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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).

Introduction HMAs are the only approved therapy for CMML. In order to improve response rates and survival, the addition of VEN has been utilized in both CMML and after blast transformation (CMML-BT) with conflicting results in small single center studies. To clarify the role of upfront HMA+VEN in CMML and CMML-BT, we performed a large multicenter analysis. Methods We included patients with HMA-naïve CMML and CMML-BT treated with combination HMA and VEN from 4 academic centers. The primary outcome was overall survival (OS) with secondary outcomes including overall response rate (ORR) by 2015 MDS/MPN IWG criteria for CMML and 2022 ELN criteria for CMML-BT. To better appreciate the benefit of adding VEN to HMA treatment, we also performed 1:1 propensity score matched (PSM) analysis of patients treated with HMA alone as upfront therapy for CMML or CMML-BT. Matching was based on age, sex, ECOG performance status, CPSS-Mol, blood counts, peripheral and bone marrow blasts. Results Eighty-nine patients were treated with frontline HMA+VEN, 51 CMML and 38 CMML-BT (5 had received HMA alone for prior CMML). The median age was 71 years, 70% were male, and 83% had ECOG 0-1. Among CMML patients, 55% had MP-CMML subtype, 65% had CMML-2 and 12%, 30%, 58% were CPSS-mol intermediate-1, intermediate-2 and high risk, respectively. In the entire cohort, ASXL1 (63%), TET2 (47%), SRSF2 (39%) and RUNX1 (27%) were the most commonly mutated genes and 55% of patients harbored a RAS mutation ( KRAS [20%], NRAS [18%], CBL [13%], NF1 [11%], PTPN11 [9%]). For HMA combination partner, 46 (52%) were treated with azacitidine, 35 (39%) with decitabine, and 8 (9%) with both during their VEN course. During a median follow-up time of 15.8 months (mos) 52 patients (58%) died: 23 (45%) with CMML and 29 (76%) with CMML-BT. Among CMML patients, the ORR was 90%, including complete remission (CR) in 22%, complete cytogenic remission (CCR) in 4%, optimal marrow response (CMR) in 50%, and partial marrow response (PMR) in 6%. The OS of CMML patients was 25.1 mo (95% CI 15.7 - NR). A PSM population of HMA patients matched to HMA+VEN treated patients showing similar baseline characteristics. ORR was significantly higher with HMA+VEN (95%) as compared those treated with HMA alone (46%), p < 0.001. However, there was no significant OS difference between HMA+VEN vs HMA alone (19.1 mos vs 19.1 mos, p = 0.85, Figure 1). For CMML-BT patients, the ORR of HMA+VEN was 81% including CR in 17%, CR with partial hematologic recover (CRh) in 3%, CR with incomplete hematologic recovery (CRi) in 28%, morphologic leukemia-free state (MLFS) in 22% and partial response (PR) in 11%. The OS of CMML-BT patients was 8 mos (95% CI 4.6 - 26.2). A PSM population of CMML-BT patients treated with HMA alone showed a significantly increased ORR with HMA+VEN (82% vs 40%, p = 0.039). However, there was no difference in OS (7.49 mos vs 9.26 mos, p = 0.83, Figure 2). Among HMA-VEN treated patients, achievement of a CR was associated with an improved OS in the CMML (HR 0.31, 95% CI 0.13 - 0.75) and CMML-BT (HR 0.37, 95% CI 0.19 - 0.74). No predictors of response were identified, including the presence of a RAS mutation. In terms of safety, 7 patients (7.9%) had a grade 3/4 (G3/4) neutropenic infection and 2 patients (2.2%) had a G3/4 hemorrhagic event while being treated with HMA+VEN. A total of 29 patients went on to receive allogeneic transplant (alloSCT): 22 (43%) with CMML and 7 (18%) with CMML-BT. CMML patients who underwent alloSCT after HMA+VEN were younger as compared to non-transplanted patients (64 vs 74 years, p < 0.0001), but had otherwise similar baseline characteristics including stage, CPSS-mol category and prevalence of RAS mutations. Similarly, CMML-BT patients bridged to alloSCT were younger (66 vs 74 years, p < 0.002) and had a better ECOG performance status (p = 0.047). Of patients who received alloSCT, 10 patients (34%) died, 5 with CMML and 5 with CMML-BT. The median OS after alloSCT was 26.7 mo. Conclusions Although the use of VEN with HMA in the upfront treatment of CMML and AML secondary to CMML results in higher response rates compared to HMA alone, this combination does not improve survival based on this multi-center PSM analysis. The current sample size of CMML-BT may limit detection of OS differences between treatment groups. The role of VEN to reduce blasts and serve as a bridge to alloSCT, particularly after HMA failure, requires further investigation in larger studies in both the CMML and CMML-BT populations.

Introduction: Germline DEAD-box RNA helicase-1 mutant (DDX41) predisposition syndrome ( DDX41-MT-GPS) is one of the most frequent hereditary predisposition syndromes in adults with myeloid neoplasms (MN-2-5%). Somatic mosaicism involving the unmutated DDX41 allele (e.g., R525H) as a potential mechanism for myeloid evolution has been reported in 39-50% of patients (pts). In addition, given the later age of onset, somatic mosaic states composed of age-related clonal hematopoiesis (ARCH) (e.g., DNMT3A, ASXL1, TET2, and TP53), with reported frequencies less than expected in MDS and AML, have been documented. We conducted this study to define the somatic mosaic landscape in DDX41-MT-GPS . Methods: After approval from the Mayo Clinic IRB the prospective GPS database was queried for pts with DDX41-MT GPS. Germline confirmation was conducted on skin fibroblast-, or hair follicle-derived DNA. Somatic CH testing was conducted using a targeted NGS panel as previously described. Given the high frequency of variants of undetermined significance (VUS), comparisons were made between pathogenic ( DDX41path) and DDX41VUS. Results: We identified 106 pts with DDX41-MT-GPS, of which 83 (78%) met criteria for MN, 16 (15%) for CH/CCUS (clonal cytopenias of undetermined significance), while 7 (6%) were unaffected carriers. Somatic mosaicism in DDX41-GPS patients without MN Sixteen DDX41-MT pts without MN met criteria for CH/CCUS ( DDX41path [n=4] and DDX41VUS [n=12]; median age 69 years (yrs) (range [R], 19-78) ( DDX41path [69 yrs] and DDX41VUS [67 yrs], p=0.85), with a male predilection (75%). Four (25%) pts had somatic DDX41 MT (R525H [n=3] and A376T [n=1]) with median variant allele frequency (VAF) 10.5% (R, 5-27); 2 (50%) pts with germline DDX41path had R525H (VAF; 5% and 13%) and 1 (8%) pt each with germline DDX41VUS had R525H (VAF 5%) and A376T (VAF 27%) somatic variants, respectively. Abnormal cytogenetics (CG) were observed in 2/13 evaluable (15%) pts ([+8 and del20q]) in the germline DDX41path group only (p= 0.05). ARCH co-mutations were observed in 11 (69%) pts, not significantly (NS) different between DDX41path (75%) and DDX41VUS (75%), p= 0.66. Individual co-mutations: DNMT3A (25% vs 16%, p= > 0.99), ASXL1 (25% vs 8%, p=0.71), TET2 (25% vs 8%, p= >0.99), and JAK2 (0% vs 8%, p= >0.99) were NS different between the two groups. Somatic mosaicism in DDX41-GPS patients with MN Eight-three pts met criteria for MN ( DDX41path [n=43] and DDX41VUS [n=40]; median age 67 yrs (range [R], 27-92), with a male predilection (58%). Fifty-four (65%) pts had MDS ( DDX41path [55%] and DDX41VUS [45%], p=0.55) and 29 (35%) had AML ( DDX41path [41%] and DDX41VUS [59%], p=0.65). Overall, 15 (18%) pts had somatic DDX41 MT; 12 (80%) pts with DDX41path and 3 (20%) pts with DDX41VUS (p= 0.003). The median VAF of somatic DDX41 MT was 12% (R, 5-37), NS different between DDX41path (10.5%) and DDX41VUS (12%) (p=0.08). The most common somatic DDX41 MT was R525H (66% [n=10]), significantly associated with DDX41path (9/10 [90%]), p=0.01. ARCH was observed in 17 (20%) pts, significantly higher in DDX41path (76%) compared to DDX41VUS (24%), p= 0.03. Abnormal CG were observed in 10/73 evaluable (14%) pts (-Y [n=2], del 20 [n=3], del 5q [n=2], del 7q [n=1], t (3;8) [n=1] and complex CG [n=1]), higher in DDX41path (70%) compared to DDX41VUS (30%) with p value >0.99. We compared the spectrum of CH between DDX41 MT CH/CCUS and MN pts ( Table 1) and found no difference in the proportion of pts with somatic DDX41 MT (p= 0.52) and ARCH/myeloid MT (p= 0.56). The individual occurrences of DNMT3A (p= 0.49), ASXL1 (p= 0.71), TET2 (p=0.59), JAK2 (p>0.99) and TP53 (p= 0.56) mutations were NS different between the two groups ( Figure 1). Conclusion: We define the somatic mosaic landscape in DDX41-MT GPS and demonstrate that somatic mutations involving the other allele, especially R525H, are seen in ~20% of pts (>80% of R525H present in MN). The frequency and composition of ARCH and myeloid driver mutations was lower than expected for the age of presentation, in comparison to de novo and secondary MDS/AML and occurred more frequently in DDX41path compared to DDX41VUS. Forty-two % (n=41), including 37% with MN, did not have somatic mosaicism, underscoring the fact that mechanisms of leukemia progression remain to be defined in subsets of affected pts.

Introduction Truncating ASXL1 mutations in chronic myelomonocytic leukemia (CMML) are associated with inferior overall survival (OS) and leukemia free survival (LFS). Conversely, loss-of-function mutations in TET2 are associated with better outcomes, with the ASXL1wt/ TET2mut genotype being associated with the best OS and LFS-rates (Coltro G, et al. Leukemia. 2020). 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) - do not consider concomitant ASXL1/ TET2 mutational status or mutational VAF. We hypothesized that the ASXL1/ TET2 genotype would better risk stratify patients in comparison to ASXL1 mutations alone, and that this effect would be proportional to the underlying mutational VAF. 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 to the respective institution. 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. All statistical calculations were performed using the BlueSky Statistics (v10.3.1) interface for R. Results The median age of the cohort was 71 years (range 20 - 94), 67% were male, 46% had proliferative CMML (pCMML), and 19% had CMML-2. The distribution of low, intermediate-1, intermediate-2, and high-risk patients was 15%, 11%, 32%, and 42% by the MMM and 9%, 23%, 40%, and 28% by the CMM, respectively. The most frequently mutated genes were ASXL1 (45%), TET2 (43%), SRSF2 (41%), and RUNX1 (17%), while 40% harbored at least one RAS pathway mutation ( NRAS 16%, CBL 15%, KRAS 8%, PTPN11 4%, FLT3 2%). The median OS and LFS of the entire cohort were 31.8 months (95% CI 28.4 - 33.5 months) and 28.4 months (95% CI 24.6 - 32.1 months), respectively. Patients were stratified into ASXL1wt/ TET2wt (28%), ASXL1mut/ TET2wt (29%), ASXL1wt/ TET2mut (26%), and ASXL1mut/ TET2mut (17%) genotypes. Those with isolated TET2 mutations had the longest median OS (53.6 months; p < 0.02 for all comparisons) whereas those with isolated ASXL1 mutations had the shortest median OS (20.9 months; p < 0.04 for all comparisons); mutations in neither or both genes performed similarly (p = 0.9224; Figure 1A). The same trend was seen for LFS. We further hypothesized that the ASXL1 or TET2 mutation VAF would be more predictive of outcomes than a binary metric. The respective median VAFs were 37% (quartiles 28% - 45%) and 45% (quartiles 40% - 49%). When treated as a continuous variable, however, there was no correlation between either VAF and OS or LFS by both Pearson linear regression and Cox proportional hazard modeling. Unlike the overall cohort, the ASXL1/ TET2 genotypes did not further stratify patients with pCMML, CMML-2, or those considered high risk by the CMM in Kaplan-Meier analyses, suggesting that the high-risk features inherent in these subgroups outweigh the benefits of the TET2 mutation. In contrast, patients considered high-risk by the MMM were further stratified by the ASXL1/ TET2 genotypes. Incorporating TET2 mutations into the MMM as a protective factor (worth -1.5 points) improved detection of low- (≤ 1 point) and high-risk (≥ 4 points) patients, while intermediate-1 and intermediate-2 coalesced into a single intermediate risk category ( Figure 1B). A similar phenomenon was observed when TET2 was incorporated into the CMM (worth -1 point, yielding low- (0 points), intermediate- (1-2 points), and high-risk (≥ 3 points) groups with median OS of 83 months (95% CI 66.2 - 104.2), 34.3 months (95% CI 31.6 - 38.7), and 17.7 months (95% CI 15.7 - 20.2), respectively. Conclusions Truncating and hypomorphic TET2 mutations are associated with better outcomes in CMML, with the ASXL1mut/ TET2wt genotype being associated with improved risk stratification in comparison to ASXL1 mutational status alone. Counterintuitively, in our data set, this stratification was not impacted by mutational VAF, supporting binary mutational assessments in prognostic models.

Abstract VEXAS (Vacuoles, E1 enzyme, X‐linked, Autoinflammatory, Somatic) syndrome is a newly identified disease caused by somatic alterations in UBA1 which produce a recalcitrant inflammatory state along with hematologic disturbances. Patients with VEXAS can have a wide spectrum of clinical symptoms and providers should be familiar with the heterogeneity of associated clinical features. While hematologic parameters may be generally non‐specific, peripheral blood features of macrocytosis, monocytopenia, and/or thrombocytopenia coupled with bone marrow vacuolization of erythroid or myeloid precursors should raise suspicion for this condition. Due to an increased mortality, prompt recognition and accurate diagnosis is paramount. Access to testing for confirmation of UBA1 variants is not yet universally available but clinicians should understand the current available options for genetic confirmation of this disease. Treatment options are limited due to lack of prospective clinical trials but cytokine directed therapies such as interleukin‐6 inhibitors and JAK–STAT inhibitors as well as hypomethylating agents such as azacitidine have shown evidence of partial effect. Though cases are limited, allogeneic stem cell transplantation holds promise for durable response and potential cure. The intent of this review is to outline the pathophysiology of VEXAS syndrome and to provide a practical approach to diagnosis and treatment.

Introduction: The mechanistic link between early coronary artery disease (CAD) and atrial fibrillation (AF) continues to be explored. Clonal hematopoiesis of indeterminate potential (CHIP) is increasingly recognized as a risk factor for cardiovascular disease. We hypothesize CHIP further increases the risk for AF in early CAD. Methods: 184 participants underwent invasive coronary angiogram with endothelial function testing. Buffy coat was used for whole exosome sequencing to identify CHIP (variant allele frequency > 2% and minor allele frequency ≤ 0.1%). Early CAD was defined by the presence of coronary endothelial dysfunction (CED) in the absence of obstructive CAD. Invasive coronary angiography with endothelial function testing characterized CED as ≥ 20% increase in coronary artery diameter or ≤ 50% increase in coronary blood flow in response to acetylcholine. AF was identified by ICD-10 code or patient survey and confirmed by an independent investigator by chart review. Cox proportional hazard regression was adjusted for age, sex, and conventional cardiovascular comorbidities (hypertension, diabetes, and total cholesterol). Results: Of the 184 participants, we identified 23 cases with both early CAD and CHIP and 62 normal controls without CHIP or CAD. Case patients were significantly older (mean 60 ±9 vs 52 ±10 years, p = 0.004 years) and predominantly female (74% vs 81%, p = 0.506). AF was more likely among case participants when compared to normal controls (29% vs 10%, p = 0.036). Mean time to AF or last follow-up was 11 ±7 years. Case patients with both early CAD and CHIP were nearly 10 times more likely to develop AF when compared to normal controls in a Cox regression model adjusted for age, sex, and conventional cardiovascular comorbidities (HR 9.52, 95% CI 1.93 - 47.02, p = 0.006). The risk for AF was not significant if we defined cases by only the presence of CHIP (n = 30) or early CAD (n = 122). Conclusions: Patients with both CHIP and early CAD may have greater risk for developing AF beyond the presence of CHIP or CAD alone. Our findings suggest an added mechanistic role of CHIP that further increases the risk for AF in early CAD.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n = 18) and a solid tumor type, ovarian cancer (OC, n = 18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell type identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n = 6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

Abstract Objective To retrospectively identify patients with VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome) among male patients with bone marrow vacuolization using a clinically applicable, targeted-screening approach. Methods Bone marrow reports from 1 May 2014 through 18 February 2022 were reviewed for documentation of cytoplasmic vacuolization. Patients with acute leukaemia, lymphoma, metastatic solid tumour, amyloidosis or POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes) syndrome were excluded, as were those without clinical records available for direct chart review. Cases were rated for suspicion of VEXAS syndrome using a 5-point scale based on the presence of laboratory findings, clinical features and treatment response. Patients with available DNA material and moderate (three patients) or high (four to five patients) suspicion were tested for somatic UBA1 variants associated with VEXAS syndrome. Results A total of 315 reports from 292 unique patients included documentation of vacuolization. Following exclusion criteria, 64 patients underwent direct medical chart review to assess likelihood of VEXAS syndrome, for which 21 patients met moderate to high suspicion. Available DNA was present in eight patients, seven (87.5%) of whom had a pathogenic somatic UBA1 variant consistent with VEXAS syndrome. The distribution of cytoplasmic vacuolization in the bone marrow biopsy reports among patients with VEXAS syndrome were erythroid and myeloid precursors (6/7), erythroid precursors only (1/7) and myeloid precursors only (0/7). Conclusion In this study, the utilization of a clinically applicable targeted-screening approach to test bone marrow specimens (with vacuolization) for the presence of previously undiagnosed VEXAS syndrome resulted in a positive detection rate of 87.5%.