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Abstract Background: Disease relapse is a major cause of mortality post allogeneic hematopoietic stem cell transplantion (alloHCT) in patients with myeloid neoplasms (MN). Cytogenetic risk classification is a key prognostic factor in predicting relapse post-transplant. Next generation sequencing (NGS) offers added prognostic insight by identifying relapse-associated mutations. We assessed the impact of mutation type and burden on post-alloHCT relapse risk and relapse-free survival (RFS) in MN patients with non-adverse-risk cytogenetics. Methods: We retrospectively reviewed patients who underwent alloHCT at Mayo Clinic Rochester between 01/01/2018 - 10/01/2023. Eligible patients had MN with non-adverse-risk cytogenetics [Acute myeloid leukemia (AML) with non-adverse-risk cytogenetics per ELN 2022, myelodysplastic syndrome (MDS) with non-high/very high-risk cytogenetics per IPSS-R criteria, and myeloproliferative neoplasms (MPN) (excluding myelofibrosis), chronic myelomonocytic leukemia (CMML), or MDS/MPN overlap with non-complex and non-monosomal karyotypes]. The primary endpoint was 3-year cumulative incidence of relapse (CIR) post-alloHCT, analyzed via competing risk models. RFS was estimated via Kaplan-Meier method. Prognostic mutations were identified using univariate (UVA) and multivariate (MVA) Cox models. A decision tree was used to define mutation-based relapse risk groups. Results: Of 585 patients, 215 (37%) met inclusion criteria; 133 (62%) were male. Median age at diagnosis was 61 years (IQR 53-65.5). Diagnoses included AML (125, 58.1%), MDS (55, 25.6%), CMML (17, 7.9%), MDS/MPN (11, 5.1%), and MPN (7, 3.3%). Median follow-up post-alloHCT was 3.2 years (IQR 1.9-5.2). Donor sources were mostly matched unrelated donors (120; 55.8%), followed by matched related donors (72; 33.5%), haploidentical donors (15; 7%), mismatched unrelated donors (6; 2.8%), and cord blood transplants (2; 0.9%). Most patients reduced intensity/non-myeloablative conditioning (144, 67%), while others received myeloablative (71, 33%). Mutation frequencies were as follows: ASXL1 58 (27.0%), RUNX1 45 (20.9%), FLT3 41 (19.1%), SRSF2 41 (19.1%), DNMT3A 39 (18.1%), STAG2 22 (10.2%), SF3B1 18 (8.4%),BCOR 18 (8.4%), WT1 16 (7.4%), U2AF1 13 (6.0%), ZRSR2 10 (4.7%), TP53 6 (2.8%), EZH2 3 (1.4%). In UVA, mutations in FLT3 (HR 2.15, p=0.018), SF3B1 (SF3B1 HR 3.08, p=0.002), PTPN11(HR 2.93, p=0.032), and WT1 (HR 2.29, p=0.069) were associated with increased risk of 3-year CIR. However, only FLT3 (HR 1.98, p=0.042) and SF3B1 (HR 4.0, p<0.001) retained significance in MVA. SF3B1 correlated with inferior 3-year RFS in UVA (HR 1.99, p=0.034) but not in MVA. Mutations in ASXL1, BCOR, EZH2, RUNX1, SRSF2, STAG2, ZRSR2, U2AF1, and TP53 had no significant impact on CIR or RFS. Harboring ≥2 or ≥3 mutations did not impact CIR or RFS. A decision tree model based on SF3B1, FLT3, DNMT3A, and WT1 mutations stratified patients into 3 risk groups. The high-risk group (n=32; 15%) had 50% 3-year relapse incidence (RI) and included patients with SF3B1 mutations or SF3B1-wildtype (WT) with concurrent FLT3 and DNMT3A mutations. The intermediate-risk group (n=36; 17%) included SF3B1-WT patients with either FLT3 mutations and DNMT3A WT (21% RI), or SF3B-1-WT and FLT3-WT patients with WT1 mutations (33% RI). The low-risk group (n=147; 67%) lacked all 4 mutations and had the lowest 3-year RI of 14%. The 3-year CIR significantly differed across these 3 risk groups (51.9%, 27.3%, and 15.8% in high, intermediate, and low-risk groups respectively; p<0.001). The 3-year RFS was significantly inferior in the high-risk group (35.55%, p=0.001), but similar among intermediate and low-risk groups (67.11% and 63.4%, respectively). Conclusion: In non-adverse risk cytogenetic MN, SF3B1 and FLT3 mutations predicted relapse post-alloHCT. Notably, mutations that were traditionally associated with adverse-risk AML per ELN 2022 (e.g, ASXL1, TP53) were not associated with relapse in this cohort. The discrepancy may reflect exclusion of patients with high-risk cytogenetics, as the prognostic impact of these mutations may depend on co-cytogenetic anomalies. In non-adverse-risk cytogenetics MN, we identified 3 groups of patients based on the presence or absence of SF3B1, FLT3, DNMT3A, and WT1 mutations. Those with mutated SF3B1 or concurrent FLT3/DNMT3A mutations experienced the highest risk of relapse post-alloHCT, translating into inferior RFS. Our findings need to be verified in a larger data registry.

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL), accounting for approximately 30% of all NHL cases. DLBCL diagnosis relies on histologic examination, immunophenotyping, molecular and fluorescence in situ hybridization (FISH) analysis to differentiate it from similar NHL subtypes with overlapping morphological features, including more high-grade lymphomas like double-hit lymphoma. Because FISH is performed in a panel of single- or dual-target test format and analyzed through microscopy and manual image analysis, assessment of multiple target biomarkers can be cumbersome and expensive. As additional biomarkers for diagnosis, subtyping, prognosis, and therapeutic decision-making become available, target panels will become larger, posing additional challenges to clinical laboratories. Patients and Methods In this study, Hi-C sequencing of FFPE samples was performed to identify all genomic rearrangements by a single sequencing analysis for an unselected DLBCL case series of 159 patients, and the results were compared to FISH for the detection of clinically important variants. Results Hi-C sequencing showed superior performance compared to FISH: standard FISH for DLBCL includes testing for MYC, BCL2, and BCL6. Between FISH and Hi-C, these biomarkers were detected 105 times, of which, FISH detected 80/105 (76%) and Hi-C detected 102/105 (97%). The cohort included 4 double-hit lymphomas by FISH but Hi-C sequencing detected five. Besides DLBCL diagnostic biomarkers (MYC, BCL2, BCL6), many additional biomarkers were detected by Hi-C sequencing including variants not tested by FISH because the clinical suspicion was ambiguous and the relevant FISH probes were not used, and those not tested because the biomarkers were not thought to be common and, hence, left out of the FISH panel. In total, Hi-C sequencing detected additional biomarkers in 42 (26%) cases, including (1) variants targeted by FISH (i.e. MYC, BCL2, BCL6) but negative, (2) classification biomarkers for other lymphomas (CCND1, MALT, IRF4), and (3) those not tested because the biomarkers are not common and not included in the FISH panel. These additional biomarkers were supportive of suspected diagnosis of DLBCL versus double-hit lymphoma (24 cases), prompted reconsideration of a different diagnosis that may have led to different treatments (6 cases, e.g. mantle cell lymphoma), or biomarkers such as PDL1, ALK, and ATM which have implications for targeted therapies and/or inclusion in clinical trials (5 cases). Conclusion The Hi-C FFPE sequencing technology has the potential to improve the workflow of pathology labs for detection of a wide range of structural variants informing precision diagnosis and treatment for lymphomas.

Abstract Introduction: Among Non-Hodgkin lymphomas (NHL), DLBCL is the most prevalent subtype (~30% of diagnoses). First-line (1L) chemoimmunotherapy cures many patients (pts), but 30-40% will relapse. Prognostic and predictive biomarkers are needed to identify pts refractory to 1L therapy or at high risk of relapse, enabling timely intervention and improved outcomes. This study evaluated tumor-informed WGS-based longitudinal ctDNA monitoring in a real-world DLBCL cohort. Methods: We retrospectively analyzed ctDNA data from 49 pts with DLBCL. Plasma samples were collected before, during, and at the end of 1L therapy (EOT, defined as ± 21 days from the last infusion (preferred when available) or ± 14 days from post-treatment PET imaging) and during surveillance/subsequent treatments. A WGS-based tumor-informed assay (Signatera™ Genome, Natera, Inc.) was used for ctDNA detection and quantification. The association of ctDNA dynamics with event-free survival (EFS) was evaluated using Cox regression analysis, restricted to pts completing at least 5 cycles of 1L therapy. Results: The median age of the cohort was 62 years (range: 56-74), and median follow-up was 22 months (range: 14-30). Pre-treatment ctDNA-positivity rate was 100% (31/31). A total of 19 pts achieved ctDNA clearance during 1L therapy (early clearance within 3 cycles, n=5; late clearance between cycle 4 and EOT, n=14). Pts with no clearance or ctDNA-positivity at EOT (n=7) had significantly inferior EFS compared to those achieving ctDNA clearance (HR 8.04, 95%CI: 1.87-34.62, p=0051). On further stratifying the clearance group and comparing with no clearances, pts with early clearance had superior outcomes (HR 13.22, 95%CI: 1.48-1741.94, p=0.0166), relative to late clearances (HR 5.65, 95%CI 1.46–25.16, p=0.0129). Notably, all 5 pts with early ctDNA clearance remained serially negative and relapse-free. Of the 49 pts, 9 proceeded to chimeric antigen receptor T-cell therapy (CAR-T). Among 6 of these pts with available ctDNA results pre and post therapy, all 3 who stayed ctDNA-positive after CAR-T relapsed (1.3, 2, and 4.5 months post-infusion); 1 pt with pre- and post-CAR-T ctDNA-negativity and 2 pts with ctDNA clearance post-CAR-T remained relapse-free for 9.6, 15.9, and 22.4 months, respectively. Taken together, the PPV and NPV for a single ctDNA time point post CAR-T infusion were 100% for predicting long-term clinical outcomes. Conclusions: Our data support longitudinal ctDNA monitoring as a valuable prognostic and predictive biomarker in DLBCL. ctDNA clearance, particularly early clearance within the first 3 cycles of 1L therapy, is associated with improved pt outcomes. Longitudinal ctDNA monitoring and dynamics can enable identification of high-risk pts who do not clear ctDNA and may benefit from earlier referral for CAR-T. Furthermore, ctDNA monitoring pre- and post-CAR-T infusion is predictive of treatment response and can identify pts at high risk of relapse.

Abstract Background Acute graft-versus-host disease (GVHD) is a major cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). High-dose steroids are standard first-line therapy for grade II–IV acute GVHD, whereas systemic treatment (tx) is generally not recommended for grade I. However, in practice many patients (pts) with grade I GVHD receive systemic steroids. A prior GITMO randomized trial (n=173) of grade I GVHD to either systemic steroid tx or “watch and wait” found that preemptive steroid tx reduced progression to grade II but not grade III/IV GVHD, increased serious infections, and resulted in similar chronic GVHD, NRM and survival (Bacigalupo, Haematologica 2017). In this study we analyzed real world evidence for early systemic steroid use and the potential impact of serum biomarkers in grade I GVHD. Methods We analyzed clinical data and serum samples from pts with grade I acute GVHD at 24 Mount Sinai Acute GVHD International Consortium (MAGIC) centers between 2014 and 2022 who were treated according to physician discretion. Pts were divided into a preemptive steroid tx group or a watch-and-wait group (systemic steroids administered only if progression to grade II–IV GVHD). Preemptive steroid use was treated as a time-dependent covariate and a frailty proportional hazards model with random effects was used to adjust for center effects to account for possible heterogeneity in clinical practices across transplant centers. Serum samples collected at the diagnosis of grade I GVHD were analyzed for ST2 and REG3α to calculate validated Ann Arbor (AA) scores (1, 2, 3). Results Grade I GVHD was diagnosed in 1143 pts; 591 (52%) received preemptive tx and 552 (48%) were treated by a watch-and-wait strategy. In the preemptive tx group, steroids were initiated at a median of 0 days and within 7 days for >90%. The median initial dose of steroids was 1 mg/kg of prednisone or its equivalent. In the watch and wait group, only 184 pts (33%) required systemic steroids for progression to grade II-IV GVHD at a median of 14 days from grade I diagnosis. Preemptive steroid tx reduced the risk of grade II–IV acute GVHD (HR, 0.71; 95% CI, 0.56–0.89; P=0.003), but not the risk of grade III/IV acute GVHD (HR, 1.03; 95% CI, 0.69–1.52; P=0.899) or chronic GVHD (HR, 1.03; 95% CI, 0.69–1.52; P=0.899), consistent with the GITMO trial. The 12-month incidence of NRM was higher for preemptive tx vs. watch and wait (13% vs 7%, P=0.002). In multivariate analysis preemptive steroid use independently predicted an increased risk of NRM (HR, 1.63; 95% CI, 1.06–2.50; P=0.026) as did several other known risk factors (older recipient age, umbilical cord blood donor, HCT-CI ≥3, and myeloablative conditioning). Greater NRM in the preemptive tx group was driven primarily by 3-fold more infectious deaths; deaths due to uncontrolled GVHD were similar for both tx groups. The risk for relapse was unexpectedly lower in the preemptive tx group (HR 0.64; 95% CI 0.47-0.88; P=0.006) and overall survival was therefore not different between groups, but risk factors for relapse such as minimal residual disease status were not available precluding further analysis of this finding. Serum was collected in 871 (76%) pts at onset of grade I GVHD for retrospective biomarker analysis. AA scores were AA1 (80%), AA2 (15%), and AA3 (5%). AA scores were highly predictive of progression to grade III/IV GVHD (AA1 vs AA2 vs AA3: 9% vs 14% vs 44%, all P<0.001) and the risk of 12-month NRM (8% vs 16% vs 39%, all P<0.001); this pattern was observed in both tx groups. The negative effect of preemptive steroid tx was most pronounced in pts with grade I/AA1 GVHD who experienced 5-fold more infectious related deaths compared to pts managed with a watch and wait approach. Conclusion Treatment of grade I GVHD with systemic steroids did not reduce the incidence of grade III/IV GVHD or deaths due to uncontrolled GVHD and instead increased the risk of NRM from infections compared to pts who were treated only after GVHD progressed. Importantly, 2/3 of the watch and wait pts never needed systemic steroid tx. These findings remained robust after adjusting for multiple confounders, including center effects, and were consistent with the GITMO trial. MAGIC biomarkers at the onset of grade I GVHD predicted outcomes and a watch and wait strategy may be best for the 80% of pts with AA1 GVHD. Better treatment options are needed for the 20% of pts with grade I and AA2/3 GVHD.

Abstract INTRODUCTION The ECHELON-2 trial for newly diagnosed CD30+ peripheral T-cell lymphomas (PTCL) demonstrated superior overall survival with brentuximab vedotin plus chemotherapy (BV-CHP) compared to CHOP. However, total enrollment was restricted to only 30% of patients with non-anaplastic large cell lymphoma (ALCL) histologies and was not powered to analyze survival benefits in this cohort. Thus, the benefit of BV-CHP in non-ALCL patients remains less clear. Adding to this uncertainty, CHOP plus etoposide (CHOEP) remains a standard regimen for many non-ALCL PTCL histologies for patients <60 years of age. Which of these three regimens offers superior outcomes for patients with CD30+ non-ALCL PTCL remains to be determined, is a critical knowledge gap in this area, and could meaningfully inform the management of these patients. METHODS We conducted a retrospective analysis of chemotherapy-naïve CD30+ (≥1%) PTCL from 7 centers in the United States. Histologies were limited to PTCL-NOS and PTCL with a T follicular helper phenotype (PTCL-TFH). Frontline regimens were defined as BV-based (BV-CHP), etoposide-based (CHOEP/EPOCH), novel-CHOP (CHOP/CHOEP + novel drug on a clinical trial), or CHOP. CD30 was considered positive if any expression was reported, and when available, stratified as ≥10% or <10% of the total lymphocyte population. RESULTS The CD30+ PTCL cohort consisted of 194 patients with a median age of 68 years at diagnosis and a male (65%) predominance. Nodal PTCL-TFH (65%) was the most common histology. Most had stage III-IV disease (89%) with a high-intermediate to high-risk IPI score of 3-5 (54%). 18% had an ECOG of ≥2, 45% had ≥2 extranodal sites, and 43% had bone marrow involvement, although biopsies were not performed in all patients. CD30 expression in ≥10% of malignant T cells was observed in 55% of all quantitatively reported cases. Frontline therapy consisted of BV-based (36%), etoposide-based (30%), CHOP (27%), and novel-CHOP (7%). Baseline characteristics and CD30 expression were similar between treatment groups. The ORR was 72% (64% CR, 8% PR), with no differences observed by treatment or histology. On multivariate analysis (MVA), a high LDH was associated with lower CR (OR 2.84, p=0.003). For the patients who demonstrated a CR to frontline therapy, 61% underwent ASCT. With a median follow-up of 26.1 months (IQR 12.3-51.6), the median PFS and OS were 11.4 (9.8-14.2) and 47 (32-60.3) months, respectively. The 2-year PFS and OS were 34.9% (28.1-41.7) and 62% (54.5-68.6), respectively. PFS and OS were similar for PTCL-NOS compared to PTCL-TFH, and AITL fared similarly to non-AITL PTCL-TFH. By treatment, novel-CHOP demonstrated the most favorable PFS (median 41.9 months) and OS (median 68.1 months). Survival curves overlapped continuously with all other treatment regimens and was not statistically significant. For patients <60, PFS and OS were equivocal with BV-based (n=17) compared to etoposide-based (n=26) regimens. On MVA, stage III/IV disease conferred an inferior PFS (HR 2.00, p=0.018) as did an IPI score of 3-5 for OS (HR 2.15, p=0.001). Neither histology nor frontline treatment impacted PFS or OS. The chosen frontline treatment also had no survival impact in key subgroup analyses including patients <60 (n=56) or with CD30-expression ≥10% (n=43). For patients with CR to frontline therapy, ASCT demonstrated a significant PFS benefit (HR 0.59, p=0.028) but no statistically significant difference in OS benefit (HR 0.58, p=0.073). CONCLUSION These data provide valuable insight into the lesser-represented patient population from the pivotal ECHELON-2 trial. In this cohort of CD30+ (≥1%) non-ALCL PTCL, there was no clearly superior standard frontline regimen. The number of patients with quantitively reported CD30 expression was too small for definitive conclusions in the ≥10% CD30+ cohort and retrospective pathology review is ongoing. ASCT for patients in CR offered superior outcomes, supporting this strategy regardless of the frontline regimen, recognizing the limitations of non-randomized, retrospective analyses. Novel-CHOP regimens demonstrated encouraging survival, and clinical trials should maintain priority over standard regimens for patients with CD30+ (1-10%) PTCL-NOS and PTCL-TFH.

Abstract Allogeneic hematopoietic cell transplantation (HCT) using an HLA mismatched unrelated donor (MMUD) with calcineurin inhibitor-based graft versus host disease (GVHD) prophylaxis results in increased incidence of non-relapse mortality (NRM) and lower survival compared to matched donor HCT. The NMDP sponsored 15-MMUD study demonstrated that post-transplant cyclophosphamide (PTCy)-based prophylaxis following bone marrow derived MMUD HCT resulted in reduced incidence of GVHD and favorable survival. Because marrow grafts can be difficult to procure and vary in quality, we conducted a phase II, prospective, multicenter study to evaluate PTCy-based GVHD prophylaxis in adult recipients of MMUD using peripheral blood stem cell (PBSC) allografts. The primary study aim was to estimate the probability of one-year overall survival (OS) post HCT. Secondary aims were to assess other key clinical endpoints and determine the rate of severe infections. Initial results of the first 70 recipients who received reduced intensity conditioning (RIC) were previously reported by Al Malki et al (JCO 2025) demonstrating a 1-year OS of 78.6% (95% CI, 67% to 86.5%). Here, we report results from a planned expansion of the study that included 123 additional RIC recipients, with updated results from the initial cohort of 70 patients (total N = 193 subjects) treated at 33 centers. Study participants received cyclophosphamide 50 mg/kg (ideal body weight) administered on days 3 and 4 post HCT with tacrolimus and mycophenolate mofetil as previously described (Al Malki, JCO, 2025). The median study participant age was 65.0 (range: 22.9-78.9); 92 (47.7%) of participants were male, and 84 (43.5%) identified as having other than non-Hispanic white ancestry. The HCT comorbidity index was high-risk (>3) in 65 (33.7%). Underlying histologic diagnosis was AML in 96 (49.7%) participants, MDS in 43 (22.3%), ALL in 20 (10.4%), lymphoma or CLL in 24 (12.4%), and CML/other leukemias in 10 (5.2%). Conditioning regimens used were fludarabine and melphalan (100 or 140 mg/m2) (N=120; 62.2%), 2-day busulfan and fludarabine (N=38; 19.7%), and fludarabine, cyclophosphamide and total body irradiation (200 cGy) (N=32; 16.6%). Donor HLA matching (considering HLA-A, -B, -C, and -DRB1) was 7/8 in 131 (67.9%) of participants, 6/8-matched in 51 (26.4%), and 4-5/8-matched in 11 (5.7%) participants. Median donor age was 25.5 years (range: 18.7-35.7). The one-year overall OS estimate was 79.6% (95% confidence interval [CI]: 73.2-84.7). Survival was similar among HLA 7/8-matched donor recipients (77.8%, 69.7-84%) and <7/8 matched donor recipients (83.7%, 71.8-90.9%). The one-year GVHD-free, relapse-free survival (GRFS) was 49.9% (42.6-56.9%) and the one-year progression-free survival was 70.2% (63.2-76.2%). The one-year cumulative incidence (CI) rates of NRM and relapse were 12.5% (8.3-17.6%) and 17.3%(12.3-23%), respectively. The 6-month CI of grade II-IV and grade III-IV acute GVHD were 40.0% (33.0-46.8%) and 7.8% (4.6-12.1%), respectively. The one-year incidence of all chronic GVHD and severe chronic GVHD were 15.6% (10.7-21.2%) and 3.7% (1.6-7%), respectively, by NIH consensus criteria. The CI of grade 1-4 cytokine release syndrome (CRS) by day 7 post HCT was 28.0% (21.8-34.9%), and grade 3-4 CRS was 0.5% (0-2.9%). Primary engraftment failure occurred in 9 (4.7%) participants. Among 145 evaluated participants, 85.5% were >95% donor in blood chimerism at day 100 post HCT. Within the first 100 days after HCT, CTCAE grade 2-5 infections occurred in 113 (58.5%) of participants and grade 3-5 infections occurred in 59 (30.6%) of participants. The most common etiologies of grade 2-5 infections were bacterial (N=78), followed by viral (N=48). The most common grade 3-5 non-infectious toxicities were cardiac disorders, which occurred in 4 (2.1%) of participants. In conclusion, the results including this expansion cohort demonstrate encouraging 1-year OS, NRM and relapse rates in RIC recipients of PTCy-based GVHD prophylaxis and PBSC allografts from MMUDs. This larger cohort of 193 recipients re-affirm the results from the initial cohort with greater robustness.Measures to mitigate infection risk are likely to further improve outcomes. Reducing the dose of PTCy is a candidate approach currently being tested in the ongoing OPTIMIZE trial (NCT 06001385).These results support the extension of PBSC MMUD using PTCy to individuals who lack an HLA-matched donor and require RIC HCT.

Abstract Introduction: Inherited & acquired bone marrow failure syndromes (BMFS) have “stress erythropoiesis” with macrocytic anemia, elevated fetal hemoglobin (HbF) & erythropoietin (EPO) levels. The degree of HbF elevation & its capacity to discriminate between patients with inherited & acquired BMFS needs to be systemically studied. Genetic testing is often delayed & it is important to establish an early clinical diagnosis to make clinical decisions such as initiation of immunosuppressive therapy. Methods: After institutional review board approval, we compiled a clinically & genetically well-annotated cohort of patients with BMFS evaluated at Mayo Clinic. All patients were required to have at least cytopenia(s) & evidence of low or variable bone marrow cellularity upon a bone marrow evaluation. Patients with pre-clonal or malignant disorders were excluded. Clinical diagnoses were independently verified for accuracy. Detailed clinical & laboratory features were recorded. Germline genetic testing was performed as clinically appropriate on peripheral blood or bone marrow samples through targeted panels. Distribution of continuous variables was statistically compared using nonparametric (Mann-Whitney or Kruskal-Wallis) tests, while nominal variables were compared using the Chi-Square test. Kaplan-Meier estimate of overall survival (OS) was computed from the date of diagnosis to date of death or last follow-up (also censored at the time of allogeneic hematopoietic stem cell transplant). Results: Between years 1998 - 2023, 129 patients met criteria for inclusion in the study. Among these, clinical diagnoses included immune-mediated aplastic anemia (AA, n=56, 43%, 21% severe or very severe as per the Camitta criteria), telomere biology disorders (TBD, n=37, 29%), Fanconi anemia (n=10, 8%), GATA2 haploinsufficiency (n=8, 6%), Diamond-Blackfan anemia (DBA, n=2, 2%), deficiency of adenosine deaminase-2 (DADA2, n=2, 2%), congenital dyserythropoietic anemia (CDA, n=1, 1%), congential amegakaryocytic thrombocytopenia (CAMT, n=1, 1%), Schwachman-Diamond syndrome (n=1, 1%) & undefined (n=11, 9%). Among the inherited group, 63% had an identifiable germline pathogenic variant (most commonly, TERT/TERC 30%, FANC group 25% & GATA2 15%) with the rest diagnosed on a clinical basis. Elevated HbF >1% was noted in 79% of all BMFS patients; 83% AA, 70% TBD, 50% in DADA2 & 67% in undefined patients (no data was available for other inherited subtypes). In patients with an elevated HbF, absolute monocyte count (p=0.04) was significantly higher; however, there were no other significant associations with variables at diagnosis such as hemoglobin (p=0.5), white blood cell count (WBC, p=0.14), absolute neutrophil count (p=0.5), platelet count (p=0.7), mean corpuscular volume (MCV, p=0.2), absolute lymphocyte count (p=0.1), absolute reticulocyte count (p=0.4), EPO level (p=0.3), presence of a PNH clone (p=0.2), or presence of a germline variant (p=0.3). When diseases were grouped into inherited & acquired (AA) subgroups, median HbF was not significantly different (median%, 4.5 versus 2.3, p=0.4), with elevated HbF (>1%) in 75% versus 83% (p=0.6) patients respectively. On the other hand, minor PNH clone (>0.1% in at least two cell types) was detectable in the acquired group only (0 versus 55%, p<0.0001). Similarly, acquired group had a shorter (<3 months) onset of cytopenias preceding diagnosis (P<0.0001), with no significant differences in MCV (p=0.2), marrow dysplasia (p=0.09) or atypia (p=0.6), & splenomegaly (p=0.9). At median follow-up of 7 years, Kaplan-Meier estimate of transplant-censored median OS was lower in inherited versus acquired group (16 years versus median not reached, p=0.02). Elevated HbF did not significantly predict adverse OS in either group. Conclusions: Fetal hemoglobin is elevated in both patients with inherited & acquired (aplastic anemia) BMFS. When compared to established variables, elevated HbF has neither diagnostic utility nor prognostic relevance in predicting adverse outcomes. On the other hand, onset of cytopenias and presence of a minor PNH clone are both useful clinical variables in differentiating patients with inherited from acquired BMFS. Our data offers practical clinical utility in making early treatment decisions for patients with BMFS.

Abstract Introduction: The DEAD-box helicase 41 (DDX41) gene, located on chromosome 5q35, is a tumor suppressor gene involved in RNA processing. Germline mutations in this gene can predispose patients to myeloid malignancies. Studies have suggested a more favorable prognosis in those with germline mutations in DDX41, with progression of disease being a frequently encountered cause of death. In this retrospective study, we explored the causes of death in a cohort of patients with pathogenic DDX41 mutations. Methods: This was a retrospective study of patients diagnosed with myeloid neoplasms between 2009-2024 and found to have DDX41 mutation (DDX41m) via next generation sequencing (NGS) at our institution. Myeloid NGS was obtained at or after the diagnosis of myeloid disease. Germline testing (when available) was performed through buccal/skin samples. Carriers of DDX41m without a myeloid neoplasm diagnosis and patients with only DDX41m VUS were excluded. Baseline characteristics at the time of myeloid neoplasm diagnosis included age at diagnosis, DDX41m variant allele frequency (VAF), blood counts (hemoglobin, white blood cell count, platelets), cytogenetics, bone marrow blast percentage, number of co-mutations, and treatment received. Unadjusted comparisons were made using continuous or categorical variables. Median overall survival (OS) was calculated from the time of diagnosis with myeloid neoplasm to the time of death, using BlueSky Statistics V.10.3.4. Results: Of the 112 patients identified to have pathogenic DDX41m, 29 (26%) patients were deceased at the time of data analysis. In this cohort of 29 patients (median age at diagnosis 72 years, 17 (59%) males), disease groups at initial diagnosis included 17 (59%) patients with MDS, 9 (31%) AML, and 3 (10%) MPN. Fourteen of the 17 (82%) of the patients with MDS were classified as MDS-EB1 or MDS-EB2. Seven of the 17 (41%) patients with MDS progressed to AML at the time of death, with 57% of the patients having more than 30% bone marrow blasts at time of progression. Blood counts at diagnosis in the cohort included a median hemoglobin of 9.8 g/dL, platelets of 83 x 109/L, and white blood cell count of 2.1 x 109/L. Twenty-one (72%) of patients had normal cytogenetics. Nine (31%) patients had both a pathogenic mutation and a variant of uncertain significance (VUS) in DDX41, with the remaining 20 patients harboring only a pathogenic DDX41 mutation. Eight (27%) patients underwent germline testing, with 7 confirmed to harbor a germline DDX41m. In the deceased cohort, 9 (31%) patients underwent HSCT. Three of the patients with AML and 1 patient with MDS were observed and did not undergo treatment. Median overall survival of the deceased cohort was 29 months. The median time to AML progression in the group with MDS was 14 months. Causes of death included infection in 11 (38%) patients, 9 (31%) with disease progression/relapse, 3 (10%) with acute gastrointestinal graft versus host disease (GVHD), and 1 (3%) from cerebral bleed. The cause of death was unknown in the remaining 5 (17.2%) patients. Infections included pneumonia, bacteremia, neutropenic enterocolitis, fungal, and unknown etiology. Of the 4 patients who were observed, 2 died from unknown cause and 2 died from disease progression. Of the 16 patients with AML at time of death, 9 (56%) died from disease progression or relapse, 3 (19%) from infection, 2 (13%) from GVHD, and 2 (13%) from unknown causes. Of the 10 deaths in the MDS group, 7 (70%) were from infection, 1 (10%) from GVHD, 1 (10%) from cerebral bleed, and 1 (10%) from an unknown cause. Three cases of infection in the MDS group occurred during remission. Infection was a more common cause of death in the MDS group compared to the AML group, where disease progression prevailed as a cause of death (70% vs 19%, p=0.01). Eight (80%) of the patients with MDS who died from infection were neutropenic prior to death, and 4 (50%) of those patients were on neutropenic antimicrobial prophylaxis. Conclusions: In this cohort of 29 deceased patients with a DDX41-mutated myeloid neoplasm, we characterized causes of death. Disease progression/relapse was a significantly more common cause of death in those with AML, compared to infection in patients with MDS. Our work underscores the need for additional studies looking into non-leukemic causes of death in patients with DDX41m myeloid diseases and potential strategies to mitigate infection-related mortality in this population.

Abstract Introduction: Multiple Myeloma (MM) is a complex hematologic malignancy, where patients often display one or more potentially high risk cytogenetic alterations associated with poor outcomes. Traditional high risk cytogenetics events in myeloma, such as NSD2 t(4;14) translocations, gain of 1q21, or deletion of 17p13, have often been studied in isolation, but emerging evidence suggests the combination of these high risk alterations has a greater negative prognostic impact than any on their own. This has been recently reflected in risk stratification guidelines from IMS and IMWG, which consider certain events, such as t(4;14), as high risk only in combination with gain of 1q21 or deletion of 1p32. Still, the mechanisms through which multi-hit high risk events disrupt the microenvironment and promote poor outcomes remains unclear. We hypothesize that myelomas with multiple high risk alterations remodel the immune environment to promote tumor progression and immune escape. Methods: To study the role of multi-hit high risk cytogenetics on the immune microenvironment, we analyzed CD138-bone marrow aspirate single-cell RNA sequencing (scRNAseq) data from 337 newly diagnosed MM patients from the MMRF CoMMpass study, generated as part of the Immune Atlas initiative. Cytogenetic alterations for these patients were derived from whole-genome and whole-exome sequencing of CD138+ bone marrow aspirates from the MMRF CoMMpass cohort. Patients were classified as multi-hit high-risk MM if they exhibited either (i) two high-risk cytogenetic events, including NSD2 t(4;14), MAF t(14;16), del(17p13), or (ii) one high-risk event with gain of 1q21. Results: Based on cytogenetic information, 33 patients in the Immune Atlas cohort were classified as 'multi-hit’ high-risk, while 34 patients had only a single high-risk alteration. These multi-hit high risk patents displayed significantly worse progression free survival compared to standard risk patients, adjusting for age, therapy, and ASCT (HR=2.34, p<7.2e-5), while single-hit patients showed a non-significant trend towards worse outcomes (HR=1.51, p=0.071). The most frequent multi-hit risk event in the cohort was NSD2 t(4;14) in combination with 1q21, observed in 23 patients. Compositional analysis of the immune populations in the scRNAseq data revealed that relative to both standard and single-hit high risk patients, multi-hit high risk patients displayed significant reductions in IFN-I stimulated CD4+ and CD8+ T cell populations, marked by decreased expression for antiviral response markers such as ISG15, XAF1, and IFI44L. Enrichment of inflammatory CD16+ non-classical monocytes was also observed. Further analysis of the most frequent multi-hit risk event, NSD2 with 1q21, revealed distinct enrichment of late-activated cytotoxic HLA+ CD8+T cells and cytotoxic CD4+.T cells. Neither alteration was observed in patients with single-hit NSD2 or 1q21. Further analysis on the interactions between high risk events revealed distinct transcriptomic profiles dependent on the high risk events involved, particularly in multi-hit events involving mutations or deletions of TP53 or gain of 1q21. Reduction in IFN-I signaling across various immune populations was primarily restricted to multi-hit risk events involving gain of 1q21. Conversely, patients with biallelic loss of TP53 displayed significant enrichment of these same IFN-I populations. Given this contrast in IFN-I signaling spanning multiple immune subtypes, we hypothesized that the malignant myeloma cells might similarly display altered IFN-I activity. Utilizing CD138+ myeloma bulk RNA sequencing data from CoMMpass, we found that IFN-I response scores strongly correlated between paired scRNA immune and bulk RNAseq tumor datasets (R2: 0.75, p=1.63e-32). Across 660 patients with bulk RNAseq and cytogenetic data, we confirmed significant reductions in IFN-I response scores in tumor cells in multi-hit high risk involving 1q21, and significant enrichment in those with complete loss of TP53 relative to standard-risk MM. Conclusions: Overall, we do observe that MM patients with multiple high risk cytogenetic alterations display distinct immunologic traits relative to standard risk or single-hit high risk patients in addition to significantly worse outcomes. Furthermore, we find variability in the immune alterations observed based on the cytogenetic events involved, further illustrating the heterogeneity in how MM shapes the immune microenvironment.

Abstract Introduction: Primary myelofibrosis (PMF), a myeloproliferative neoplasm (MPN), features bone marrow fibrosis, splenomegaly, and abnormal blood cell production. Aberrant JAK/STAT signaling, elevated cytokines or hyperactive NFκB are implicated in MPN pathogenesis; however, their interplay remains unclear. JAK inhibitors (JAKi) alleviate PMF symptoms, but they don't fully address disease progression or mitigate cytokine storms. NFκB inhibitors have shown promise in MPN models but are associated with off-target effects and toxicities. This study aims to elucidate the mechanisms of JAKi resistance and develop novel therapeutic strategies targeting hyperactive NFκB in vitro and in vivo, offering potential benefits beyond current JAKi therapies. Methods: To investigate JAKi resistance in PMF, ruxolitinib-resistant HEL cells were generated and NFκB/JAK2/STAT signaling was examined by Western blot. Biomarkers associated with JAKi response were identified in PMF patients treated with BMS-9115437 (Gangat et al, Blood Cancer J, 2023) via Western blot. Molecular mechanisms of MPN pathogenesis were further elucidated using Affymetrix HG-U133Plus2 assay on RNA from treatment-naïve PMF, essential thrombocythemia (ET), polycythemia vera (PV) patients and healthy donors, followed by KEGG and gene set enrichment (GSE) analysis. In vitro, NFκB was inactivated by shRNA, Ixazomib or emetine followed by Western blot for NFκB/JAK2/STAT phosphorylation as well as flow cytometry and clonogenic assays for cell growth. NFκB DNA binding activity was determined by electrophoretic mobility-shift assays (EMSA). The therapeutic and preventive effects of Ixazomib and emetine were evaluated in HEL-engrafted nude mice by assessing tumor formation, development, and underlying mechanisms using Western blot, H&E and IHC staining. Results: Short-term JAKi (BMS-911543, ruxolitinib) exposure altered JAK2 phosphorylation, increasing Tyr1008 phosphorylation and decreasing Tyr221, Tyr570, and STAT5 phosphorylation. Notably, JAK2, STAT3 and STAT5 were phosphorylated in ruxolitinib-resistant HEL cells or after treatment cessation, implying sustained signaling activity. Western blot analysis and clinical data in PMF patients (n=3) receiving BMS-911543 showed no hyperphosphorylation of JAK2 Tyr1008 with diverse responses independent of JAK2V617F status. A lack of correlation between JAK2 and STAT5 expression/phosphorylation suggests that other factors contribute to hyperactive STAT5 in PMF. Symptomatic improvement in a JAK2V617F-positive patient correlated with decreased STAT5 protein expression and phosphorylation, potentially serving as a predictor for JAKi response. Transcriptomic analysis in MPN patients (PMF (n=11), ET (n=6), PV (n=4), HD (n=5)) pinpointed the transcriptomic dissimilarities between PMF and PV or ET, and highlighted the higher NFκB activity in PMF compared to PV or ET. The importance of NFκB in PMF was further evidenced by the network analysis for the overproduction of circulating cytokines in PMF patients (Tefferi et al, JCO, 2011). In vitro studies revealed that NFκB overexpression promotes cell proliferation and inhibits apoptosis through enhanced IL-6/IL-6R expression and JAK2/STAT5 phosphorylation. Inactivating NFκB using shRNA, emetine, or ixazomib reversed these effects. Further, IL-6 treatment increased JAK2 phosphorylation. In vivo studies showed that Ixazomib significantly reduces tumor volume (734 ±133 to 98 ±14 mm3; P <0.001) and weight (582 ±156 to 87 ±22 mg; P =0.0005) compared to controls. Emetine similarly suppressed tumor growth dose-dependently without affecting spleen weight and even prevented tumor formation when administered prior to cell engraftment. Mechanistically, both Ixazomib and emetine inhibited NFκB and JAK2 phosphorylation while increasing cleaved caspase-3 in tumors. Conclusions: We discovered that STAT inactivation independently predicts improved outcomes in PMF and that NFκB hyperactivity holds particular significance for PMF compared to other MPN subtypes. We identified a NFκB-IL-6-JAK2-STAT axis in regulating PMF growth regardless of JAK2V617F status. Targeting NFκB with novel inhibitors Ixazomib and emetine disrupts the NFκB-IL-6-JAK2-STAT axis, suppressing PMF growth and offering potential therapeutic alternatives to overcome current JAKi limitations.