Tefferi, Ayalew

SummaryMastocytosis constitutes the neoplastic proliferation of mast cells and is broadly classified into systemic mastocytosis (SM), cutaneous mastocytosis and mast cell sarcoma. SM is further partitioned into advanced (AdvSM) and non‐advanced (SM‐non‐Adv) subcategories. AdvSM includes aggressive SM (ASM), SM with an associated haematological neoplasm (SM‐AHN) and mast cell leukaemia (MCL). In 2022, two separate expert committees representing the 5th edition of the World Health Organization (WHO5) and the International Consensus (ICC) classification systems submitted revised classification criteria for SM, highlighted by the ICC‐proposed incorporation of mast cell cytomorphology in the diagnostic criteria for MCL and myeloid‐lineage restriction for the AHN component in SM‐AHN. Recent developments in SM also include the introduction of KIT‐targeting tyrosine kinase inhibitors (KITi), including midostaurin and avapritinib, both drugs have shown potent activity in reducing mast cell and mutant KIT burden and alleviating mast cell‐associated organopathy and mediator symptoms; however, their overall impact on survival or superiority over pre‐KITi era treatment options (e.g. cladribine) has not been studied in a controlled setting. In the current review, we provide a summary of recent changes in disease classification and an analysis of recent clinical trials and their impact on our current treatment approach in AdvSM.

Background: The 2022 European LeukemiaNet (ELN) genetic-risk stratification for acute myeloid leukemia (AML), assigns favorable prognosis to t(8;21)(q22;q22.1)/ RUNX1:: RUNX1T1, inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/ CBFB:: MYH1, NPM1 mutations without FLT3-ITD or adverse karyotype, and bZIP in-frame CEBPA mutations (Döhner , Blood, 2022 ). This risk stratification was derived from intensively treated patients; accordingly, prognostication in ELN favorable genetic risk AML, following venetoclax and hypomethylating agent (VEN-HMA) therapy, remains unclear and was the subject of the current study. Methods: Under an institutional review board approved minimum risk protocol, the Mayo Clinic database was searched to identify AML patients with favorable genetic risk, who received treatment with VEN-HMA in the frontline setting. All patients received at least one cycle of either azacitidine 75 mg/m 2 days 1-7 or decitabine 20 mg/m 2 days 1-5 with VEN dose adjusted based on azole antifungal prophylaxis. Genetic risk stratification and treatment response were assessed according to the 2022 ELN criteria. Survival was estimated from the time of VEN-HMA treatment start to last follow-up or death. Results: Patient characteristics A total of 46 newly-diagnosed AML patients with ELN 2022 favorable genetic risk [ NPM1 mutation, n=36 (78%), CEPBA bZIP mutation, n=7 (15%) and t(8;21)(q22;q22.1)/ inv(16)(p13.1q22), n=4 (9%)] received a median of 4 cycles (range 1-32) of VEN-HMA. Median age was 71 years, with 63% males, and 87% de novo AML. Additional mutations frequently involved TET2 in 17/43 (40%), DNMT3A in 14/45 (31%), SRSF2 in 10/43 (23%), K/NRAS in 10/45 (22%), IDH1 in 8/46 (17%), FLT3-TKD in 7/46 (15%), ASXL1 in 6/43 (14%) and IDH2 in 6/46 (13%),of informative cases.Myelodysplasia (MDS)-related gene mutations ( ASXL1, RUNX1, BCOR, EZH2, SRSF2, U2AF1, SF3B1, STAG2, and/or ZRSR2) were present in 15 of 40 (38%) of patients (93% males). Predictors of response Of the 39 (85%) patients that achieved complete remission, with (CR) ( n=24; 52%), or without count recovery (CRi) ( n=15; 33%); CR/CRi rates were not significantly higher in patients with NPM1 mutations (89%), t(8;21)(q22;q22.1)/ inv(16)(p13.1q22) (75%), or CEPBA bZIP mutations (67%) ( p=0.37). Among patients achieving CR/CRi, measurable residual disease by multiparametric flow cytometry was negative in 15 of 16 (94%) of evaluable cases after cycles 1 or 2. In addition, NPM1 qPCR was negative in 15 of 24 (63%) informative patients. Median time to response was 1.2 months with median response duration of 9.4 months. CR/CRi rates were significantly higher in females vs males (100% vs 76%, p=0.01), and in the absence vs presence of MDS-related gene mutations (96% vs 60%, p=0.004). Multivariable analysis confirmed superior response in the absence of MDS-related gene mutations ( p=0.04). Predictors of survival At a median follow-up of 10.6 months (range; 1.1-51 months), 21 patients (46%) have died, 9 relapsed (23% of those achieving CR/CRi), and 6 (13%) underwent allogeneic hematopoietic stem cell transplant. Median overall survival was 21 months with 1/2/3-year survival rates of 65%/41%/41% (Figure 1a); median relapse-free survival (RFS) was not reached; 1/2/3-year RFS rates were 81%/66%/57% (Figure 1b). Overall survival was superior in patients achieving CR/CRi (23 months vs 5.6 months; p<0.01), in females vs males (not reached vs 9.7 months; p<0.01), and in the absence of MDS-related gene mutations (not reached vs 5.6 months; p=0.01) (Figure 1c). Subsequent multivariable analysis identified female gender ( p=0.01), and absence of MDS-related mutations ( p=0.17) as independent predictors of superior survival. Similar results were obtained when analysis was restricted to NPM1 mutated patients. Conclusions: In the current retrospective study, VEN-HMA therapy in elderly/unfit AML patients with ELN 2022 favorable genetic risk, resulted in response rates that were comparable to those previously appreciated from published reports of patients receiving intensive induction therapy; however, follow-up in the current study was too short to accurately assess long-term overall and relapse-free survival impact. Our study also highlights the adverse prognosis retained by MDS-related gene mutations.

Background: Non-transplant treatment options in myelofibrosis MF are palliative in scope and include Janus kinase 2 (JAK2) inhibitors (JAKi). FDA-approved JAKi for the treatment of MF include ruxolitinib, fedratinib, and pacritinib. A fourth JAKi, momelotinib, is currently under review. We have previously reported long-term survival data on momelotinib and drug survival that was significantly longer, retrospectively compared to ruxolitinib ( AJH. 2022;97:E433). In the current study, we focused on molecular predictors of drug survival (i.e., drug retention) and longevity without allogeneic hematopoietic stem cell transplant AHSCT (transplant-censored survival). Methods : The current study included data from Mayo Clinic patients who participated in a phase-1/2 core study of momelotinib (NCT00935987). Study design, drug doses and schedule, and additional details on treatment effect and side effects have previously been published. Drug survival was calculated from time of initiation of momelotinib to time of treatment discontinuation. Survival analysis required censoring of patients at time of AHSCT. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results : 79 JAKi-naïve patients with MF were considered: median age 67 years; males 54%; PMF 65%, post-PV MF 20%, post-ET MF 15%; JAK2 71%, type 1/like CALR ( CALR-1) 14%, CALR-2 3%, MPL 8%, triple-negative 5%. Risk category according to DIPSS was high/intermediate in 80% of the patients and unfavorable karyotype in 29%. Non-driver mutation information was available in a subset of patients and included ASXL1 22% and SRSF2 18%. Median time from diagnosis to study entry was 25.2 months; patients were enrolled between November 2009 and March 2011 with follow-up information updated through July 2023; during this period, 68 (86%) deaths, 13 (16%) leukemic transformations, and 7 (9%) AHSCTs were recorded. Median follow-up for alive patients was 12.5 years (range 11.3-13.1). Anemia response rate was 48% and spleen 47%. Median treatment duration was 25 months (range 0.1-157); the proportion of patients remaining on active therapy was 68% at 1 year, 30% at 3 years, 16% at 5 years, and 7% at 10 years (Figure 1a). At the time of this writing, 5 (6.3%) patients were still receiving momelotinib therapy with treatment durations of 152 to 157 months. In univariate analysis, molecular predictors of drug survival included CALR-1 (p=0.006) but not ASXL1 (p=0.6) or SRSF2 (p=0.3) mutation, or unfavorable karyotype (p=0.08). The 3-, 5-, and 10-year drug survival rates for CALR-1-mutated patients were 64%, 40%, and 18%, respectively, and for CALR-1 wild-type 25%, 11%, and 4% (p=0.01, Figure 1b). CALR-1 independent association with longer drug survival was also appreciated for younger age (p<0.01) and absence of baseline transfusion dependency at time of study entry (p=0.02); all three risk factors remained significant during multivariable analysis with HR (95% CI) rates of 0.4 (0.2-0.87) for CALR-1, 0.5 (0.3-0.89) for age <63 years, and 0.6 (0.3-0.9) for absence of transfusion-dependency. Spleen response (p<0.01) but not anemia response (p=0.23) was also associated with longer drug survival; in multivariable analysis all 4 risk factors retained significance. Multivariable analysis of genetic variables for transplant-censored survival identified absence of CALR-1 (HR 3.5, 95% CI 1.2-10.5; p=0.03) and presence of high molecular risk (HMR; ASXL1/ SRSF2) mutations (HR 2.3, 95% CI 1.1-4.6; p=02) to independently predict inferior survival. Among 11 patients with CALR-1 mutation, median transplant-censored survival was 125 months with 10-year survival rate of 60% (Figure 1c); by contrast, the corresponding figures were 37 months and 12%, in the 68 patients not harboring CALR-1 mutation (Figure 1c). In multivariable analysis, absence of CALR-1 (HR 2.9, 95% CI 1.02-8.1), age ≥63 years (HR 3.0, 95% CI 1.5-6.0), and absence of spleen response (HR 2.6, 95% CI 1.5-4.5) were identified as being most significant in predicting inferior transplant-censored survival. Conclusions : CALR-1 mutation is the most important risk factor for both drug survival and longevity without AHSCT, in momelotinib-treated patients with MF. Such information might help identify MF patients who might benefit from treatment with momelotinib and in whom AHSCT might be deferred or not.

Background Venetoclax (Ven) in combination with hypomethylating agent (HMA) is the standard treatment for newly diagnosed elderly/unfit patients with acute myeloid leukemia (AML). Treatment options for patients with relapsed/refractory (RR) disease following Ven-HMA are limited in the absence of targetable mutations. In a recent phase 2 study, the addition of Ven to cladribine (CDA) plus low-dose cytarabine (LDAC), in newly-diagnosed elderly/unfit AML, resulted in complete remission with (CR) or without (CRi) count recovery rate of 93% ( J Clin Oncol 20;40(33)). In the current study, we share our experience with this regimen in patients relapsed or refractory to Ven-HMA, treated outside of clinical trials. Methods Under an institutional review board approved minimum risk protocol, the Mayo Clinic (MN, AZ, FL) database was searched to identify patients with AML who failed treatment with Ven-HMA and subsequently received at least one cycle of salvage therapy with Ven-CDA-LDAC: Ven 100-400 mg, dose adjusted, d1-21, CDA 5 mg/m 2 d1-5, LDAC 20 mg twice daily d1-10, outside of clinical trials. Cytogenetic and molecular studies were performed at the time of Ven-CDA-LDAC initiation in most cases, by conventional karyotype and next-generation sequencing, respectively. The 2022 European LeukemiaNet (ELN) criteria were applied to define risk and response. Survival was calculated from the time of Ven-CDA-LDAC treatment start to last follow-up or death. Results Patient characteristics A total of 39 RR AML patients (median age 65 years, 67% male, 64% de novo), with prior exposure to Ven-HMA, received Ven-CDA-LDAC (median 1 cycle; range 1-5). Study patients had received one ( n=16), two ( n=14), three ( n=5), four ( n=2), five ( n=1) or nine ( n=1) prior therapies, including Ven-HMA. Nine (23%) patients had relapsed following allogeneic hematopoietic stem cell transplant (AHSCT). ELN cytogenetic risk included intermediate (46%, n=18), and adverse (54%, n=21). Mutations involved TP53 in 9 patients (23%), K/ NRAS in 8 (21%), RUNX1 in 7 (18%), TET2 in 6 (15%), ASXL1 in 5 (13%), and STAG2 in 5 (13%). Response and toxicity Eleven (28%) patients achieved CR ( n=2; 5%) or CRi ( n=9; 23%); median time to response was 1 month and median response duration 3.4 months. In addition, 2 (5%) patients each experienced partial remission (PR) and bone marrow aplasia without fulfilling criteria for morphologic leukemia free state. Measurable residual disease (MRD) was negative by multiparametric flow cytometry in 2 of 7 (29%) informative CR/CRi cases. CR/CRi rates were higher in females (54% vs 15%; p=0.01), de novo vssecondary AML (40% vs 7%; p=0.02), and absence of adverse karyotype (50% vs 9%; p<0.01), K/NRAS (35% vs 0%, p=0.01), ASXL1 (32% vs 0%, p=0.05), or STAG2 mutations (32% vs 0%, p=0.05) (Table 1). Multivariable analysis confirmed superior response in de novo AML (p<0.01), absence of adverse karyotype, (p<0.01), and absence of K/NRAS mutations (p=0.02). CR/CRi rates were not significantly affected by relapsed vs refractory setting ( p=0.95), number of prior therapies ( p=0.51), prior AHSCT ( p=0.64), or TP53 mutations ( p=0.64) (Table 1). Treatment-emergent toxicities included infections (49%, n=19), comprising bacteremia ( n=11), pneumonia ( n=10), peri-rectal abscess ( n=6), and laboratory evidence of tumor lysis syndrome (3%, n=1). Survival At a median follow-up of 3.9 months, from initiation of Ven-CDA-LDAC, 31 deaths (79%), 6 relapses (15%), and 7 AHSCT (18%; including 5 in CR/CRi, 1 in aplasia) were documented. Median survival following Ven-CDA-LDAC was 4.6 months, and superior in the presence vs absence of CR/CRi (8.1 vs 2.9 months; p=0.01), and in patients receiving AHSCT (10.5 vs 3.4 months; p=0.02). Absence of adverse karyotype was also associated with superior survival (6 vs 3.4 months; p=0.05). Multivariable analysis confirmed the favorable survival impact of CR/CRi ( p=0.03) and AHSCT ( p=0.05); survival impact was not apparent for secondary AML ( p=0.29) or K/NRAS ( p=0.70) or TP53 ( p=0.10) mutations. Conclusions The current study suggests transplant bridging value for Ven-CDA-LDAC in AML patients relapsed or refractory to Ven-HMA, especially in the absence of adverse karyotype or K/NRAS mutation. AHSCT and achievement of CR/CRi appear to be indispensable for favorable post-Ven-CDA-LDAC survival.

INTRODUCTION: The Stromal Antigen 2 ( STAG2), on Xq25, is the most common cohesin complex gene mutated in myeloid neoplasms (MN) (5-10%). STAG2 mutations ( STAG2m) were incorporated in the ELN2022 as AML-MDS defining genetic mutations inferring poor prognosis and is a genetic marker in IPSS-M risk stratification. The PHD-finger protein 6 ( PHF6) gene, on Xq26.3, is a tumor suppressor gene, mutated in 3% of MN, and is more common in T-ALL. PHF6m is believed to be a late event in MN with little data on its prognostic implications in MN. Both STAG2m and PHF6m are more prevalent in males, but their implications on the clinical presentation of MN have not been compared prior. METHOD: Our study is a multicenter study at Mayo Clinic (Rochester, Florida, Arizona). After IRB approval, we retrospectively analyzed charts of 7935 patients who had NGS between 2016-2022. STAG2m was found in 96 patients, and PHF6m in 116 (including 3 with concurrent STAG2m and PHF6m). We excluded 4 PHF6m patients who had T-ALL. Data was captured at time of NGS. MDS risk stratification was by IPSS-M and AML by ELN2022. Overall survival (OS) was calculated from date of NGS to date of last follow-up. BlueSky Software was used for statistical analysis. RESULTS: Characteristics: We compared 96 STAG2m vs 112 PHF6m MN patients. Of STAG2m cases, 78% were male vs 76% in PHF6m MN. Median age was 72 vs 73 years, respectively. Diagnoses included AML (29% of STAG2m vs 34% of PHF6m MN), MDS (54% vs 28%), MDS/MPN (10% vs 21%) and MPN (3% vs 9%). MDS was more common among STAG2m MN (p<.001) while MDS/MPN was more common among PHF6m MN (=.05). MDS-IB was the most common subtype in both (69% vs 52% in STAG2m vs PHF6m MDS). MDS-LB and 5q subtypes were only found in PHF6m MDS, and there was more PHF6m MDS-RS (13% vs 2%). Median IPSS-M score was 0.9 vs 0.8 in STAG2m vs PHF6m MDS (both high-risk). Majority of STAG2m AML were adverse risk (96%) while PHF6m AML were intermediate (79%) (p<.001). AML cases included 43% vs 30% secondary AML (sAML) in STAG2m vs PHF6m AML. Among STAG2m and PHF6m MN, 17% of each were tMN. Cytogenetic abnormalities were more common in PHF6m MN (50% vs 31%, p=.005). The median bone marrow (BM) blasts count among STAG2m MN was 7% vs 4% in PHF6m MN (p=.03). Genetic characteristics: STAG2m were nonsense, frameshift and splice site (54%, 36% and 11%). PHF6m were nonsense, frameshift, splice site, missense and start loss variant mutations (44%, 33%, 12%, 10% and 1%). The median variant allele frequency (VAF) of STAG2m MN was 50% compared to 32% in PHF6m MN (p=.008). Co-mutations: The median number of co-mutations was 3 in STAG2m and 4 in PHF6m MN (p=.04). The most common were TET2m (37% in STAG2m MN vs 45% in PHF6m MN), RUNX1m (31% in both), SRSF2m (39% vs 20%), ASXL1m (66% vs 38%), BCORm (19% vs 6%), U2AF1m (13% vs 14%) and IDH2m (18% vs 7%). ASXL1m, SRSF2m, BCORm, IDH2m and SF3B1m were more common among STAG2m MN (p=<.001, .003, .006, .02, .09). JAK2m, IDH1m are more common among PHF6m MN (p=.02, .06). Survival: Of STAG2m and PHF6m MN, 70% and 72% of patients received treatment. HSCT was performed in 23 (24%) and 23 (21%) of STAG2m and PHF6m patients, including AML (n=9, n=10), MDS (n=12, n=8), MDS/MPN (n=2, n=2). OS among STAG2m and PHF6m MN was 20.5 and 22.1 months, respectively. OS among AML cases was 13.5 vs 21 months (p=.6) in STAG2m vs PHF6m, was NR vs 22 months (p=.4) in MDS, 11.7 vs 17.6 months in MDS/MPN (p=.4), and 10.4 vs 27.4 months (p=.5) in MPN. There was no difference between males and females in either STAG2m or PHF6m MN. HSCT led to longer OS among both STAG2m (NR vs 19.9 months, p=.01) and PHF6m MN patients (NR vs 17.6 months, p=.07). Among AML patients, HSCT did not significantly impact OS (18 vs 9.6 months, p=.3 and 25.1 vs 21 months, p=.2, in STAG2m and PHF6m). HSCT improved OS in STAG2m MDS (NR vs 20.5 months, p=.03). In PHF6m MDS, OS was 22.1 vs 13.1 months in HSCT vs non-HSCT (p=.3). CONCLUSION: STAG2m and PHF6m are 2 X-linked mutations that occur in MN. Both commonly occur in elderly males. MDS was the most common diagnosis in STAG2m MN. STAG2m co-occurred particularly with SRSF2m ASXL1m and BCORm, which are associated with poor prognosis. In our cohort, STAG2m AML carried a poorer prognosis than PHF6m AML. Our study confirms the ELN2022 AML risk stratification. MDS risk classification and OS did not differ between STAG2m and PHF6m in our cohort. A larger cohort is required to confirm our findings and may provide stronger validation.

Introduction The advent of venetoclax (VEN) based chemotherapy has revolutionized the treatment of elderly acute myeloid leukemia (AML), with improvement in remission rates from historical 10%-20% to 60%-70%. However, remission duration is short-lived in patients (pts) with high-risk AML with consequent dismal outcome. As our knowledge of genomic landscape of AML expands and more immunotherapies become available, exploration of the immune tumor microenvironment (TME) of AML may allow identification of subsets of pts that may benefit from immune based therapy. We conducted an investigator initiated prospective study to evaluate TME of pts receiving VEN plus hypomethylating agent (HMA; azacitidine or decitabine) combination. We hypothesize that these pts will cluster into immune-infiltrated vs. immune-depleted sub-types by high-definition immune cell profiling. Methods In 26 newly diagnosed AML pts, who were planned to receive VEN plus HMA therapy, we isolated bone marrow (BM) and peripheral blood (PB) derived mononuclear cells (MC) by density gradient centrifugation and cryopreserved. We performed CyTOF (cytometry time-of-flight) analysis to study their immune signatures. Thawed cells were labeled with metal-conjugated antibodies that bind CD33, CD196, CD19, CD127, CD11b, CD4, IgD, CD11c, CD14, CD56, CD123, TCRgd, CD185/CXCR5, CD45RA, CD27, CD28, CD66b, CD183 (CXCR3), CD161, CD45RO, CD197 (CCR7), CD8a, CD25, CD3, CD20, CD38, HLA-DR, CD194 (CCR4), CD57, CD16 and CD45. We performed a Rphenograph clustering on 25 paired BMMC and PBMC samples (one patient only had PBMC) to create 35 unique clusters for each ( Figure 1). Then we broke down the distribution of cluster by two different groupings: pts with TP53 mutation (m) and those who achieve complete remission. Statistical significance of clusters calculated using t-test. We performed Pearson correlation test on the paired BMMC and PBMC samples to assess correlation between BMMC and PBMC samples. Results Baseline characteristics The median age of the pts was 74 years (range, 53-88), and 17 (65%) pts were male. Ten (37%) pts had secondary AML, and 12 (46%) pts had ELN adverse risk disease (n=6; TP53m). There was no significant difference in median age (p= >0.99), proportion of pts with secondary AML (p= 0.64), acute myelomonocytic leukemia (p= 0.14) white blood cell count (WBC) ≥ 100 (10 9/L) (p= >0.99), and frequency of commonly occurring co-mutations ( ASXL1 [p=0.35]), FLT3 ITD [p=0.54], splicing [p=0.29], IDH1/2 [p=0.28], DNMT3A [p=0.29], NPM1 [p=0.29], TET2 [p=0.35]) in TP53m and TP53 wild-type group. Response 12/20 (60%) evaluable pts achieved complete remission with or without count recovery (CR/CRi). There was no significant difference in median age (p= 0.06), proportion of pts with secondary AML (p= 0.16), therapy related AML (p= 0.60) WBC ≥ 100 (10 9/L) (p= 0.40), and frequency of commonly occurring mutations ( TP53 [p=0.60], ASXL1 [p > 0.99]), FLT3 ITD [p=0.54], splicing [p > 0.99], IDH1/2 [p=0.60], DNMT3A [p=>0.99], NPM1 [p=0.61], TET2 [p >0.99]) in CR/CRi and no-CR/CRi group. CyTOF analysis and Rphenograph clustering on 25 paired BMMC and PBMC samples We found significant clustering with high expression of B-cell activated (cluster 2; p= 0.005), NK cell rich (cluster 1; p= 0.005, cluster 35; p= 0.0009), CD8 T-cells (cluster 31; p= 0.01) and CD4 effector cells (cluster 17; p= 0.005) phenotype among TP53m AML ( Table 1). Among pts who achieved CR/CRi, significant clustering with low expression of B-cell activated (cluster 29; p= 0.01), CD8 cytotoxic (cluster 14; p= 0.01) CD4 activated (cluster 21; p= 0.03) phenotypes observed. Correlation between BMMC and PBMC clustering Among clusters which showed significance for a particular phenotype, we performed Pearson correlation (R 2) and observed strong correlation between BMMC and PBMC samples clustering in cluster 1 (R 2 = 0.99), cluster 2 (R 2 = 0.95), cluster 17 (R 2 = 0.96), cluster 21 (R 2 = 0.99) and cluster 29 (R 2 = 0.93). Conclusion In this single institutional study using CyTOF analysis we have identified novel clustering with immune- infiltrated phenotype among pts with TP53m AML and conversely immune-depleted phenotype among AML pts who responded to VEN plus HMA. Moreover, we found strong correlation in CyTOF analysis conducted from BMMC and PBMC samples. Future studies with larger sample size needed to validate our findings.

Introduction Chronic myelomonocytic leukemia (CMML) can be classified into dysplastic (dCMML) and proliferative (pCMML) subtypes based on a white blood cell count (WBC) ≥ 13 x 10 9/L for pCMML. Though this cutoff may seem arbitrary, there is evidence that pCMML is a unique biological entity with a distinct molecular profile and worse outcomes (Carr RM, et al. Nat Commun. 2021). We hypothesized that contemporary CMML prognostic scoring systems - such as the Mayo Molecular Model (MMM; Patnaik MM, et al. Leukemia. 2014) and the CPSS-Molecular Model (CMM; Elena C, et al. Blood. 2016) - would not adequately risk-stratify pCMML patients. Moreover, while truncating ASXL1 mutations are considered a high-risk feature in CMML, their prognostic relevance in pCMML is unknown. Methods After IRB approval, we compiled a large (n = 888), molecularly-annotated database of CMML patients seen at two US medical centers (Mayo Clinic and MD Anderson) via retrospective chart review. All statistical analyses considered the clinical and laboratory parameters obtained at the time of presentation. The cohort was divided into dCMML and pCMML subgroups, which were analyzed independently alongside the entire cohort. Categorical variables were compared by Fisher exact or Pearson χ 2 tests and continuous variables by Mann-Whitney U tests. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. Survival was assessed via the Kaplan-Meier method. P-values < 0.05 were considered significant. Calculations were performed using the BlueSky Statistics (v10.3.1) interface for R. Results We identified 474 (54%) dCMML cases and 410 (46%) pCMML cases with median age 71 years and 67% males. Patients with pCMML were more likely to have higher AMC (5.1 vs 1.5 x 10 9/L, p < 0.0001), circulating immature myeloid cells (IMC; 65% vs 36%, p < 0.0001) and abnormal karyotypes (37% vs 30%, p = 0.0187). They were also overrepresented in higher-risk categories of the MMM and CMM compared to dCMML (p < 0.0001 for each model). The number of somatic mutations was higher in pCMML than dCMML (mean 3.0 vs 2.6, p = 0.0050). Mutations in ASXL1 (53% vs 39%), NRAS (21% vs 11%), CBL (18% vs 12%), SETBP1 (14% vs 7%), JAK2 (10% vs 3%), CEBPA (5% vs 2%), GATA2 (3% vs 0%), and FLT3 (3% vs 1%) were more frequent while mutations in TET2 (38% vs 48%), SF3B1 (2% vs 8%), ZRSR2 (2% vs 8%), and NPM1 (1% vs 3%) were less frequent in pCMML compared to dCMML, respectively (p < 0.05 for all). Though the MMM and CMM effectively stratified the dCMML group, they did not provide meaningful risk stratification in the pCMML group (MMM p < 0.05 overall but could not distinguish between low and intermediate-1/2 or intermediate-1 and high risks; CMM p > 0.05). Likewise, there was no difference in the median overall survival (OS) or leukemia free survival (LFS) when pCMML patients were stratified by truncating ASXL1 mutations( Figure 1A). We then sought to better risk stratify pCMML. Univariate analysis identified increasing age, male gender, abnormal karyotype, and elevated WBC, ANC, and AMC as adverse predictors for OS. In addition, the presence of IMC and peripheral blood (PB) blast % were adverse factors for LFS, while age was not significant for LFS. None of the aforementioned mutations were significant in the univariate model for OS, whereas GATA2 and NPM1 mutations were significant for LFS. In multivariate analysis for OS, only male gender and AMC ≥ 10 x 10 9/L retained significance. In multivariate analysis for LFS, male gender, AMC ≥ 10 x 10 9/L, and peripheral blasts ≥ 5% remained significant; GATA2 and NPM1 mutations were not significant. These variables risk-stratified pCMML patients based on the presence of 0, 1, or ≥ 2 risk factors ( Figure 1B). In comparison, OS was 40.7 months (95% CI 35.2 - 50.1 mo.) for dCMML patients, 20.1 months (95% CI 18.2 - 22.7 mo.) for pCMML patients, and 31.8 months (95% CI 28.4 - 33.5 mo.) for the entire cohort. At last follow up, 586 deaths (66%) and 168 AML transformations (19%) were recorded in the cohort. Deaths in dCMML vs pCMML were 294 (62%) vs 291 (71%; p = 0.0067), with AML transformations in 83 (18%) vs 84 (21%; p = 0.2055) patients, respectively. Conclusions Truncating ASXL1 mutations, along with ASXL1-mutation weighted CMML prognostic models failed to effectively risk stratify pCMML patients. In pCMML, male gender and AMC ≥ 10 x 10 9/L, effectively risk stratified for OS, while these factors along with PB blasts ≥ 5% effectively risk stratified for LFS.

BACKGROUND: Recent World Health Organization (WHO) classifications (5 th edition), International Consensus Classification (ICC) recognized TP53-mutated ( TP53mut) myeloid neoplasms (MNs) as a distinct subcategory, acknowledging their uniformly poor outcomes. However, there are critical differences between the two guidelines in how TP53 allelic status is assigned. Both WHO-5 and ICC acknowledged the poor outcome of multi-hit TP53mut but excluded single-hit TP53mut. These changes are driven by a pivotal study demonstrating favourable outcome of single-hit TP53mut, however this study predominantly included de novo myelodysplastic syndromes (MDS) cases (Bernard et al. Nat Med 2020). In contrast, we previously reported poor outcome of TP53mut therapy-related myeloid neoplasm (t-MN) irrespective of the allelic status of TP53 (Hiwase et al Blood 2022andShah et al BCJ 2023). Finally, the ICC removed the subcategory of “therapy-related,” substituting it with diagnostic qualifiers instead. The WHO has grouped t-MN with secondary MN and renamed it as “myeloid neoplasm post cytotoxic therapy,” with the assertion that only multi-hit T P53mut MDS occurring post-cytotoxic therapy have poor outcome compared with single-hit. METHODS: We analyzed one of the largest international cohorts of TP53mut MN (n=544) with full characterization of TP53mut allele status utilizing uniform ICC criteria and similar large numbers of de novo and t-MN cases managed at Mayo Clinic Enterprise (USA) and South Australian MDS Registry, Adelaide (Australia) with clinical, integrated genomic profiling, and long-term follow-up. RESULTS: In our cohort of TP53mut MN (n=544), 475 (87.3%) MN had TP53mut variant allele frequency (VAF) of ≥10% and thus satisfied ICC criteria for TP53mut MN. Majority of TP53mut t-MN presented with MDS (bone marrow (BM) blasts 0-9%) compared de novo MN (62% vs. 48%; P=0.0012). While acute myeloid leukemia (AML) (BM blasts ≥20%) were more prevalent in TP53mutde novo MN compared to t-MN (38% vs. 24%; P= 0.001) and there was no difference in frequency of MDS/AML (BM blasts 10-19%) between the two cohorts (14% vs. 14%; P=0.8) ( Figure 1A). Majority of TP53mut were observed in the DNA-binding domain in t-MN and de novo MN (95% vs. 96.2%; P=0.48). However, mutations in Tetramer Domain were more frequent in t-MN (4.3% vs. 0.3%; P=0.002), whereas those in the TAD2 domain were more frequent in de novo TP53mut MN (2.4 vs. 0%; P =0.006). Integrating the mutation, conventional G-banding cytogenetic, copy number variation (CNV), FISH, and SNP array analysis, TP53mut MDS (BM blast 0-9%; n=260) were classified as multi-hit (n=179, 68.9%) or multi-equivalent (n=35, 13.5%) and single-hit (n=46, 17.7%) (Arber et al. Blood 2022). Striking enrichment of multi-hit/multi-hit equivalent was observed in TP53mut t-MDS compared to de novo MDS (91% vs. 72%; P<0.0001). While single-hit TP53mut were prevalent in de novo MDS compared to t-MDS (28% vs. 9%; P<0.0001). Importantly, median overall survival (OS) of single-hit was significantly longer than multi-hit de novo MDS (25.4 vs. 7.2 months; P=0.0002). In contrast to this we did not observe OS difference between single-hit and multi-hit TP53mut t-MDS (11.6 vs. 10.9 months; P =0.73) ( Figure 1B). CONCLUSION: Our study based on a large international cohort of T53mut MN provides compelling evidence that in contrast to de novo MDS, single hit TP53mut t-MDS is associated with poor outcome. Our data supports the consideration of single hit t-MDS as a multi-hit equivalent.

Background: In addition to the driver mutations JAK2, CALR and MPL, patients (pts) with primary myelofibrosis (PMF) may also express mutations in a number of genes frequently associated with myeloid neoplasms. These genes encode regulators of chromatin modification and DNA methylation, RNA splicing and transcription, as well as oncogenes and signal transduction. In pts with PMF, including those with prefibrotic (pre-)-PMF, selected mutations were shown to deserve prognostic relevance, and led to the development of molecular integrated, or exclusive, scores (such as MIPSS70, MIPSS70-plus, GIPSS and others). Aim: The aim of the study was to correlate the type of mutations, clustered in relation to their known functional classes, with hematological and clinical correlates, and outcome in a large population of molecularly annotated pts with PMF. Patients and Methods: All pts with a confirmed ICC 2022 diagnosis of PMF from the database at CRIMM (Florence), for whom mutation analysis of a panel of 40 myeloid neoplasm-associated genes performed by NGS (Oncomine, ThermoFisher) was available, were included in the study. Pts were stratified in 6 categories based on known function of mutated gene: spliceosome (cat. 1: SF3B1, U2AF1, SRSF2, ZRSR2), DNA methylation gene (cat. 2: TET2, DNMT3A, IDH1, IDH2, SETBP1), histone modification gene (cat. 3: ASXL1, EZH2), RAS pathway (cat. 4: NRAS, KRAS, CBL, NF1, PTPN11), transcription factor (cat. 5: RUNX1, NFE2, TP53) and “other” gene mutations (cat. 6: LNK/SH2B3). Results: A total of 286 pts with at least 1 myeloid neoplasm-associated gene mutation were included (110 prePMF, 38.5%; 176 overt PMF, 61.5%). Of these, 69 have mutations in spliceosome genes, 81 in DNA methylation genes, 95 in histone modification genes, 19 in the RAS pathway, 13 in transcription factor genes and 9 in “other” gene category. Analysis of clinical characteristics (Table) showed that Cat. 1 pts were significantly older (80.6% aged >60y, P=0.003), no difference regarding gender. There was no difference in blood counts but circulating blasts >1%, that were higher in cat 3 and 1 compared to the others (P=0.009). A G3 fibrosis was more common in cat 3 and 5 (43.5% and 50%, p<0.001). No significant difference was noticed as regarded driver mutations and karyotype abnormalities. Splenomegaly was more common in cat 3 and cat 6 (86.5% and 88.9%, respectively, p=0.015), while constitutional symptoms were enriched in cat 5 (69.2%, p=0.015). The proportion of pts with transfusion dependency was lower among those with mutations in DNA methylation genes (cat. 2) and “other” mutations (cat. 6) (21% and 22.2%, respectively, p=0.014). There was no significant difference among gene categories as regarded IPSS score, MIPSS70 score, thrombosis and bleeding events, progression to overt MF (in case of pre-PMF), transformation to AML and rate of secondary neoplasms. The median overall survival (OS) for all pts was 7.1 years (IC: 5.7-8.5), with categories 2, 4 and 5 showing the longest survival (10.7y (6.3-15.1), 11.5y (4.4-18.7), and 10.5y (2-18.9) respectively). In particular, pts in cat 2 showed significantly longer OS than pts in cat 1 (5.4y; HR 1.7, 95% CI, 1.1-2.6; p=0.021) and cat 3 (10.7 vs 6.1y; HR 1.5, 95% CI, 1-2.3). Considering that cat 1, 3, and 6 on one side, and cat 2, 4, 5 on the other, did not differ significant each to the other, we combined those categories to assess respective impact on survival. We found a statistically significant difference between the 2 groups, with pts harboring mutations in spliceosome, histone modifiers and “other” genes having significantly shortened survival (5.8y, CI 4.1-7.5; HR 1.57, CI 1.13-2.18) compared to the other categories (median survival, 10.8y, CI 7.2-14.3) (P=.007). Conclusion: Current analysis revealed distinctive clinical characteristics and survival outcomes associated with specific categories of mutated myeloid genes in pts with PMF, largely overlapping with the ICC defined “myelodysplasia-related gene mutations” category.

Progression of myeloproliferative neoplasms to blast phase (BPMPN) is associated with lack of response to conventional therapies and dire clinical outcomes. Consequently, there is a major unmet need to develop new therapies for BPMPN. Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a key contributor to somatic variation in cancer, but this phenomenon has not yet been described in BPMPN. More broadly, whether chromothripsis might result in actionable molecular events that are amenable to targeting remains an open question. To characterise the contribution of structural variants to BPMPN, we first performed integrated copy number and mutation profiling in 64 BPMPN patients by SNP karyotyping and targeted next generation sequencing. We observed a recurrent pattern of chromothripsis that involved chromosome 21, which together with other structural variants led to amplification of a common region of chromosome 21 (‘chr21amp‘) in ~25% of patients (GISTIC q-val<0.01, Fig 1A). Chr21amp was associated with TP53 mutations and a higher number of copy number alterations. Patients with chr21amp had a particularly aggressive and treatment-resistant phenotype, with 0% surviving 12 months compared to 46% in the non-chr21amp pts ( p=0.0007), retaining significance in multivariate analysis including after correction for TP53 mutation status. Whole genome sequencing confirmed that the chromosomal rearrangements resulting in chr21amp occurred by different mechanisms, ranging from simple amplification to highly complex chromothriptic events involving multiple chromosomes. There were no recurrent translocation partners or mutations. The minimally amplified region (MAR) spanned 2.7Mb and contained 24 genes, with a median copy number of 3.5 (range 2.7-8.3) Single-cell transcriptomics combined with allelic resolution genotyping revealed that chr21amp was present in the dominant subclone and occurred subsequent to JAK2V617F and mut TP53 acquisition. Chr21amp was detectable in phenotypic HSCs and throughout early stages of hematopoiesis, but not in mature erythroid cells, consistent with a differentiation block. Of the 24 genes in the minimally amplified region, only one gene, DYRK1A, a serine threonine kinase linked to cell proliferation and survival, was both differentially expressed (single-cell and bulk RNAseq) and differentially accessible (ATACseq). To explore the functional role of DYRK1A in BPMPN we performed shRNA and CRISPR-mediated DYRK1A-knockdown and knockout (KO) in BPMPN cell lines (HEL and SET-2), which led to impaired cell proliferation. The DYRK1A inhibitors EHT1610 and GNF2133 also led to dose-dependent growth inhibition. DYRK1A-KO BPMPN HEL or SET2 cell clones showed a reduced ability to propagate leukemia in vivo with a significant survival advantage vs. wild type control mice. BPMPN chr21amp+ primary patient CD34+ cells were highly sensitive toDYRK1A inhibitors, while healthy control CD34+ cells were unaffected Prior studies have shown that DYRK1A activates the DREAM complex, a transcriptional repressor of DNA-repair pathways. In chr21amp patient cells, the DREAM DNA repair gene signature was significantly downregulated (NES -1.74, q-val <0.001), while conversely in CRISPR DYRK1A KO SET2 cells the transcriptional DNA repair signature was upregulated (NES 1.76, q-val <0.001). In functional assays, CRISPR DYRK1A KO was protective against DNA damage, with a reduction in γ-H2AX foci after etoposide treatment or irradiation ( p<0.01 for both). A second mechanism of leukemogenesis emerged from geneset enrichment analyses, which suggested enhanced JAK-STAT signaling in chr21amp BPMPN cells and downregulation in the CRISPR DYRK1A KO context. We validated this by showing that DYRK1A overexpression activates and potentiates STAT5B transcriptional activity in a luciferase reporter assay. Finally, we noted that the STAT target BCL2 was selectively upregulated in chr21amp cells. BCL2 inhibition showed strong synergy with DYRK1A inhibitors for induction of BPMPN cell apoptosis (Bliss synergy score 15). Collectively, these findings define the chr21amp event as a novel prognostic biomarker in BPMPN. We pinpoint DYRK1A amplification as a central driver of genomic instability and exacerbated JAK-STAT signalling, for the first time linking chromothripsis to a specific druggable target ( Fig 1B).