Patnaik, Mrinal M.

Abstract Background: The DEAD-box helicase 41 (DDX41), an RNA helicase, have been described as a component of the RNA spliceosome (Cheah et al. International Journal of Hematology 2017). Although DDX41 mutations predispose to late-onset higher grade myeloid neoplasms (MN), these patients may have a trend toward favorable prognosis and outcomes. In this work, we describe the clinical characteristics and survival outcomes of isolated and co-mutated DDX41 patients (pts). Methods: We retrospectively analyzed 4,524 consecutive pts who underwent next-generation sequencing (NGS) (OncoHeme 42 genes panel, Mayo Clinic) testing and included 32 pts harboring pathogenic DDX41 mutation and one pt with proven DDX41 germline variant of unknown significance (VUS). Chart review of DDX41-mutated (m) cases between 2009 and 2021 was conducted after IRB approval. We compared overall survival (OS) of unmatched 27 t(8;21)AML and 40 inv(16) AML pts with 10 m DDX41-AML pts. JMP® Pro 14.1.0 Software was used for statistical analysis. Results: DDX41 mutations characteristics: Our cohort included 19 (58%) myelodysplastic syndromes (MDS), 10 (30%) acute myelogenous leukemia (AML), 2 (6%) myeloproliferative neoplasms (MPN), one clonal cytopenia of undetermined significance (CCUS) (3%) and one (3%) germline carrier. Germline testing was carried out in 10 pts, 9 of whom (90%) were confirmed to be germline). The start-loss variant (p.M1I) was the most common mutation type (N=10, 31%). Other types were frameshift (N=9, 28%), missense mutation (N=8, 25%), nonsense (N=3, 9%), and splice site mutation (N=2, 2%). Twenty-one (65.6%) DDX41 mutations clustered in the N-terminus (NT), 7 (22%) in the helicase-C domain (HCD), and 4 (12.5%) in the DEAD-box domain. Compared to NT mutations, patients with HCD mutation had no family history of solid tumors and were more likely to have an accompanying additional DDX41-VUS (0% vs 70%; p=.001) and (N=6, 86% vs. N=2,10%; p=.0001); respectively. I solated vs. co-mutated DDX41: Twenty (60%) pts were isolated-DDX41 and 13 (40%) were co-mutated. The median DDX41-VAF was 48% vs. 45% (p= .2) in the isolated compared to the co-mutated cases, respectively. The median number of co-mutations in the 13 co-mutated cases was 1 (range,1-3) with DNMT3A (38%), ASXL1 (30%), JAK2 (N=3, 23%), and EZH2 (N=2, 15%) were the most common co-mutations detected. Isolated DDX41 had more males (85% vs. 54%, p=.05), the p.M1I variant (47% vs. 8%, p=.02), normal cytogenetics (100% vs. 91%, p= .02), and less family history of solid tumors (77% vs. 33%, p= .02) compared to their co-mutated counterparts. However, there was no difference in OS (p=.99). Comparison of clinical characteristics and hematological features of isolated and co-mutated DDX41 pts are reported in (Table 1). Treatment and survival outcomes in DDX41-MDS/AML : Twenty-three (80%) patients were treated, MDS pts received hypomethylating agents (HMA) (N=10, 71%), HMA plus Venetoclax (HMA+VEN) (N=1, 7%), erythropoiesis-stimulating agents (N=2, 14%) and lenalidomide (N=1 ,7%). AML pts were treated with induction chemotherapy (N=6, 67%) and HMA+VEN (N=3, 33%). Overall response rate of MDS/AML patients was 77% and 100% of AML pts achieved complete remission (CR) when treated with induction chemotherapy or HMA+VEN regimen. There was no significant difference in OS between responders vs. non-responders 2-yr-OS (90% vs. 50%; p=.38) and treated vs. untreated 2-yr-OS (83% vs. 100%; p=.52). Comparing m DDX41-AML vs. core binding factor-AML: After a median follow-up of 33.3 months, all m DDX41-AML patients were alive. There was a significantly better OS in mDDX41-AML patients compared to pts with t(8;21) AML with 2-yr-OS (100% vs. 51%; p=.024) and a trend of better survival when compared to inv(16) AML 2-yr-OS (100% vs. 84%; p=.2). Conclusion We describe the characteristics and outcomes of m DDX41 patients. We demonstrated that isolated and co-mutated m DDX41 patients have different features. Isolated DDX41 patients had male predominance, more p.M1I variant, normal cytogenetics and less family history of solid tumors. In this study we found that m DDX41 AML has high response to treatment and has comparable (if not possibly better) OS compared to other "favorable risk" AML. This study, although limited by the small number of patients, supports the universal testing for DDX41 mutation in adults with MN diagnosis. Figure 1 Figure 1. Disclosures Al-Kali: Astex: Other: Research support to institution; Novartis: Research Funding. Foran: abbvie: Research Funding; OncLive: Honoraria; boehringer ingelheim: Research Funding; trillium: Research Funding; pfizer: Honoraria; takeda: Research Funding; revolution medicine: Honoraria; bms: Honoraria; gamida: Honoraria; actinium: Research Funding; aptose: Research Funding; novartis: Honoraria; servier: Honoraria; taiho: Honoraria; syros: Honoraria; sanofi aventis: Honoraria; certara: Honoraria; kura: Research Funding; h3bioscience: Research Funding; aprea: Research Funding; sellas: Research Funding; stemline: Research Funding. Badar: Pfizer Hematology-Oncology: Membership on an entity's Board of Directors or advisory committees. Salama: Mayo Clinic: Current Employment, Other: Mayo Clinic had the contractual work for the central pathology review for this study and I was one of the reviewing pathologists; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Litzow: Astellas: Research Funding; Biosight: Other: Data monitoring committee; Amgen: Research Funding; AbbVie: Research Funding; Actinium: Research Funding; Pluristem: Research Funding; Jazz: Other: Advisory Board; Omeros: Other: Advisory Board. Patnaik: Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Research Funding; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Mutations involving isocitrate dehydrogenase 1/2 (IDH) are known oncogenic drivers in hematological malignancies, conferring neomorphic enzymatic activity to IDH 1/2, resulting in the oncometabolite, 2-hydroxyglutarae (2-HG). 2-HG in turn suppresses TET activity, making IDH and TET2 mutations synthetically lethal and almost mutually exclusive. The frequency of IDH mutations in CMML is <10% and their prognostic impact remains unclear. We carried out this study in a large database of molecularly annotated CMML patients to better define the clinical profile and prognostic impact of these mutations. Methods: After IRB approval, CMML patients from the Mayo Clinic, Minnesota and the Moffitt Cancer Center (MCC), Tampa, Florida, were included in the study. All patients had bone marrow (BM) biopsies with cytogenetics and molecular genetics done either at diagnosis, or at first referral. Clinical and mutational data were abstracted and retrospectively analyzed. Overall survival (OS) was calculated from date of CMML diagnosis to date of death/last follow, while AML-free survival (AML-FS) was calculated from date of CMML diagnosis to date of leukemic transformation (LT). Patients that had undergone allogeneic HCT were excluded from the study (n=3). Statistical analysis was carried out using the Blue Sky software. Results: Six hundred and forty four patients were included in the study (Mayo Clinic-357, MCC- 287), median age 71 years (range, 20-95 years), 67.8% being male. Forty-three (6.7%) patients had IDH mutations, 35 (82%) IDH2 and 8 (18%) IDH1; of which, 34 (97%) involved the IDH2R140 hotspot and 5 (62.5%) involved the IDH1R132 hotspot, respectively. The median variant allele fractions (VAF) for IDH1 mutations was 41% (range, 8-46%) and for IDH2 mutations was 46% (range, 7-70%). There were no significant demographic or clinical differences between IDH mutant and wild type CMML patients, with the exception that IDH mutant CMML patients were less likely to be thrombocytopenic (p=0.006), were less likely to have TET2 co-mutations (14% vs 53.2%; p<0.001) and were more likely to have SRSF2 co-mutations (69.8% VS 40.3%; p<0.001). Importantly there were no differences in proliferative or dysplastic subtypes (p=0.3), cytogenetic (p=0.12) and molecular risk stratifications (p=045). There were also no significant demographic or clinical differences between IDH1 vs IDH2 mutant CMML patients. Six (14%) IDH mutant CMML patients had TET2 co-mutations; 5 (83%) with IDH2R140Q (median VAF-28%;all male) and 1 (17%) with IDH1R132H (VAF-44%;female) (Figure 1). Five (11%) IDH2 mutant patients were treated with enasidenib (IDH2 inhibitor), none with a durable response, while none of the IDH1 mutant patients received targeted therapy. At last follow up (median 18 months), 337 (52%) deaths and 119 (18.5%) LT have been documented, with IDH mutant patients having a higher LT rate (30.2% vs 17.6%, p=0.04) compared to wildtype patients. The median OS of the entire cohort was 35 months, with no difference in OS between IDH mutant and wild type patients (34.5 vs 35 months, p=0.12), with IDH1 mutant patients having a shorter OS in comparison to IDH2 mutant patients (31 vs 37 months; p=0.005- Figure 1). IDH mutant CMML patients also had a shorter AML-FS in comparison to wild type patients (36.6 vs 210 months, p=0.005), with there being no differential impact on AML-FS of IDH1 vs IDH2 mutations (p=0.26, Figure 1). Conclusions: IDH mutations are infrequent in CMML (7%), with IDH2 mutations being more common than IDH1 mutations (80 vs 20%). IDH mutations co-occur very infrequently with TET2 mutations (14%), with IDH mutant patients being less likely to have thrombocytopenia and more likely to have SRSF2 co-mutations. IDH mutations negatively impacting AML-FS without a significant impact on OS. Prospective clinical trials testing the safety and efficacy of IDH1/2 inhibitors in CMML are much needed. Figure 1 Figure 1. Disclosures Komrokji: AbbVie: Consultancy; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Speakers Bureau; Acceleron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Consultancy. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution. Padron: BMS: Research Funding; Stemline: Honoraria; Taiho: Honoraria; Kura: Research Funding; Incyte: Research Funding; Blueprint: Honoraria. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Abstract Background Approximately 100 high oxygen affinity (HOA) hemoglobin (Hgb) variants have been reported to date; review of the Mayo Clinic laboratory database (1974-2018) identified 80 distinct variants including 12 novel variants (60 β, 20 α). One-third of HOA Hgb variants result in secondary erythrocytosis, provoking concern regarding increased risk for thrombosis. Current management guidelines lack supporting evidence regarding the utilization of phlebotomy in such cases. We describe presenting features, treatment strategies and follow-up events involving 41 consecutive cases seen at our institution. Methods Study patients were recruited from our institutional laboratory and clinical Hgb variant database. Initial evaluation for Hgb variants was conducted by capillary electrophoresis and high-performance liquid chromatography. Additional testing which included mass spectrometry and isoelectric focusing was pursued as necessary. Since the majority of variants were difficult to identify by protein studies, DNA sequencing of HBB, or HBA1, HBA2 genes was performed for confirmation. Symptoms and thrombosis, both at presentation and during follow up, were recorded. Therapeutic interventions were based on physician discretion and mostly included phlebotomy and/or aspirin therapy; a careful response assessment was performed to determine the impact of each therapy on symptoms and/or thrombosis. Results A total of 41 patients with HOA Hgb variant-associated erythrocytosis (median age 39 years, range 1-81; 54% males) were seen at our institution between January 1973 and February 2020. The majority of the patients carried β-chain variants (n=34; 83%), common variants being Hgb Malmo (n=13), Olympia (n=4), San Diego (n=3), and Wood (n=2). Among the 7 patients with α-chain variants, Hgb Dallas was the most frequent (n=4). Presenting median values (range) for Hgb/Hct, serum erythropoietin and p50 were 18 g/dl/52.9%(16-21.9g/dl/48-66%) 10.4 mIU (4-36.3 mIU), and 20 mmHg (12-25 mmHg), respectively. Family history was documented in 34 patients, of which 24 (71%) reported one or more affected family members. Family history of thrombosis was documented in 7 patients (21%). CV risk factors were present in half of the patients; by contrast, history of thrombosis prior to or at diagnosis was documented in only two patients (5%). Of 23 pregnancies reported in 12 women, live birth rate was 78% (n=18); none of the fetal losses were attributed to erythrocytosis. Active therapies at the time of initial referral consisted of phlebotomy (n=12), aspirin (n=11) and systemic anticoagulation (n=1). At a median follow-up of 10 years (range; 0.04-44), 23 patients had reported one or more symptoms, attributed to hyper-viscosity, such as headaches, fatigue, and lightheadedness. Neither Hct level at diagnosis (p=0.32) nor phlebotomy (p=0.16; 75% patients on vs 52% not on phlebotomy) or aspirin therapy (p=0.75; 55% patients on vs 60% not on aspirin) appeared to influence the occurrence of symptoms. Phlebotomy relieved symptoms in 7 (42%) symptomatic patients; however, 7 (30%) of 23 patients on phlebotomy reported one or more adverse symptoms that were attributed to phlebotomy-induced iron deficiency. Ten patients (24%) experienced thrombosis prior to or following diagnosis: 6 arterial and 4 venous. Median age at thrombotic event was 51 years and median hematocrit 52%; active therapies at the time of event included phlebotomy in 5 patients, aspirin in 4, and systemic anticoagulation in 2. Hct level at diagnosis (p=0.10) or the time of event (p=0.67) did not correlate with occurrence of thrombosis. Additionally, the incidence of thrombosis was no different among patients receiving or not receiving phlebotomy (5/23(22%) vs 5/18(28%), respectively; p=0.66). The presence of CV risk factors was predictive of arterial events (p=0.002). Two of the 4 venous events developed in the context of concomitant thrombophilia. Eight patients (20%), median age 28 years, without CV risk factors, were observed without therapy for a median of 9.5 years (range; 0.4-21) and have not experienced any thrombosis to date. Conclusions We found no association between Hct level and either thrombotic or non-thrombotic symptoms in HOA hemoglobinopathy-associated erythrocytosis; furthermore, implementation of aggressive phlebotomy did not provide a clear benefit with respect to thrombosis risk reduction. Figure 1 Figure 1. Disclosures Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Abstract Background Clonal cytopenia of undetermined significance (CCUS) is a risk factor for development of myeloid neoplasms (MN) such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Therapy-related myeloid neoplasm (t-MN) is defined as the development of a MN in the context of prior DNA damaging therapies including chemotherapy, radiation, and immunosuppressive therapies. CCUS diagnosed in the setting of prior DNA damaging therapy (t-CCUS) and its outcomes are not known. The aim of this study was to study clinicopathological features and outcome of t-CCUS and compare it with t-MN. Methods Patients who had received DNA-damaging therapy and subsequently determined to have CCUS or t-MN were identified. The diagnosis of CCUS was made if the patient had non-diagnostic bone marrow biopsy evaluation combined with evidence of pathogenic myeloid somatic genetic alterations using conventional cytogenetics or next generation sequencing (NGS) panel. Pathogenic/likely pathogenic variants (PV) were stratified by DTA (DNMT3A, TET2, and ASXL1) genes vs. others. Event-free survival (EFS) was calculated using interval from t-CCUS to t-MN with death as a competing risk. Overall survival (OS) for t-CCUS and t-MN patients was calculated using interval from t-CCUS or t-MN diagnoses respectively to the last follow up. Statistical analysis was performed using JMP (v14.1, SAS Institute). Results We identified 30 patients with t-CCUS and 259 patients with t-MN. Eleven (37%) t-CCUS patients developed t-MN and were included in both cohorts (Fig. A). Clinical characteristics of t-CCUS patients are shown in Table 1. Briefly, age (median, interquartile range or IQR) at the diagnosis of primary malignancy and t-CCUS was not different between those who developed t-MN and those did not (P=0.09 and 0.378 respectively). There was no difference in hemoglobin, white blood cells, or ANC between the two groups. Patients who developed t-MN had a significantly lower platelet count (68 vs. 130, P<0.01) and higher bone marrow blast percent (2 vs. 0, P=0.04) compared to those who did not. At t-CCUS diagnosis, cytogenetics was abnormal in 11 (37%) patients and none had complex karyotype (CK). Paired cytogenetic analysis at t-CCUS and t-MN was available for 10 patients: 4 had unchanged karyotype at t-MN, whereas 6 had cytogenetic evolution. In contrast, 213 (83%) of 256 t-MN patients with available cytogenetics had abnormalities and 126/256 (49%) had CK. Thus, a significantly higher proportion of patients had CK t-MN compared to t-CCUS (χ 2 26.4, P<0.01). A 42-gene NGS panel identified 36 PV in 21 patients (median 1, range 1-4) at t-CCUS. The most common PV were in TET2 (37.1%), TP53 (11.4%), DNMT3A (8.6%), and ZRSR2 (8.6%) genes. Two patients had paired NGS available at t-CCUS and t-MN: one had BCOR and U2AF1 PV at t-CCUS and acquired an additional NRAS PV at t-MN. The second patient had 2 PV in TP53 at t-CCUS diagnosis, that remained the only PV identified at t-MN. In the t-MN cohort, median number of PV was 2 (range 1-9). The most common PV identified were TP53 (21.5%), TET2 (6.7%), DNMT3A (6.4%), and ASXL1 (6.4%), whereas 4.8% patients had no PV. Three of 22 patients in the t-CCUS cohort had TP53mut, compared to 55 of 157 in the t-MN cohort (χ 2 4, P=0.05). Management strategies for t-CCUS included observation (20), growth factors (4), treatment of primary malignancy (2), immunosuppressives (1), hypomethylating agent followed by allogeneic SCT (1), and others (2). Following t-CCUS, 9 patients progressed to MDS and 2 to AML with a median of 3.5 months (IQR 1.7-43.4). EFS was 43.4 months (IQR 6.8-80.9). The presence of abnormal cytogenetics (Fig. B) and the absence of DTA-mutations (Fig. C) predicted shorter EFS. At last follow up, 12 (40%) deaths were noted. Four (33%) patients died without developing t-MN. Causes of death in the t-CCUS cohort were: t-MN (3), primary malignancy (1), progressive cytopenia without MN (1), infection (1), graft-vs.-host-disease (1), and undetermined (5). t-MN patients had a significantly shorter survival compared to t-CCUS (Fig. D). Conclusion More than 10% of t-MN patients had a precursor t-CCUS. On the other hand, t-CCUS patients are at a high risk of progression to MN. t-MN is characterized by the acquisition of TP53mut, genomic instability, and a significantly shorter survival compared to t-CCUS. The presence of abnormal cytogenetics and the absence of DTA mutations at t-CCUS predict shorter EFS. Figure 1 Figure 1. Disclosures Litzow: Omeros: Other: Advisory Board; AbbVie: Research Funding; Jazz: Other: Advisory Board; Amgen: Research Funding; Astellas: Research Funding; Pluristem: Research Funding; Actinium: Research Funding; Biosight: Other: Data monitoring committee. Patnaik: Kura Oncology: Research Funding; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution.

Abstract Introduction: Factors contributing to the antitumor activity of systemic oncolytic virotherapy (OV) include (i) generalized innate immune activation due to virus infusion; (ii) direct oncolytic and/or immune-mediated destruction of OV infected tumor cells due to intratumoral spread of the virus infection; (iii) collateral boosting of tumor antigen-specific T cell responses that target uninfected tumor cells. We investigated the relative contributions of these 3 factors T cell lymphoma (TCL) patients (pts) responding to a single intravenous infusion of Voyager-V1, an oncolytic vesicular stomatitis virus (VSV) encoding IFNβ and sodium iodide symporter (NIS) on NCT03017820. Of relevance, the virally encoded IFNβ enhances tumor specificity, boosts proinflammatory activity & serves as a convenient serum biomarker of infection. Methods: In a first in human trial, a single infusion of Voyager-V1 was administered to 15 pts with relapsed refractory hematologic cancers; 4 dose levels (DL) were tested (5x10 9, 1.7x10 10, 5x10 10, 1.7x10 11 TCID50); 7 pts had TCL. Cytokine levels and CRS symptoms were determined as a measure of generalized immune activation; serum IFNβ levels were monitored as a pharmacodynamic biomarker of virus infection; antitumor T cell responses were measured using ELISPOT assays. Results: Of 8 TCL pts, 3 had RECIST responses: PR at DL 2, a CR & PR at DL 4. Most pts experience short lived CRS typically resolving within 24 hours of virus infusion. Serum IFNβ levels increased post-therapy in all pts, typically peaking by 24 hours post virus infusion and returning to baseline by median day 10. T cell responses could not be accurately determined in most cases due to suboptimal T cell viability and/or function after thawing of frozen PBMC. Factors contributing to tumor regression in each of the responding pts appeared to differ significantly, as exemplified by the case reports below. Pt (I) with CTCL experienced grade 1 CRS followed by brisk regression of multiple cutaneous lesions. The PR lasted 3 mo, after relapse of an eye lesion, but responses at other sites deepened after the relapsed lesion was irradiated; pt remained off active therapy for the next 2 yrs. Peak serum IFNβ 4 hours post infusion was only 182 pg/ml suggesting that disease response may have been driven by nonspecific innate immune activation associated with the virus infusion. Pt (IV) (CD30+ PTCL/ cutaneous relapse) had short lived grade 2 CRS, then developed signs of IFNβ toxicity with AST elevation and thrombocytopenia. Oral ruxolitinib was administered for 5 days as IFNβ toxicity mitigation leading to normalization of AST and platelets. All PET-avid tumors regressed completely. This CR continues 16 months post virus infusion. Peak serum IFNβ was 18,213 pg/ml strongly suggesting that response was driven by extensive intratumoral amplification of the virus and oncolytic tumor destruction. Robust CTL responses to tumor neoantigens were detected in this pt at baseline indicating that collateral boosting of tumor-specific T cells may have been responsible for the long term maintenance of this CR Pt (V) (CD30+ PTCL) had short lived grade 2 CRS followed by response at all disease sites (including complete normalization of an enlarged FDG-avid spleen). Her PR ended after 6 months due to relapse in cervical lymph nodes. Peak serum IFNβ level was modest, 600 pg/ml & sustained over 48 hours suggesting slower intratumoral spread of the virus infection. The deepening of the response long after resolution of the oncolytic phase points strongly to a contribution from antitumor CTL but the assay was technically unsatisfactory in this pt. Conclusion: Analysis of the correlative data supports that intravenous Voyager-V1 therapy can recruit diverse antitumor effector mechanisms to impact tumor response in pts with heavily pretreated TCL. Importantly, intratumoral virus amplification, which results in high circulating concentrations of IFNβ, does not necessarily lead to tumor regression perhaps because of defective immune cell recruitment and/or activation in some tumors but not in others. This will be further evaluated in future study participants by performing CTL assays on fresh, rather than frozen PBMC. In conclusion, a single IV infusion of Voyager-V1 shows highly promising activity against treatment refractory TCL and should be further advanced as a new single-shot or combination therapy for this therapeutically challenging indication. Figure 1 Figure 1. Disclosures Witzig: Karyopharm Therapeutics, Celgene/BMS, Incyte, Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS, Acerta Pharma, Kura Oncology, Acrotech Biopharma, Karyopharm Therapeutics: Research Funding. Paludo: Karyopharm: Research Funding. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding. Dispenzieri: Takeda: Research Funding; Alnylam: Research Funding; Pfizer: Research Funding; Oncopeptides: Consultancy; Sorrento Therapeutics: Consultancy; Janssen: Consultancy, Research Funding. Bennani: Kymera: Other: Advisory Board; Vividion: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Verastem: Other: Advisory Board. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Gertz: Aurora Biopharma: Other: Stock option; Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring; Ionis Pharmaceuticals: Other: Advisory Board. Dingli: Janssen: Consultancy; Sanofi: Consultancy; GSK: Consultancy; Apellis: Consultancy; Novartis: Research Funding; Alexion: Consultancy. Kumar: Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Merck: Research Funding; Oncopeptides: Consultancy; Roche-Genentech: Consultancy, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Tenebio: Research Funding; Novartis: Research Funding; Bluebird Bio: Consultancy; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Bergsagel: Oncopeptides: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Genetech: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties: human CRBN mouse; Janssen: Consultancy, Honoraria. Kapoor: Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; AbbVie: Research Funding. Lin: Sorrento: Consultancy; Vineti: Consultancy; Legend: Consultancy; Novartis: Consultancy; Bluebird Bio: Consultancy, Research Funding; Merck: Research Funding; Juno: Consultancy; Celgene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Gamida Cell: Consultancy; Janssen: Consultancy, Research Funding; Takeda: Research Funding. OffLabel Disclosure: VSV-IFNβ-NIS is an oncolytic virus in clinical trial for systemic administration in hematologic malignancies

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aging disease that arises on the background of clonal hematopoiesis, commonly involving multiple TET2 mutations or TET2/SRSF2 co-mutations. In a prior study of 1084 CMML patients (Leukemia 2020; 34:1407-1421), we demonstrated a high prevalence of TET2 mutations (56%) in CMML, with approximately 30% of patients having >1 TET2 mutation. The cell-type and allele specific distribution of these mutations remains of great interest and are thus far poorly defined. To visualize the genotypic and immunophenotypic correlates, we performed single-cell DNA analysis and simultaneous protein profiling on 2 CMML patients with multiple TET2 mutations. Methods: After Mayo Clinic IRB approval, two CMML patients with dual TET2 mutations and available peripheral blood mononuclear cells were included in the study. Patient samples were labeled with TotalSeq TM -D Heme Oncology Cocktail (BioLegend, San Diego, CA) consisting of 45 unique oligo-conjugated cell surface markers. The samples were then washed and loaded into a Tapestri microfluidics cartridge, where single cells (3,500 cells/µL) were encapsulated, lysed and barcoded using the MissionBio Myeloid custom amplicon panel consisting of 312 amplicons in 45 genes relevant to myeloid disorders (MissionBio, San Francisco, CA). The individual barcoded DNA and protein for each cell was purified, recovered, and labeled with Illumina i5/i7 indices, quanitifed by Agilent Bioanalyzer and pooled for sequencing. The libraries were sequenced via Illumina NovaSeq by the Mayo Genomics Core, and data was recovered and analyzed using MissionBio Tapestri Insights v3.0.2. Results: Two dual TET2 mutated CMML patients were included in the study, both male, ages 79 and 83, and both with CMML-0. Case 1 had TET2 p.G1172Efs*53 (variant allele fraction/VAF-6%) and p.H1732Tfs*13 (VAF-13%) mutations, while case 2 had TET2 p.Y620Ffs* (VAF-85%), p.G898* (VAF-82%), and SRSF2 p.95H (VAF-45%) mutations, respectively. In both patients, TET2 mutations were found to be preferentially distributed in monocytes (CD11b+, CD14+, CD64+, CD13+, CD33+, HLA-DR+), with very few classically defined granulocyte (CD16+,CD13+.CD33+, HLA-DR neg), B (CD19+,CD22+)/T (CD2+,CD4+,CD5+,CD7+) lymphocytes, NK cells (CD16+/CD56+/HLA-DR neg) and plasma cells (CD38+/CD138+), demonstrating mutant TET2. Among monocytes, TET2 mutations were enriched in classical/M01 (CD11b+, CD14+,CD16-) and intermediate/M02 monocytes and were infrequent in the MO3 fraction, commensurate with monocyte repartitioning seen in CMML. Interestingly, a large number of single cells with TET2 mutations also demonstrated an aberrant surface phenotype, with universal expression of CD14+ and CD11b+, along with a mixture of other markers (CD163+, CD11c, CD4+, CD2+, and/or CD56+). With regards to allelic distribution, while several permutations and combination were seen involving heterozygosity and homozygosity for the TET2 mutations identified on bulk sequencing, data was corroborative for both TET2 mutations being located on trans alleles. Conclusions: In this study, we demonstrate that TET2 mutations in CMML are highly enriched in classical circulating monocytes and infrequently occur in classical granulocytes, B/T and NK cells; with multiple TET2 mutations most commonly co-existing on trans alleles. Analyses on additional samples along with copy number variation assessments are ongoing. Figure 1: Lollipop plot of TET2 exons with dual TET2 mutations labeled above and oxygenase domain labeled in green; below are number of cells identified with each genotype for Case 1 (A) and Case 2 (B). Mutations are labeled proximal (yellow) and distal (red) and alleles are in grey. The directionality of the arrows to different genotypes is based on decreasing cell number and genotype. Uniform manifold approximation and projection (UMAP) plot of Case 1 (C) and Case 2 (D) with cells clustered by immunophenotype and labeled by genotype (non TET-2 mutated purple, heterozygous TET2 in green, homozygous TET2 in yellow). Relative protein expression of T-cell phenotype (CD14-, CD3+, CD4+/-,CD8+/-, CD5+, CD7+, CD2+), or classical monocytes (CD14+/CD16-) for Case 1 (E & F) and Case 2 (G & H) overlayed on UMAP. Royal blue represents non TET2-mutated cells, and mutated cells are labeled in pastel blue, green and light orange by mutation type. Figure 1 Figure 1. Disclosures Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Abstract Objective : We describe the Mayo Clinic experience in 797 newly-diagnosed patients with acute myeloid leukemia (AML) serially treated with 7+3 induction chemotherapy that included 3 days of daunorubicin at a daily dose of 60 mg/m2 (dauno-60; n=239) or 90 mg/m2 (dauno-90; n=52), or idarubicin 12 mg/m2 (IDA-12; n=506). Our objective was to compare overall (OS) and relapse-free (RFS) survival outcome. Methods : Newly-diagnosed AML patients seen at our institution and received intensive induction chemotherapy were identified from the Mayo Clinic AML database. Treatment period spanned from January 2004 through May 2021. Follow-up information was updated as of June 2021. Conventional criteria were used to diagnose AML, assign cytogenetic risk category, and classify treatment response. Results : The study group included 797 patients (median age 60 years, range 18-88; 58% males): 506 (63%) patients received IDA-12, 239 (30%) dauno-60, and 52 (7%) dauno-90. The respective median (range) ages were 60 (18-88), 61 (19-82), and 53 (22-72) years (p=0.01) (Table). Primary, secondary, and therapy-related AML accounted for 65%, 25% and 10% of patients treated with IDA-12, 69%, 22%, and 9% of those treated with dauno-60, and 75%, 23% and 2% of patients treated with dauno-90, respectively (p=0.1). The corresponding frequencies of adverse karyotype were 34%, 25% and 25% (p=0.05). CR/CRi was documented in 78% (620/793) of all evaluable patients: IDA-12 80% (400/503), dauno-60 75% (175/238), and dauno-90 87% (45/52) (p=0.1). 210 (34%) patients underwent allogenic hematopoietic stem cell transplant (AHSCT), including 125 (25%), 59 (25%) and 26 (50%) patients, in the three treatment groups, respectively (p=0.0004). After a median (range) follow up 19 (0.2-203) months, 348 (54%) relapses and 518 (65%) deaths were documented. Median (range) survivals for IDA-12, dauno-60 and dauno-90 groups were 21 (0.3-243), 14.5 (0.45-198), and 27.7 (0.2-180) months (p=0.07; figure 1). The respective 1-, 3-, and 5-year OS rates were 67%, 42%, and 34% (IDA-12); 66%, 37%, and 30% (dauno-60); and 78%, 51%, and 49% (dauno-90), respectively; the trend favoring dauno-90 was no longer apparent during age-adjusted analysis (p=0.33). Multivariable analysis that accounted for age, cytogenetic risk category, FLT3-ITD/NPM1 status and AML subtype confirmed the lack of additional contribution from IDA-12 vs dauno-60 vs dauno-90 (p=0.2) while affirming the independent prognostic value of the other four variables; AHSCT carried an additional predictive value for superior survival without altering these results. A total of 348 (54%) relapses were documented: 228 (57%) in the IDA-12; 99 (57%) in the dauno-60; and 21 (47%) in the dauno-90 cohorts (p=0.7); RFS was similar in the three treatment groups (p=0.1; figure 2). Conclusion :In the current large single-institution study of consecutive adult patients with AML, neither the choice of anthracycline (idarubicin vs daunorubicin) or the dose of daunorubicin (60 vs 90 mg/m2) appeared to effect outcome in terms of remission rates or overall or relapse-free survival. The study otherwise confirms the independent favorable effect of younger age, non-adverse karyotype, FLT3-ITD-/NPM1+ status, and AHSCT. Figure 1 Figure 1. Disclosures Patnaik: Kura Oncology: Research Funding; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees. Al-Kali: Astex: Other: Research support to institution; Novartis: Research Funding. Litzow: Pluristem: Research Funding; Actinium: Research Funding; Amgen: Research Funding; Jazz: Other: Advisory Board; Biosight: Other: Data monitoring committee; Omeros: Other: Advisory Board; AbbVie: Research Funding; Astellas: Research Funding.

Abstract Introduction: Therapy-related myeloid neoplasm (t-MN) is defined as the development of a myeloid neoplasm in the context of prior DNA damaging therapies. t-MN is an aggressive disease with no effective therapies. BCL2-inhibitor venetoclax (VEN) represents a novel treatment for acute myeloid leukemia (AML) patients including those with high-risk features. Available VEN studies included only a small portion of t-AML patients, and its efficacy in t-MDS is not known. Our aim was to study the efficacy and outcomes of VEN in t-MN. Methods: Patients diagnosed with t-MN based on the WHO criteria who received VEN were identified. Minimal residual disease (MRD) assessment was performed in a subset of patients by multiparametric flow cytometry or molecular marker as available. Best response to VEN was divided into 3 categories: 1) MRD(-) complete remission (CR); 2) other CR [including complete morphological remission, MRD(+) CR, and CR with incomplete count recovery]; and 3) partial response/progressive disease/stable disease (PR/PD/SD). Progression-free (PFS) and overall survival (OS) were calculated from the time of initiation of VEN therapy using Kaplan-Meier analysis using Wilcoxon test. Logistic regression analysis was performed using Cox Proportional Hazard method. Statistical analysis was performed using JMP (v14.1, SAS Institute) and significance was defined as P<0.05. Results: Of 332 t-MN patients identified (248 at Mayo Clinic, 84 at the Central Adelaide Health Network), 65 (19.6%) received VEN. Clinical and pathological characteristics of patients that received VEN and did not receive VEN (non-VEN) were matched except: a higher proportion of VEN patients had t-AML (49.2% vs. 32.6%, P=0.004), higher peripheral (2% vs. 0%, P=0.02), and bone marrow blasts (18% vs. 8%, P=0.02). VEN patients had a higher proportion of abnormality of chromosome 17, complex karyotype, and monosomal karyotype compared to non-VEN patients. The proportion of patients with pathogenic/likely pathogenic variant (PV) in TP53 or RAS genes was not different. The proportion of patients receiving stem cell transplant for t-MN was not different (Table 1). The indication for VEN was treatment naïve t-MN, relapsed/refractory (R/R) disease, and maintenance in 31 (47.7%), 32 (49.2%), and 2 (3.1%) patients respectively. Prior to VEN, patients received a median of 1 (range 0-4) lines of therapy. The median duration of VEN exposure was 3 cycles (IQR 1-4). VEN was administered with azacitidine, decitabine, low dose cytarabine, intensive chemotherapy, single agent, and >1 agents in 37.9%, 40.9%, 12.1%, 4.5%, 4.5%, and 2.9% respectively. The best and the final responses to VEN are shown in Table 2. Median PFS and OS from initiation of VEN was 5 months (IQR 2-9.5, Fig. A) and 6.5 months (2-14.5, Fig. B) respectively. Female sex (vs. male, χ 2 9.6, P=0.03), VEN as the 1 st line of therapy (vs. other line of therapy, χ 2 8.3, P=0.02), and hypomethylating agent (HMA)-backbone (vs. other, χ 2 12.1, P=0.02) were associated with a significantly higher likelihood of achieving CR. Abnormality of chromosome 7 was associated with a shorter PFS (3.5 vs. 6 months, P=0.03) and OS (4.7 vs. 10.5 months, P=0.02). Using VEN as the 1 st line therapy was associated with an improved PFS (5.5 vs. 2.7 months, P<0.01) and a trend towards improved OS (9.3 vs. 5.1 months, P=0.06). Using HMA (n=51), Ara-C based (n=8), and single agent VEN (n=3) was associated with median PFS of 5.5, 4.2, and 1.5 months (P<0.01) and OS of 9.1, 4, and 1.5 months (P<0.01, Fig. C) respectively. Achieving MRD(-) CR (n=7), other CR (n=17), and PR/PD/SD (n=36) was associated with a median PFS of 7.7, 9.5, and 2 months (P<0.001) and median OS not reached, 12.9, and 2.9 months respectively (P<0.01, Fig. D). Finally, best response rate, PFS, and OS did not differ based on morphological diagnosis [t-MDS vs. t-AML] at diagnosis or bone marrow blast percent [<20% vs. ≥20%] at the start of VEN therapy. On multivariate analysis, only the best response remained an independent predictor of PFS and OS. Conclusion: VEN induced remission, including MRD(-) remission, in a subset of patients. However, progression was noted in 83% patients with continued use, and PFS and OS were overall short. The morphological diagnosis of t-MDS/t-AML or blast percentage at the start of VEN therapy did not impact outcome. Earlier use of VEN, using HMA-based therapies, and achieving deeper responses were associated with improved outcomes. Figure 1 Figure 1. Disclosures Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding. Wei: Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tiong: Pfizer: Consultancy; Amgen: Speakers Bureau; Servier: Consultancy, Speakers Bureau. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: venetoclax is not approved in the treatment of myelodysplastic syndrome.

Abstract Introduction: VEXAS syndrome (vacuoles, E1 ubiquitin ligase, X-linked, autoinflammatory, somatic) is a newly recognized inflammatory disorder caused by somatic mutations in the UBA1 gene. Bone marrows from these patients reveal a range of morphological changes in hematopoietic precursor cells. In this study, we aim to assess the laboratory indices and morphologic spectrum of bone marrow pathology in VEXAS syndrome. Methods: We identified 16 cases of VEXAS syndrome. All cases had confirmed UBA1 mutation. We reviewed bone marrow biopsies corresponding to the date of diagnosis. This study was approved by the Mayo Clinic Institutional Review Board. Results: All patients were male with a median age of 73 years - associated autoimmune disorders included Sweet syndrome, inflammatory arthritis, relapsing polychondritis and granulomatosis with polyangiitis. 14/16 patients had anemia with median hemoglobin of 10.4 (Range: 6.7- 14.1 g/dL). 15/16 had macrocytosis with median MCV 110.4 (Range: 94.8- 123.1 /fL). 5/16 had thrombocytopenia with median platelet count 174 (Range: 20- 500 x10^9/L). 7/16 had leukopenia with median WBC 3.65 (Range: 2.4- 11.6 x10^9/ L). The ESR and CRP medians were 61.0 mm/hr and 81.5 mg/L, respectively. Karyotype was performed in 12 patients of which 11 were normal and the remaining case showed a complex karyotype. An NGS panel targeting the most frequent myeloid disorder associated gene mutations was negative in 10/15 cases. GS for myeloid mutations revealed pathogenic mutations in 5 patients, involving genes TET2 (2/5), DNMT3A (2/5), and TP53 (1/5). Conclusions: Bone marrow findings in VEXAS syndrome, in this series of 16 patients, are individually non-specific, yet when taken altogether in the overtly abnormal cases, are very suggestive when the clinical index of suspicion is high. In such scenarios, the combined clinical and bone marrow findings should prompt discussion and consideration for UBA1 mutation testing given the significant clinical implications for patient management and prognosis. Disclosures Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding. Warrington: Eli Lilly: Research Funding; Kiniksa: Research Funding.