Gangat, Naseema

Background: Sodium-glucose co-transporter-2 inhibitors (SGLT-2I) are FDA-approved for diabetes mellitus, heart failure and chronic renal disease. Clinical trials on their anti-diabetic and cardiorenal protective effects showed an association with erythrocytosis. We have previously reported on the benign nature of SGLT-2I associated JAK2-unmutated erythrocytosis ( Blood 2021). Recently, we described the same phenomenon in the setting of a previously unrecognized myeloproliferative neoplasm (MPN), which led to unmasking of underlying MPN and thrombosis in 3 (33%) of 9 informative cases ( AJH 2023). The main objective of the current study was to assess the erythropoietic and thrombotic impact of SGLT-2I therapy in patients with a well-established MPN diagnosis, prior to treatment initiation. Methods: The Mayo Clinic MPN database was queried to identify patients prescribed SGLT2-I after diagnosis of essential thrombocythemia (ET), polycythemia vera (PV) or myelofibrosis (MF). Baseline Hgb/Hct at SGLT-2I initiation, peak Hgb/Hct, absolute change in Hgb/Hct levels, and thrombosis during treatment were recorded. Results: SGLT-2I use in essential thrombocythemia Eleven patients with ET (median age; 72 years, 55% males, 73% JAK2 mutated, 64% with thrombosis history) received SGLT-2I. Active treatments included aspirin ( n=10), cytoreductive therapy ( n=10) and systemic anticoagulation ( n=4). All 11 patients displayed increase in Hgb/Hct following SGLT-2I treatment. Baseline median (range) values for Hgb/Hct were 12.8 g/dL (9.5-14.9)/38.9% (30.3-44.6); median (range) peak Hgb/Hct levels during SGLT-2I therapy, were 14.5 g/dL (11-16.4)/44.4% (34.8-51.1), with median (range) increase in Hgb/Hct of 1.5 g/dL (1-4)/5.1% (2.6-13.8); 2 patients (18%), exceeded the proposed ICC Hgb/Hct diagnostic threshold for PV (Patient 7 and 8) (Figure 1). JAK2 mutation/VAF (p=0.54/ 0.20), or serum erythropoietin (Epo) (p=0.48) did not correlate with changes in Hgb/Hct levels. One patient experienced thrombosis, despite treatment with hydroxyurea and aspirin and Hgb/Hct levels below the threshold of concern (11.4 g/dL/37.7%). SGLT-2I use in polycythemia vera Nine patients with PV (median age 74 years, 66% males, JAK2 median VAF 14%, two patients with thrombosis history) received SGLT-2I. Concomitant treatments included aspirin ( n=9), cytoreductive therapy ( n=8), and systemic anticoagulation ( n=3). Phlebotomy intensity, defined as the number of phlebotomies, six months prior to and after SGLT-2I initiation, ranged from 0-3 and 0-12, respectively. Documented Hgb/Hct levels showed an increase in all 9 cases with baseline median (range) values for Hgb 14.1 g/dL (10.5-15.9) and Hct 42.7% (33.3-49.2) and peak median (range) values for Hgb/Hct, 15.4 g/dL (11.2-19.9)/46.7% (35.9-63.3). JAK2 VAF (p=0.85), and serum Epo (p=0.43) did not correlate with increase in Hct. Figure 2 highlights five patients (56%) with Hct exceeding 45% while on SGLT-2I; two patients (22%) had increased phlebotomy requirements; 6-month pre/post SGLT-2I phlebotomies for patient 5 and 9 were 3 and 6, and 0 and 12, respectively. Patient 9, experienced thrombosis in the context of uncontrolled Hgb/Hct level at 19.9 g/dl/63.3% and absence of cytoreductive therapy. SGLT-2I use in myelofibrosis Eight patients with myelofibrosis (primary 6, post-ET 2; median age 71 years; 50% males; 63% JAK2 mutated; MIPSS-70 2.0 int/high 6; on ruxolitinib 4),received SGLT-2I. Baseline median (range) values for Hgb/Hct were 10.6 g/dL (8.2-14.7)/27.5% (33.8-45.9); two (25%) patients displayed anemia with Hgb 8-10 g/dl. Peak median (range) values for Hgb/Hct were 11.5 g/dL (10.2-16.1)/35.3% (31.8-49.8), with median (range) baseline increase in Hgb/Hct of 1.6 g/dL (0.7-3.8)/4.3% (1-7.5). Patient 2 and 4 with baseline Hgb 8.2 g/dl and 9.4 g/dl, and not receiving ruxolitinib, had Hgb increase of 2 g/dl and 1 g/dl, respectively (Figure 3). One thrombotic event occurred during therapy with Hgb/Hct level of 11.6 g/dl/37.3%. Conclusion: The current study suggests that SGLT-2I therapy in MPN patients leads to increase in Hgb/Hct levels, regardless of disease subtype, and in some cases surpass the PV diagnosis and thrombosis risk thresholds. These observations mandate increased awareness to secure patient safety and diagnostic accuracy. The erythropoietic effect of SGLT-2I in MF suggests its potential use for MF-associated anemia.

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with myelofibrosis (MF) and anemia. Chronic elevations in hepcidin contribute to the onset and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin, a co-receptor in the bone morphogenetic protein-signaling pathway driving hepcidin expression. Preclinical studies have shown that DISC-0974 suppresses hepcidin and increases serum iron. A healthy volunteer study has demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously, and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In this Phase 1b/2a, open-label, multiple-ascending dose study (NCT05320198), we are assessing the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with MF and anemia. Eligible participants include patients over 18 years of age with intermediate-2 or higher-risk MF and anemia. MF is required to be confirmed using the World Health Organization 2016 criteria. Anemia is defined per protocol as Hgb < 10 g/dL or transfusion dependence, as defined by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT). A stable dose of hydroxyurea and/or Janus kinase (JAK) inhibitor is allowed on trial. Major exclusion criteria include: liver iron concentration ≥ 7 mg/g dry weight; splenectomy; nutritional, genetic, infectious, or autoimmune causes of anemia. Dose escalation is based on a Bayesian optimal interval design with accelerated titration. Safety Review Committee assessments gate escalation decisions. Dosing occurs monthly for a total of 6 doses. Primary endpoints include safety and tolerability assessments of adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include anemia response as defined by the IWG-MRT, transfusion response as defined by a decrease of 50% in transfusion requirement over any 8-week period, standard PK parameters and PD metrics including serum hepcidin-25, iron, transferrin saturation, ferritin, and hematology biomarkers. All data are summarized using descriptive statistics. Initial data show a dose-related increase in pharmacokinetic parameters. DISC-0974 treatment led to dose-dependent reductions in serum hepcidin-25 and increases in serum iron levels. There have been no serious AEs or AEs leading to study withdrawal. Most AEs were mild, transient, and unrelated to DISC-0974. As of August 1, 2023, enrollment and dose escalation are ongoing. Safety, PK, and PD data from at least the first two cohorts will be presented.

Background: Venetoclax (Ven) in combination with hypomethylating agent (HMA) is the preferred treatment for elderly/unfit patients with newly-diagnosed acute myeloid leukemia (AML). Current prognostication for AML is based on European LeukemiaNet (ELN) genetic risk stratification which has limited applicability in patients treated with Ven-HMA ( Blood, 2022). Accordingly, our primary objective was to determine predictors of treatment response and survival outcomes in treatment-naïve AML patients receiving Ven-HMA. Methods: Our study population was recruited from Mayo Clinic (MN, FL, AZ), after institutional review board approval and based on documentation of newly-diagnosed AML treated with Ven-HMA outside of clinical trials between November 2018 and April 2023. Cytogenetic and molecular studies were performed by conventional karyotype, and next-generation sequencing, respectively. Response was assessed according to the 2022 ELN criteria Results: Patient characteristics A total of 301 newly-diagnosed AML patients (median age 73 years, 66% male, 62% de novo) received a median of 3 cycles (range 1-48) of azacitidine 75 mg/m2 days 1-7 ( n=100) or decitabine 20 mg/m2 days 1-5 ( n=201) with Ven. ELN 2022 cytogenetic risk included favorable (1%, n=4), intermediate (61%, n=184) or adverse (38%, n=113). Mutations involved TP53 in 77/301 (25%), ASXL1 in 54/279 (19%), RUNX1 in 54/296 (18%), NPM1 in 44/299 (15%), DNMT3A in 40/296 (13%), K/NRAS in 41/296 (14%), IDH1 in 20/301 (6%), IDH2 in 30/301 (10%), FLT3-ITD in 25/301 (8%) and DDX41 in 9/247 (4%) of informative cases. Predictors of response Complete remission, with (CR) or without (CRi), count recovery, was documented in 182 (60%) patients, including CR in 36% and measurable residual disease (MRD) negative by flow cytometry in 77% of 105 informative cases that achieved CR/CRi. Median time to CR/CRi was 1.4 months. In univariate analysis, CR/CRi was more likely to occur in the presence of NPM1 mutation, (86% vs. 56%; p < .01), IDH2 mutation, (80% vs. 58%; p = .02), DNMT3A mutation, (78% vs. 57%; p = .01), or DDX41 mutation, (100% vs. 58%; p <.01), and in the absence of adverse karyotype, (71% vs. 42%; p < .01), TP53 mutation, (67% vs. 40%; p <.01), FLT3-ITD mutation, (63% vs. 36%; p = .01), or RUNX1 mutation, (64% vs. 44%; p = .01); in multivariable analysis, adverse karyotype, ( p < .01), FLT3-ITD ( p < .01), RUNX1 ( p < .01), NPM1 ( p = .03), IDH2 ( p = .04), and DDX41, ( p = .01) mutations remained significant. Predictors of survival After a median follow-up of 8.5 months (range 0.5-56), 13.2 months for living patients, 174 (58%) deaths, 73 relapses (40% of those achieving CR/CRi), and 41 (14%; including 35 in CR/CRi) allogeneic hematopoietic stem cell tranplants (AHSCT) were documented. In univariate analysis of pre-treatment variables, thrombocytopenia < 100 x 10 9/l, (HR 1.5, 95% CI 1.03-2.2; p = .03), adverse karyotype, (HR 2.8, 95% CI 2.1-3.8; p < .01) TP53 mutation, (HR 2.5, 95% CI 1.8-3.5; p < .01), or absence of IDH2 mutation, (HR 3.5, 95% CI 1.7-7.6; p < .01) predicted inferior survival. Failure to achieve CR/CRi (HR 4.5, 95% CI 3.3-6.1; p < .01) was also associated with inferior survival and in multivariable analysis, failure to achieve CR/CRi, (HR 3.4, 95% CI 2.5-4.8; p< .01), presence of adverse karyotype, (HR 1.6, 95% CI 1.1-2.6; p = .02), TP53 mutation, (HR 1.6, 95% CI 1.002-2.4; p = .04), and absence of IDH2 mutation (HR 2.2, 95% CI 1.004-4.7; p = .04) were identified as risk factors for survival. Subsequent HR-weighted scoring resulted in three-tiered risk stratification: low (0-1 point; n= 130), intermediate (2-3 points; n= 105), and high (4-5 points; n= 66), with respective median survival (3-year rate) of 28.9 (48%), 9.6 (6%), and 3.1 (0%) months ( p < .01) (Figure). AHSCT had an independent favorable impact on survival (HR 0.3, 95% CI 0.1-0.6; p < .01), most apparent in low ( p =0.04), and intermediate ( p <.01), as opposed to high ( p = .06) risk. Conclusions: In the current single institutional series of Ven-HMA treated newly-diagnosed AML, response to Ven-HMA was the foremost predictor of survival. Additional risk factors for survival included adverse karyotype, presence of TP53 and absence of IDH2 mutations. These observations allowed for a three-tiered genetics-enhanced survival model with CR/CRi as a backbone, and also confirmed survival advantage from AHSCT, regardless of risk category.

Background: Non-transplant treatment options in myelofibrosis (MF) are palliative in scope and include JAK2 inhibitors (JAKi): ruxolitinib, fedratinib, pacritinib, and momelotinib. All four JAKi are effective in reducing spleen size and alleviating symptoms. In addition, pacritinib and momelotinib exhibit erythropoietic effect, attributed to alleviation of ineffective erythropoiesis through inhibition of activin A receptor type-I. In the current study, we sought to identify molecular or clinical predictors of response to pacritinib in MF patients treated outside of clinical trials. Methods: The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patient records (Mayo Clinic, Rochester, MN; Scottsdale, AZ; and Jacksonville, FL). Anemia response assignment was restricted to patients with baseline hemoglobin level of <10 g/dL and required, for transfusion dependent cases, achieving transfusion independence and, for transfusion-independent cases, an increase in hemoglobin of ≥1.5 g/dL, both sustained for a minimum 2 months; spleen response was clinically vetted but was also supported by radiologic imaging when available and required a ≥50% decrease in palpable splenomegaly for spleen size >10 cm or the spleen becoming not palpable for a spleen size 5-10 cm at base line. Symptom response was adjudicated by central review and required a marked or moderate improvement to qualify as response; because of the retrospective nature of the study, symptom burden assessment by formal criteria was not attempted. Leukocyte cutoff level used for response analysis was determined by receiver operating characteristic curve analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: We identified 36 NGS-annotated patients with MF who received pacritinib therapy between April,2022 and June,2023 (median age 66 years; 56% males). Twenty-five (69%) patients had failed previous JAKi therapy while 11 (31%) were JAKi-naïve. Eighteen (50%) patients were transfusion-dependent while the median (range) counts were 106 x 10(9)/L (14-578) for platelets, 6.6 x 10(9)/L (1.2-112.8) for leukocytes, and 9 x 10(9)/L (5-12.4) for hemoglobin; leukocyte count was ≥15 x 10(9)/L in 11 (31%) patients and hemoglobin <10 g/dL in 29 (81%). Karyotype was unfavorable in 12 (34%) patients. Driver mutation distribution was JAK2 55%, CALR 27%, MPL 11%, and triple-negative 5%. NGS revealed mutations involving ASXL1 36%, U2AF1 13%, IDH1 13%, SF3B1 13%, K/NRAS 5%, SRSF2 8%, and EZH2 5%. Pacritinib was started at 200 mg BID in 9 (25%) patients and at lower doses in the remaining. Median duration of pacritinib therapy was 4 months (range 1-15). Anemia response was documented in 5 (17%) of the 29 patients with baseline hemoglobin <10 g/dL, spleen response in 12 of 28 evaluable (42%) and symptom response in 18 (50%). Improvement in symptoms was always accompanied by both anemia (p=0.008) and spleen (p=0.01) response; by contrast, there was no significant correlation between anemia and spleen responses (p=0.9) (Table 1). Significant correlations with molecular or clinical variables were mostly apparent for spleen response, which was more likely to occur in the absence of ASXL1 mutation (61% vs 10%; p=0.006), with leukocyte count <15 x 10 (9)/L (55% vs 0%; p=0.005), and in JAKi-naïve vs previously-exposed cases (75% vs 30%; p=0.03; Table 1); significance was sustained by all three risk factors during multivariable analysis; the prognostic impact of ASXL1 (p=0.02 vs 0.01) and leukocytosis (p=0.01 vs 0.07) was apparent in both JAKi-naïve vs JAKi-exposed cases. Anemia response was non-significantly (p=0.27) higher in transfusion-independent (27%) vs dependent (11%) cases, at baseline, and was more likely (p=0.04) to occur in patients starting at lower dose levels. Treatment-emergent adverse events included diarrhea 44%, nausea/vomiting 19%, and grade-3/4 thrombocytopenia 38% or neutropenia (11%). Conclusions: ASXL1 mutation and leukocytosis (>15 x 10 9/L) are independently associated with inferior spleen response to pacritinib, in both JAKi-naïve and JAKi-exposed myelofibrosis. These observations are important for individualized treatment selection.

Background: According to the international consensus classification (ICC), diagnosis of myelodysplastic syndromes (MDS) with del(5q) requires presence of i) del5q, alone or associated with no more than one other cytogenetic abnormality, exclusive of -7/del(7q), ii) <5% bone marrow (BM) and <2% circulating blasts, and iii) the absence of “multi-hit” TP53 ( Arber et al. Blood 2022;140: 1200). We have recently reported on TP53 variant allele frequency (VAF) ≥22% being the sole genetic predictor of inferior survival in MDS-del(5q) (Fleti. et al. AJH 2023;98:E76). In the current study, we examined the additional prognostic impact of therapy-related setting, in the context of TP53 VAF and other risk factors. Methods : The current study included patients with MDS-del(5q) who were seen at the Mayo Clinic or Moffitt Cancer Center, USA. Diagnosis of MDS-del(5q) and therapy-related qualification was according to ICC criteria ( Arber et al. Blood 2022;140: 1200), with the exception that patients with biallelic or multi-hit TP53 were included to allow unbiased analysis of impact on prognosis. Patient selection was based on availability of mutation information from next-generation sequencing (NGS). The latter was assessed either at diagnosis or at different time points during the disease course, but at least 3 months before leukemic transformation. Otherwise, all analyses, including estimation of overall and leukemia-free survival, were based on variables collected at time of initial diagnosis of MDS-del(5q). TP53 mutation VAF threshold of 22% was considered based on our previously published report cited above. Age cutoff at 67 years was determined by ROC analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: A total of 210 patients (median age 72 years; 65% females), meeting the above-stipulated criteria for MDS-del(5q), were considered: 172 primary and 38 therapy-related. Table 1 outlines clinical and laboratory characteristics obtained at the time of diagnosis: 42% were transfusion-dependent, 69% had hemoglobin level <10 g/dL, 30% platelet count <150 x 10(9)/L, and 15% carried additional cytogenetic abnormality. NGS data performed either at diagnosis or at least 3 months prior to leukemic transformation was available in 146 patients; the most frequent mutations were TP53 (21%), SF3B1 (16%), DNMT3A (12%), TET2 (12%), ASXL1 (10%), and JAK2 (7%) (Table 1). At a median follow-up of 3.9 years (range 0.002-18.5) for living patients, 95 (45%) deaths and 34 (16%) leukemic transformations were recorded; treatment undertaken included lenalidomide in 162 (77%) cases. NGS data-exclusive multivariable analysis that included all 210 patients identified therapy-related setting (p<0.01; HR 2.9; Figure 1a), age >67 years (p=0.01; HR 1.8), transfusion-dependency (p=0.04; HR 1.5), and platelet count <150 x 10(9)/L (p=0.03; HR 1.6), as independent predictors of inferior survival. The significant effect of age and platelet count was sustained when analysis was limited to patients with primary MDS-del(5q) but lost when applied to therapy-related disease. NGS data-inclusive multivariable analysis, which was applicable to the subset of 146 patients, identified, as risk factors, therapy-related setting (p<0.01; HR 2.3) and TP53 VAF ≥22% (p<0.01; HR 2.4; Figure 1b) while age >67 years sustained borderline significance (p=0.07; HR 1.8). Figure 1c depicts survival data based on the presence or absence of TP53 VAF ≥22% and therapy-related qualification; low-risk with no risk factors (N=112; median survival 11.7 years; 5/10-year survival 73%/52%) and high-risk with one or both risk factors (N=34; median survival 4 years; 5/10-year survival 42%/14%); leukemia-free survival was affected by TP53 VAF ≥22% (p<0.01) but not therapy-related setting (p=0.13). Conclusions : The current study highlights TP53 VAF and therapy-related setting as the most prominent risk factors for survival in MDS-del(5q) and proposes a simple risk model that distinguishes patients with a projected 10-year survival that exceeds 50%, in the absence of both risk factors, and plummets to 14%, in the presence of one of the two risk factors. Accordingly, allogeneic stem cell transplant might be deferred in the former instance while it should be pursued sooner than later, in the latter instance.

Background: Biallelic TP53 alterations result from either sequence variations or deletions involving TP53 and are not necessarily synonymous with “multi-hit TP53”. According to the 2022 international consensus classification (ICC) for myeloid neoplasms (MN), multi-hit TP53 signifies i) the presence of two or more distinct TP53 mutations, each with variant allele frequency (VAF) ≥10%, ii) a single TP53 mutation with VAF ≥50%, or iii) a single TP53 mutation with VAF ≥10% accompanied by a cytogenetically-apparent del(17p13.1), copy-neutral loss of heterozygosity (LOH) at the 17p TP53 locus, or, in the absence of LOH information, complex karyotype ( Arber et al. Blood 2022;140: 1200). The current study is focused on MN with multi-hit TP53 and examines the additional prognostic impact of morphologic subtype designation, bone marrow (BM) or peripheral blood (PB) blast percentage, TP53 VAF, and MN with diagnostic qualifiers (i.e., therapy-related, or secondary progressing from myelodysplastic/myeloproliferative/overlap syndromes/neoplasms). Methods : The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Multi-hit TP53 was defined as per ICC criteria as outlined above ( Arber et al. Blood 2022;140: 1200). Morphologic subtype designations of MN were according to ICC criteria and assigned at the time of TP53 detection. NGS and cytogenetic information was available in all study patients. Survival analyses were calculated from time of TP53 mutation detection. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results : Initial screening flagged 143 patients with biallelic TP53 abnormalities derived from formal laboratory reports of NGS data and cytogenetic studies; of these, 130 met ICC criteria for multi-hit TP53: pure erythroid leukemia (PEL; N=24), acute myeloid leukemia (AML)-not PEL (N=54), myelodysplastic syndromes (MDS; N=36), MDS/AML (N=11), and other MN (N=5). Of these 130 informative cases, 128 (98%) harbored complex/monosomal karyotype (CK/MK). Further analysis excluded patients with “other” MN (N=5) and those without CK/MK (N=2), the latter to mitigate the confounding effect of CK/MK on survival and the former because of small sample size and disease heterogeneity. Clinical and laboratory characteristics of the 124 study patients are outlined in table 1. Survival analysis stratified by ICC-defined MN subtypes revealed the prognostic relevance of morphological distinction between PEL vs “AML-not PEL” (p<0.01; HR 2.4) and PEL vs “ TP53-muated MDS/AML” (p=0.02; HR 2.3) while survival was similar between “AML-not PEL” and “ TP53-mutated MDS/AML” (p=0.9; Figure 1a). Survival in “ TP53-mutated MDS” was significantly longer, compared to PEL (p<0.01; HR 0.2), “ TP53-mutated AML-not PEL” (p=0.01; HR 0.5), and “ TP53-mutated MDS/AML” (p=0.07; HR 0.5), the latter with borderline significance (Figure 1a). Multivariable analysis confirmed the independent prognostic relevance of ICC subtype designation (p<0.01) and also revealed additional negative prognostic contribution from advanced age (p=0.02), male gender (p=0.02), MN, secondary (p<0.01), MN, therapy-related (p=0.03), and DNMT3A mutation (p=0.02). Significance was retained in all instances, with the exception of MN, therapy-related (p=0.15), when analysis was repeated after excluding PEL cases (Figure 1b). Conclusions : The current study confirms the prognostic validity of the ICC morphologic subtype designation and TP53 VAF classification threshold, in the context of multi-hit TP53. The study also highlights the prognostic distinction between PEL and “ TP53-mutated AML-not PEL” and the prognostic alignment between the latter and “ TP53-muated MDS/AML”, both of which displayed inferior survival, compared to “ TP53-mutated MDS”. The study also suggests additional prognostic contribution from secondary and therapy-related qualification while the observation regarding DNMT3A mutation requires validation with higher number of informative cases.

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.

BACKGROUND: Recently proposed World Health Organization (WHO, 5 th edition) and International Consensus Classification (ICC) recognized myeloid neoplasms (MN) harboring TP53-mutated ( TP53mut) as a separate entity. However, there are critical differences between the two classifications regarding allelic-status, variance allele frequency (VAF) cut-off and blast categories, that may lead to under- or overestimation of the risk. METHODS: We analyzed MN harboring TP53mut (VAF ≥2%, n=536) managed at Mayo Clinic (USA) and South Australia Health Network (Australia). ICC (Arber et al, Blood 2022) and WHO (Bluebook, Beta v2) definitions were used to stratify MN harboring TP53mut. RESULTS: Of 536 cases, 379 (71%) had myelodysplastic syndromes (MDS) (<20% blasts) and 157 (29%) had acute myeloid leukemia (AML). Using WHO, 160 (42%) of 379 were classified as MDS-bi TP53 as they harbored 2 TP53mut (n=115; 30%) or 1 TP53mut plus confirmed loss of heterozygosity (LOH) or copy neutral LOH (cnLOH) of TP53 locus (n=45; 12%). Sixty-two (16%) TP53mut MDS with VAF >49% were classified as presumptive MDS-bi TP53. Of the 157 cases with 1 TP53mut VAF ≤49%, 17 confirmed to have an intact wild-type TP53 copy and were classified as monoallelic. While 140 (89%) cases did not have copy number variation (CNV) analysis. We next evaluated the number of TP53mut cases classified differently by WHO and ICC. As WHO does not recognize TP53mut AML as a separate entity, 157 (29%) TP53mut AML were not classifiable. 46% (174/379) of the TP53mut MDS including 48 (30%), 12 (19%) and 79 (65%) of bi TP53, presumptive bi TP53 and monoallelic TP53mut MDS according to WHO were classified differently by ICC ( Figure 1A). Discordance between the two classifications were predominantly driven by: (i) The difference in VAF cut-off. Sixty patients (11%) with TP53mut VAF <10% were excluded from the ICC classification of TP53mut MN as it mandates TP53mut VAF cut-off ≥10%. 11 (18%) and 41 (68%) of these had bi TP53, monoallelic TP53mut MDS by WHO criteria while 8 (13%) had AML. Importantly, within the VAF <10% cohort, median overall survival (OS) of 2 TP53mut or 1 TP53mut with 17p13.1 (hereafter 17p) deletion or complex karyotype (CK, n=30) was significantly poor compared to 1 TP53mut VAF <10% (11.6 vs 32.4 months; P =0.002, Figure 1B), suggesting that ICC may underestimate the poor prognosis of multi-hit or multi-hit equivalent TP53mut MN with VAF <10%. (ii) The difference in bone marrow blast cut-off. The underlying assumption by WHO is that prognosis of bi TP53mut MDS is poor irrespective of blast percentage category (0-9% vs 10-19%). In contrast, ICC considers that TP53mut MDS/AML (blasts 10-19%) is associated with poor prognosis irrespective of TP53 allelic status. Median OS of TP53mut MDS with 10-19% blasts was significantly poor compared to blast 0-9% (6.4 vs 11.6 months, P <0.0001). Second, median OS of bi TP53mut was significantly poor than monoallelic TP53mut in MDS- blast 0-9% (10.1 vs 15.9 months; P =0.0001) but not in blasts 10-19% (6.8 vs 6.2 months; P =0.19). Hence, exclusion of monoallelic TP53mut MDS with blast 10-19% as TP53mut MN from the WHO classification may underestimate the poor prognosis. (iii) The difference in the definition of biallelic and multi-hit TP53 loss. 35 cases with 1 TP53mut VAF ≤49%, had 17p deletion but did not have CNV analysis to confirm the loss of TP53 locus and could not be classified further using WHO. ICC classified 18 (50%) of these cases as multi-hit. Median OS of 1 TP53mut VAF ≤49%, and 17p deletion on karyotype was significantly poor than 1 TP53mut (VAF ≤49%without 17p deletion on karyotype (6.4 vs 14.6 months, P =0.001). (iv) The difference in the definition of multi-hit equivalent. 32% (33/104) WHO defined monoallelic cases without available CNV data, were classified as multi-hit-equivalent by ICC as they harbored CK. However, WHO does not consider CK as presumptive evidence of bi TP53. Median OS of 1 TP53mut VAF ≤49% with CK was significantly poor compared to 1 TP53mut without CK (8.2 vs 26.9 months, P =0.0008). CONCLUSION: Our study provides evidence that poor prognosis of ‘multi-hit’ TP53 with VAF <10% is underestimated by ICC. On the other hand, poor prognosis of monoallelic TP53 MDS with 10-19% blasts as well as 1 TP53mut VAF ≤49% in the presence of CK is underestimated by WHO. These data suggest that both classifications can be further refined.

572 Background: The DEFeNDR (Decreasing the burden of Febrile Neutropenia through Dynamic RPM) program leverages in-home technology and centralized nurse monitoring to facilitate ambulatory management as an alternative to hospital-based care for cancer patients with neutropenic fever (NF). The objective of this study is to assess 30-day care utilization outcomes and patient satisfaction at 12 months after program implementation. Methods: Adult cancer patients hospitalized with NF were assessed daily for DEFeNDR program eligibility including MASCC score ≥21 (per ASCO guidelines) and no cellular therapy (CAR-T, BMT) in the prior 100 days. Patients agreeing to participate in the 30-day program received a cellular-enabled tablet with pre-connected devices for twice-daily vital signs and symptom assessments. Patient generated data were EHR-integrated in real-time. Embedded decision trees facilitated alerts for adverse trends. Virtual RPM nurses responded to alerts and utilized predefined care pathways. The primary endpoint was the mean proportion of evaluable days spent inpatient (mPEDI) within 30 days of eligibility comparing those who participated in the program (RPM) with those who declined (no RPM). Results: Of 85 eligible patients, 62 were offered DEFeNDR participation. Of those, 34 (54.8%) participated and 28 (45.2%) declined. Baseline characteristics and care utilization outcomes are summarized in Table 1. RPM patients experienced significantly lower mPEDI compared with unmonitored patients [5.1% (Standard Error [SE], 1.9%) vs 12.4% (SE, 2.1%); Wilcoxon Rank Sums p-value, 0.032]. RPM patients also experienced a trend toward fewer total hospital days and lower 30-day readmission rate, as well as shorter mean length of stay and lower rate of ICU utilization when readmitted. Non-visit care activities (EHR clinical communications, documentation, and patient messaging) were decreased from pre-hospital baseline for 58.8% of RPM patients versus 46.6% of those unmonitored. All RPM patients completing a patient satisfaction survey (n=9) indicated that they would recommend the program to patients with a similar condition. Conclusions: Cancer patients with NF participating in the DEFeNDR program experienced a significant reduction in mPEDI and care utilization compared with those who declined. Further studies are warranted to assess comparative effectiveness of this model vs usual care.[Table: see text]