and the size of genome are also associated with prognosis. Meanwhile, cases with ASXL1, DNMT3A, NRAS, SETBP1, CBL and RUNX1 mutations may have inferior prognoses, but only ASXL1 mutations were confirmed to be independent predictors of the patient outcome and were included in three prognostic models. Novel treatment targets related to the various gene mutations are emerging. Therefore, this review provides new insights to explore the correlations among gene mutations, clinical phenotypes, prognosis, and novel drugs in CMML.

The Use of Monocyte Subset Repartitioning By Flow Cytometry for Diagnosis of Chronic Myelomonocytic Leukemia

Introduction Patients with chronic myelo-monocytic leukaemia (CMML) have been reported to have a relative predominance of classical or MO1 monocytes (CD14+/CD16-) at the expense of MO2 (CD14low/CD16+) and MO3 (CD14-/CD16+) monocytes (Selimoglu-Buet. Blood 2015). These authors suggested that an MO1 percentage cut-off of &amp;gt;94% could predict the diagnosis of CMML with high sensitivity and specificity (both &amp;gt;90%) from other causes of monocytosis. Since then, several independent groups have attempted to reproduce their protocol with variable results. Most recently, the Mayo Clinic (Pophali. Blood Cancer J. 2019) found that an MO1 cut off of &amp;gt; 94% in peripheral blood identified CMML with a sensitivity of 75% and a specificity of 95.4%. These figures were lower than previously reported - calling into question, the utility of flow cytometry to distinguish the aetiology of monocytosis in a real-world setting. Objective Our retrospective audit aimed to establish if monocyte subset repartitioning could be used to reliably diagnose CMML in a real-world sample of patients with monocytosis. Methods In this study, we assessed peripheral blood samples from 35 patients presenting with a monocytosis (absolute monocyte count &amp;gt; 1 x109/L) in a tertiary referral hospital in Brisbane, Australia between June 2015 and Sep 2019. The patients' final clinical diagnosis was extracted from the medical record by two clinicians (AM, PM) blinded to the results of the flow cytometry. Peripheral blood samples were subjected to MFC at a median of &amp;lt;24 hours (range, &amp;lt; 24 hours to 160 hours) after collection. Flow cytometry was performed using the BD FACS Canto II flow cytometer. Monocyte subsets were identified using Kaluza software (Beckman Coulter, USA). A CD45/ side scatter gate was set to locate the monocyte population and specific antibody combinations were used to identify and exclude other lineages; these were - CD24 to exclude granulocytes and B cells, CD16 to exclude neutrophils, CD2 to exclude T cells and CD56 to exclude NK cells. Based on the CD14 and CD16 expression, the monocytes were then divided into: MO1 (CD14+/CD16-), MO2 (CD14low/CD16+) and MO3 (CD14-/CD16+). Results are reported descriptively and Fishers Exact Test (IBM® SPSS® Statistics Version 26) was used to establish if there was any correlation between the percentage of classical monocytes and the diagnosis of CMML. Results Of the 35 patients included, 13 patients had CMML and four patients were diagnosed with another underlying myeloid neoplasm: one patient with myelodysplastic syndrome (MDS-RCMD), one patient with myeloproliferative neoplasm - not otherwise specified (MPN-NOS), one patient with multiple myeloma and one patient with acute myeloid leukaemia with myelodysplasia related changes (AML-MRC). Eighteen cases had non-clonal monocytosis. Six patients had a reactive monocytosis in the setting of autoimmune disease including: granulomatosis with polyangiitis (n=2), rheumatoid arthritis (n=1), IgG4 disease (n=1), mixed connective tissue disease (n=1) and polyarticular gout (n=1). Twelve cases of reactive monocytosis were due to infective and inflammatory causes. Among our 13 patients with CMML, seven cases (53.85%) had an MO1 percentage &amp;gt; 94%. In comparison, only four (18.18%) among the 22 cases of non-CMML were identified to have an MO1 percentage &amp;gt; 94%. There was no correlation between an MO1 percentage cut off of &amp;gt; 94% and a diagnosis of CMML (p = 0.057). Our study also examined the utility of an MO3 percentage cut off of &amp;lt; 1.13%, established by Hudson et al (Am J Clin Pathol 2018). Among our 13 cases of CMML, six patients (46.15%) were noted to have an MO3 percentage &amp;lt; 1.13%. Meanwhile, only eight, out of 22 non-CMML patients (36.36%) had MO3 percentage &amp;lt; 1.13%. There was no association between MO3 percentage and the diagnosis of CMML (p = 0.724). Conclusion Using the MO1 and MO3 percentage cut offs previously established, we were unable to reliably diagnose CMML. Given these findings, we suggest that more research with larger sample sizes is required before monocyte subset analysis can be applied in the clinical laboratory to discriminate reactive monocytosis from CMML. Disclosures Mollee: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees.

Donor Selection May Predict Improved Survival Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation in Chronic Myelomonocytic Leukemia - Experience from a Tertiary Care Centre

Background - Allogeneic Hematopoietic Stem Cell transplant (HSCT) is the only potential curative treatment in patients with chronic myelomonocytic leukemia (CMML). Predictors of outcomes of Allo HSCT in CMML vary across studies and include achievement of complete remission, year of transplant, splenomegaly, performance status, prognostic scores and graft source. Inclusion of patients who progressed to AML also confounds outcome comparisons in many previous studies. The factors predictive of outcome with the use of newer GVHD prophylactic regimens including anti thymocyte globulin (ATG) and post-transplant cyclophosphamide (PTCy) are largely unknown. We report our experience of HSCT in CMML in remission and try to identify predictors of survival. Methods - We retrospectively reviewed all cases of CMML who underwent HSCT at our centre from January 2005 to June 2020. We collected data for demographic characteristics, cytogenetic and molecular characteristics of CMML, prior CMML treatment, latent period before transplantation, prognostic scores, transplant details (donor details, conditioning regimens, GVHD prophylaxis) as well as post-transplant complications (transplant related mortality, occurrence and severity of acute and chronic GVHD, CMV and EBV reactivations). Primary outcome evaluated was overall survival and secondary outcomes were relapse rate and relapse free survival (RFS). Cox-proportional hazards regression model was used to identify predictors of survival. Results - A total of 31 patients underwent allogenic HSCT for CMML during the study period. 58% (n=18) were males. Patients who had progressed to AML were not included in this study. Median age at HSCT was 61 years (range 33-71). Baseline characteristics are summarized in Table 1. Cytogenetic analysis was abnormal in 43.3% (n=13) with high risk cytogenetics (deletion 17p, complex cytogenetics) present in 12.9% (n=4) patients. The median time from diagnosis of CMML to transplantation was 12.4 months. Pre transplant performance status was ECOG Score 0-1 in 73.3% (n=22) patients and Score≥ 2 in 26.7% (n=8) patients. Donors were matched related (MRD), matched unrelated (MUD) and mismatched unrelated MMUD) in 32.3% (n=10), 54.8% (n=17), 12.9% (n=4) transplants respectively. Donor age was significantly lower in MUD when compared to related donors (Median donor age 27 vs 54.5 years; p=0.01). Conditioning regimens used were myeloablative in 25.8% (n=8) and reduced intensity in 74.2% (n=23) patients. The most common GVHD prophylactic regimens used were ATG-PTCy based in 45.2% (n=14) patients. Transplant related mortality (TRM) was 9.67% (n=3). Acute and chronic GVHD occurred in 38.7% (n=12) and 48.4% (n=15%) respectively. After a median follow-up of 12.1 months, 25.8% (n=8) patients relapsed. Estimated 1-year Relapse free survival (RFS) and overall survival (OS) were 52.4% and 59% respectively (Figure 1A). Having a 10/10 MUD was the only significant predictor for improved OS (median OS in MUD vs MRD vs MMUD = 140 vs 10 vs 4 months; p=0.014) and RFS (median RFS in MUD vs MRD vs MMUD = 140 vs 9 vs 2.3 months; p= 0.01) after Cox Regression Analysis (Figure 1B). Presence of a MUD also predicted for a lower cumulative incidence of relapse when compared to MRD or MMUD at 1-year post HSCT (6.8% vs 40% vs 25% respectively; p=0.049) (Figure 1C). GVHD prophylactic regimens containing Alemtuzumab or ATG-PTCy showed a trend towards improved RFS and OS when compared to other GVHD prophylactic regimens (median RFS 17.4 vs 10 months; p=0.19). Cytogenetic risk stratification, donor age, and CMML prognostic scores (Mayo/MDACC/CPSS) were not predictive of survival. Conclusions - Allogeneic hematopoietic stem cell transplantation remains the only curative modality for patients with CMML. Use of matched unrelated donors may improve outcomes after allogeneic HSCT in patients with CMML, primarily by reducing the cumulative incidence of relapse. Younger donor age and increased use of in vivo T cell depletion in unrelated donor transplants may have contributed to the improvement in outcome. Disclosures Lipton: Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Honoraria; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Mattsson:Gilead: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; ITB: Honoraria; Mallinkrodt: Honoraria; Jazz Pharmaceuticals: Honoraria.

Diagnostic Approach To Oligomonocytic Chronic Myelomonocytic Leukemia

Abstract Casestudy: Pathologic diagnosis of chronic myelomonocytic leukemia (CMML) is typically straightforward with the majority of patients presenting with persistent monocytosis (&amp;gt;1x109/L, &amp;gt;=10%) and bone marrow dysplasia. The diagnosis may be challenging in patients with unusual features such as lack pf peripheral blood monocytosis and non-diagnostic bone marrow morphology. In this abstract, we present a 67-year-old female with a 5-year history of anemia of unclear etiology. At the time of initial presentation, the laboratory work-up of normocytic anemia was non- contributory, the bone marrow was reported as normocellular with maturing trilineage hematopoiesis and no significant dysplasia. Over the course of the disease, the peripheral blood monocyte count fluctuated from 11% to 18% with absolute monocyte count ranging from 0.4 to 0.7x109/L. The most recent bone marrow was markedly hypercellular with increased trilineage hematopoiesis with left shift, dysgranulopoiesis and dysmegakaryopoiesis. Blasts (including promonocytes) constituted 10% of the differential count and were immunophenotypically abnormal with uniform expression of CD117, dim to negative CD13, and partial CD15. Monocytes were elevated at 12% and were strongly positive for CD64 and partially for CD14. They were negative for CD16 consistent with classical monocytes, and showed partial loss of CD13. The karyotype was normal. Molecular testing revealed TET2, RELN and SRSF2 mutations at high allelic frequencies. This case illustrates a value of flow cytometric immunophenotyping and molecular genetic studies in diagnosing challenging cases of CMML. While the patient’s absolute monocyte count remained below the diagnostic threshold of 1x109/L throughout the course of the disease, peripheral blood and bone marrow monocytes showed skewed classical immunophenotype, immunophenotypic abnormalities of myeloid series and high allelic frequency mutations. These findings should raise a differential diagnosis of oligomonocytic CMML, even when morphologic abnormalities and monocyte count threshold are not diagnostic.

Chronic myelomonocytic leukemia genomic signature correlates with the degree of bone marrow fibrosis: A single-institutional retrospective study.

7559 Background: Chronic myelomonocytic leukemia (CMML) has features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. The median overall survival (OS) in most series is 20-40 months. CMML is a relatively rare entity, and there is limited understanding of prognostic molecular markers. CMML associated with bone marrow fibrosis grade 1, appear to have shorter progression free survival. In this study we investigated the correlation of mutations with bone marrow reticulin fibrosis in patients with CMML. Methods: We investigated a cohort of 41 consecutive patients diagnosed with CMML 0, 1, 2, and CMML-AML from 2014 to 2019 at our institute. The median age of 41 patients was 68 years (range, 34-82). 27% were females and 73% were males. This cohort consists of 8 (20%) patients with CMML0, 19 (46%) with CMML1, 8 (20%) with CMML2 and 6 (15%) with CMML-AML. Genomic DNA was extracted from the bone marrow aspirates and targeted mutation NGS libraries were prepared from 200 ng of genomic DNA using the SureSelect target enrichment system (Agilent Technologies Inc.). The gene panel consists of 73 genes focused on myeloid neoplasms. The data were curated on the basis of our molecular pathology and national databases. Additionally, clinical data and bone marrow (BM) reticulin fibrosis grades (n = 27 available) were retrieved from the patient’s medical record. Results: The mutational profile frequency in our cohort showed that the most common mutations were TET2 (31%), ASXL1 (31%), and SRSF2 (23%), with frequencies very similar to those reported in the literature. Of the 27 cases with an available reticulin stain, only low grade fibrosis (MF-0, n = 18. MF-1, n = 9) were identified in our cohort. The frequencies of mutations in ASXL1, U2AF1, TP53, JAK2 and RUNX1, positively correlated with low grade fibrosis (MF-1). Additionally, patients with higher frequencies of SRSF2, TET2, and SETBP1 mutations showed no fibrosis (MF-0). Conclusions: This study is the first to correlate the degree of fibrosis with the frequency of mutations in CMML. We found similar mutations spectrum reported in the literature in patients with CMML. The mutational profile associated with CMML cases appears to affect the degree of the bone marrow fibrosis at the time of diagnosis.

How I investigate chronic myelomonocytic leukemia.

The 2016 revised 4th edition of the World Health Organization classification of hematopoietic neoplasms updated the diagnostic criteria for chronic myelomonocytic leukemia (CMML). Persistent peripheral blood monocytosis of at least 1×109 /L and a percentage of monocytes ≥10% of the circulating white blood cell count (WBC) are both prerequisite criteria for this diagnosis. CMML represents the prototype of "overlapping" myeloid neoplasms with concurrent myeloproliferative and myelodysplastic features. However, clinical presentation is heterogeneous, with cases showing prevailing "dysplastic" features and others a predominant "proliferative" phenotype. Accounting for this diversity, two variants of CMML are recognized: "dysplastic" CMML defined by WBC<13×109 /L and "proliferative" CMML with WBC≥13×109 /L often showing features mimicking a myeloproliferative neoplasm. Although not an official WHO category, the "oligomonocytic" variant of CMML is defined by relative monocytosis with an absolute monocyte count of 0.5-0.9×109 /L. It can be considered a "pre-phase," as it frequently anticipates the development of an overt, classic CMML. In an attempt at improving disease prognostication, the blast count based grading system for CMML of the WHO 2008 Classification has been expanded in 2016 to include a new "CMML-0" category. Lastly, the large body of knowledge on the molecular events occurring in CMML has been used to assist diagnosis and assess prognosis. Despite the step forwards, diagnosis of CMML still remains one of exclusion as no clinical, pathologic or molecular findings are specific for this disease. The current review brings insight into the spectrum of CMML and provides practical advice to approach suspected cases of CMML.

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.

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.

Integrated Transcriptomic and Proteomic Analyses of Inflammasome in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.