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Abstract Introduction: Somatic mutations in splicing factors (SF) - SF3B1, SRSF2, U2AF1, and ZRSR2 – represent one of the most common drivers of premalignant clonal hematopoesis. While SF mutations are potent drivers of clonal expansion (Fabre et al., Nature 2022), multiple SF mutations are rarely observed in the same individual (negative epistasis). Prior studies have identified rare cases of concurrent SF mutations (<4%) and described specific hotspot combinations that escape negative epistasis (Taylor et al., Blood 2020). We conducted this study to further characterize the frequency and clonal relationship between concurrent SF mutations using a larger patient cohort. Methods: After IRB approval, we assessed 3,369 myeloid next generation sequencing (NGS) results that identified somatic variants in SF3B1, SRSF2, U2AF1, or ZRSR2, in 2,500 adult patients from 08/2015 to 07/2025. Patient demographics, ICD-9/10 codes, and complete blood counts with differentials were obtained, retaining only those datapoints assigned 30 days before or after NGS testing. To allow comparison of clone size between autosomal and the X-linked SF ZRSR2, VAFs were multiplied by two to estimate the cancer cell fraction (CCF), except for ZRSR2 mutations in males. For patients harboring concurrent SF mutations, we categorized them as co-dominant if the difference in respective CCFs was < 10% of the greater CCF. Specific SF variants were categorized into common hotspots (n=13), except for ZRSR2 variants, which do not occur in defined hotspots. Fisher's exact testing was used to assess the enrichment of SF hotspot variants in patients with concurrent SF mutations, as well as combinations of SF variants in patients with co-dominant SF mutations. Results: We identified 2,331 patients with at least 1 pathogenic SF mutation, of which 6.7% (n=156) had > 1 SF mutation (multi-SF MT). The total cohort comprised cases of AML (n=549, 24%), MDS (n=977, 42%), MDS/MPN (n=233, 10%), MPN (n=228,10%), and uncategorized ICD codes (“Other”, n=326), of which the largest category was clonal cytopenia(s). Notably, the prevalence of multi-SF cases was higher than previously described and our cohort included 5 patients with 3 SF mutations and 1 with 4 SF mutations. Of the multi-SF MT patients, we identified 42 (27%) with co-dominant CCFs among their respective SF mutations. Thirty-three multi-SF MT patients had multiple NGS sampling timepoints (median 2, range 2-4) spanning a median of 0.97 years (range 0.06-5.69 years); we found that 17 of these 33 demonstrated correlative SF CCFs over multiple timepoints, suggesting their SF mutations occurred in the same clone (“co-clonal”). Of these 17 patients, 82% had co-dominant CCFs at the single-timepoint level, whereas none of the 16 non-co-clonal demonstrated co-dominance. Thus, we used SF co-dominance as a surrogate for cases likely to be co-clonal. ZRSR2 (p=8.8x10-29), U2AF1Q157 (p=9.4x10-9), SF3B1K666 (p=1.4x10-4) and infrequent SF3B1 (“SF3B1-Other”, p=.0029) mutations were overrepresented in the multi-SF MT cohort compared to patients with a single SF mutation. Within the multi-SF MT cohort, 3 SF mutation combinations showed overrepresentation in codominant cases – SRSF2P95R/H/L+ ZRSR2 (p=0.014), SRSF2P95R/H/L+ SF3B1K666 (p=0.044), and U2AF1S34 + U2AF1Q157 (p=0.024), although not significant after multiple hypothesis correction. Dual ZRSR2 mutations were the most common combination seen in the multi-SF MT cohort but were underrepresented in codominant cases (p=0.025). Although there was no disease subtype uniquely associated with total or co-dominant multi-SF MT cases, occurrence of multiple SF mutations was more common in males (8% vs. 3% in females), an effect driven by ZRSR2 mutations. Hemoglobin and WBC were lower in multi-SF MT cases, with the lower hemoglobin strongly driven by the codominant multi-SF MT cases (median 8.3 g/dL vs 9.4 g/dL, p=0.00046). Conclusions: These findings suggest that SF mutation co-occurrence in myeloid neoplasms is more prevalent than previously estimated (~7%). Furthermore, we identified 6 individuals harboring > 2 SF mutations, which has not been previously described. We validate specific SF variant pairs that appear to exist in the same clone and are working to confirm the clonal architecture and characterize global splicing aberration in selected cases with single-cell studies to fully uncover the mechanism by which negative epistasis is overcome.

Abstract Introduction: Although therapies targeting Bruton Tyrosine Kinase (BTK), either as monotherapy or in combination have revolutionized the treatment in chronic lymphocytic leukemia (CLL), the disease remains incurable. Continuous treatment with these agents frequently leads to the development of resistance and disease progression, mostly associated with mutations in BTK or PLCG2. However, a significant fraction of BTKi-resistant patients does not harbor mutations in these genes, revealing that multiple distinct mechanisms of resistance can independently develop in these patient cohorts. Identifying novel molecular pathways associated with drug resistance in the relapsed/refractory (RR) setting of CLL requires more exploration. Materials and Methods: Using the Mayo Clinic CLL Database and tissue bank, we identified 30 RR patients, who had had disease progressionon a BTKi. The median lines of prior therapies were four. The median age at start of treatment was 57.9 (range, 36-78), 77% were male. Rai stage at diagnosis was 0 in 10% of patients, stage I in 37%, stage II in 20%, stage III in 6%, and stage IV in 27%. IGHV unmutated was observed in 87% of cases. Five patients subsequently evolved to Richter's Transformation (RT). A total of 64 blood samples were collected at different timepoints: baseline (before BTKi therapy), during treatment and at disease progression from BTKi (Ibrutinib/Acalabrutinib/Pirtobrutinib/Nemtabrutinib). The CD5+/CD19+ tumor population was enriched by flow sorting. We performed whole genome sequencing (WGS) in tumor/germline pairs with an average sequencing depth of 60X. Copy number variants (CNVpytor), structural variants – SV (Manta and DELLY2), single nucleotide variants (SNV) and indels (MuTect2, Strelka) were calculated. Mutational signature analysis was conducted independently on each disease timepoint with sigProfiler. De novo signatures were extracted with non-negative matrix factorization, then decomposed and mapped to known signatures from the COSMIC Human Cancer Signatures database. Results: Prior to start of BTKi treatment, the most common abnormality was del13q (65%), followed by abnormalities in TP53 (mutation and/or del17p) in 59% of patients, del8p, del11q (23% each), and mutations in SF3B1 (35%), NFKIE, NOTCH1 and MAPK signaling pathway (BRAF, KRAS, NRAS, PTPN11) in 23% of cases, each. Complex karyotype, defined as >3 copy number abnormalities was found in 59% of cases. Disease progression was observed 4.4 years (median; range 1.9-10.8 yrs) after starting BTKi therapy. At disease progression, there was an overall increase in genomic complexity. Overall, 17 cases (56%) showed mutations in BTK (p.C481S, p.C481Y, p.L528W, p.A428D) and/or PLCG2 (p.P844H). Four cases showed mutations in both genes or two or more independent mutations in BTK. Del17p and/or TP53 mutations were found in 20 (67%) cases, 9 of them with biallelic abnormalities. A third of patients (N=10) were found to have MAPK mutations, and 9 (30%) cases had mutations in the epigenetic regulators CREBBP, SETD2, SETD1A andKMT2D. Loss of heterozygosity, including 11q and 17p was found in 13% of cases. Chromothripsis was found in 23% of cases. In patients without BTK or PLCG2 mutations (N=13), TP53 abnormalities were present in 92% of cases and mutations in the MAPK pathway in 46% of these patients. Finally, we identified several SVs involving driver genes at disease progression, including inversions in RB1 (N=1), NOTCH1 (N=1) and translocations in MYC (N=1) and IGH (N=2). Analyzing the mutational signatures, baseline timepoints were dominated heavily by aging signatures (SBS5, SBS1). However, in cases that progressed after therapy, we found enrichment of mutational signatures associated with defective DNA mismatch repair (SBS44, 6%), chemotherapy treatment (SBS25, 14%), homologous and nucleotide excision repair deficiency (SBS8, 48%), and indirect effects of AID-induced somatic mutagenesis (SBS85, 7%). Additionally, the Melphalan signature (SBS99, 10%) was found in patients treated alkylating agents. Conclusions: Disease progression of CLL on BTKi confirmed the existence of multiple driver genes beyond BTK, PLCG2 andTP53, including the high prevalence of mutations in MAPK pathway and epigenetic regulators, and increased genomic complexity. Ongoing efforts are focused on integrating these findings with clinical demographics, epigenetic modifications, and preclinical drug sensitivity profiles.

Abstract Background: Sickle cell crisis (SCC) remains a leading cause of emergency hospitalization and acute morbidity in patients with sickle cell disease (SCD). With improved survival in SCD, long-term complications such as cardiovascular disease are emerging as critical determinants of morbidity and mortality. While right-sided heart failure has been adequately explored in SCD, the specific contribution of the other cardiovascular conditions to in-hospital mortality during sickle cell crisis remains unclear. This study evaluates the independent impact of cardiovascular comorbidities on in-hospital mortality among adults hospitalized for SCC using a large, nationally representative database. Methods: This is a retrospective analysis involving adult hospitalizations (≥ 18 years old) for SCC in the United States (U.S) using 2016-2020 National Inpatient Sample (NIS). The study was exempt from institutional review board approval as the NIS database contains deidentified patient information. ICD-10 codes used for SCC were: D570, D5721, D5741, D5743, D5745, D5781. Regarding cardiovascular co-morbidities, the study considered age, race (African American, Hispanic and Caucasian), dyslipidemia, hypertension, diabetes mellitus (both type 1 and type 2), systolic and diastolic heart failure, rheumatic valvular heart disease, atrial fibrillation/flutter, pulmonary hypertension, chronic coronary artery disease and ischemic stroke. Univariable Cox regression was performed to identify independent predictors associated with in-hospital mortality among patients admitted for SCC. All factors from the univariable analysis with p<0.20 were included in a multivariable Cox regression model. All the outcomes were adjusted for age, sex, and Charlson Comorbidity Index. P-value of ≤0.05 was used to determine statistical significance. Results: The current study analyzed 424,540 SCD patients admitted for SCC (mean age 32.4 years, 55.4% females), from 2016 to 2020. Each additional year of age beyond 18 was associated with a linearly increasing in-hospital mortality among study patients hospitalized for SCC. This observation was confirmed in both univariate (HR: 1.06, 95% CI: 1.05–1.07, p<0.001) and multivariate (HR: 1.05, 95% CI: 1.04–1.06, p<0.001) analyses. On univariable analysis of cardiovascular co-morbidities in patients admitted for SCC, a higher in-hospital mortality was conferred by: hypertension (HR: 2.40; 1.95 – 2.94, p <0.01) dyslipidemia (HR: 2.38; 1.54 – 3.65, p <0.01), diabetes mellitus (HR: 2.15; 1.51 – 3.04, p <0.01, systolic (HR: 5.20; 3.70 – 7.31, p <0.01) and diastolic heart failure (HR: 2.11; 1.44 – 3.08, p <0.01), atrial fibrillation/flutter (HR: 5.43; 4.04 – 7.32, p <0.01), pulmonary hypertension (HR: 2.34; 1.75 – 3.13, p <0.01) and ischemic stroke (HR: 5.75; 3.30 – 10.03, <0.01); a lower in-hospital mortality was associated with the African American race (HR: 0.43; 0.20 – 0.91, p = 0.027). In multivariable analysis of cardiovascular comorbidities that demonstrated nominal significance (p < 0.20) in univariable analysis, only the following remained significantly associated with in-hospital mortality: systolic heart failure (HR: 2.65; 1.77 – 3.98, p <0.01), atrial fibrillation/flutter (HR: 2.01; 1.41 – 2.8), p <0.01) and ischemic stroke (HR: 3.25; 1.85 – 5.71, p <0.01). Although not statistically significant in univariable analysis (p = 0.065), chronic coronary artery disease (CAD) emerged as a significant predictor of reduced in-hospital mortality in the multivariable analysis (HR: 0.52; 0.29–0.91, p = 0.021). Conclusions: Among adults hospitalized for sickle cell crises, select cardiovascular comorbidities, including systolic heart failure, atrial fibrillation/flutter, and ischemic stroke, were independently associated with significantly increased in-hospital mortality. Interestingly, CAD was linked to reduced mortality in adjusted models. This may be attributed to closer medical surveillance and management in patients with established CAD, along with the potential protective mechanism(s) in SCD that limit atherosclerotic progression — warranting further investigation

Abstract Background Neurolymphomatosis (NL) is a rare and frequently underrecognized manifestation of lymphoma, characterized by infiltration of peripheral or cranial nerves by malignant lymphocytes. Limited data exist to guide optimal management. We present the largest single-enterprise (included the 3 Mayo Clinic sites) cohort to date, evaluating disease characteristics, treatment patterns, and outcomes in patients (pts) with NL. Methods We conducted a retrospective analysis of pts ≥18 years (yrs) diagnosed with clinical, radiographic, or biopsy-confirmed NL at Mayo Clinic between January 2002 and December 2024. Baseline characteristics were compared using independent t-tests for continuous variables and Fisher's exact or Pearson's chi-square tests for categorical variables. Primary endpoints were overall survival (OS) and progression-free survival (PFS). Objective response rate (ORR) was a secondary endpoint. Kaplan–Meier methods, log-rank tests, and Cox proportional hazards regression were used to assess survival outcomes and associated factors. Results We included 149 pts. Median age at diagnosis was 64 yrs (range 22–83), and 23% were female. Most presented with stage IV disease (89%), and NL was part of the initial lymphoma presentation in 42% (primary NL). Diagnostic confirmation was based on histopathology and imaging in 55%, imaging alone in 26%, histopathology alone in 16%, and clinical features in 4%. Diffuse large B-cell lymphoma was the most common histology (61%), followed by marginal zone lymphoma (7%), and T-cell lymphoma (7%). Clinical presentation included painful neuropathy (65%), concurrent CNS involvement (37%), systemic disease (43%), elevated LDH (46.9%), and ECOG ≥2 (38%). Pattern of involvement included cranial nerves (29%), peripheral nerves (28%), and spinal nerve roots (27%). Median follow-up was 92 months (95% CI, 63–119). Among 137 treatment-evaluable pts, first-line regimens included methotrexate-based (MTX) (62%), bendamustine-based (B) (11%), cytarabine-based (Ara-C) (4%), and rituximab-based (R) (excluding MTX, B, or Ara-C) (15%) therapies. Radiation was used in 18%, and 21% underwent autologous stem cell transplant (ASCT). Among 103 response-evaluable pts, the ORR was 67% (52% complete response [CR], 16% partial response [PR]). ORRs by regimen were 78% (MTX), 92% (B), and 86% (R). On univariate analysis, primary NL (OR 4.3) and frontline ASCT (OR 8.47) were associated with higher odds of response (p<0.05), while ECOG ≥2 (OR 0.24) and localized disease (OR 0.33) were associated with lower odds. On multivariable analysis (MVA), primary NL (OR 5.0 [1.5–12.1], p=0.02) and ECOG >1 (OR 0.2 [0.06–0.79], p=0.018) remained significant predictors of response. Median PFS was 13 months (95% CI, 9–36), with a 2-yr PFS of 43% (95% CI, 35–53). On MVA, concurrent CNS involvement (HR 2.4, p=0.05), systemic disease (HR 2.7, p=0.02), and ECOG >1 (HR 2.4, p=0.02) were associated with inferior PFS, while primary NL was associated with improved PFS (HR 0.3, p=0.002). Patients undergoing frontline ASCT had a median PFS of 108 months (95% CI, 58–not reached) with a 7-yr PFS of 62% (95% CI, 46–84). Among those receiving second line and beyond (2+) therapy, 24 received MTX-based regimens, 10 received CAR-T, and 8 received tyrosine kinase inhibitors. Post-ASCT median PFS was 30 months (95% CI, 15–NR). Among CAR-T recipients, evaluable responses (n=8) included 4 CR, 1 PR, and 3 PD. Median OS was 56 months (95% CI, 29–133), with a 5-yr OS of 50% (95% CI, 41–60). On MVA, ECOG ≥2 (HR 2.5, p=0.02) predicted worse OS, while receipt of ASCT (HR 0.4, p=0.04) and primary NL (HR 0.5, p=0.04) were associated with improved OS. Conclusion This study represents the largest single-enterprise cohort of pts with NL, providing comprehensive insight into its clinical presentation, treatment patterns, and outcomes. Primary NL and preserved PS were independently associated with improved response rates and survival, while CNS and systemic involvement at diagnosis portended poorer PFS. MTX and bendamustine-based regimens achieved high ORR, and frontline ASCT was associated with significantly prolonged remission duration and OS. These findings highlight the prognostic importance of baseline disease burden and functional status, support the early incorporation of high-intensity regimens in eligible patients, and underscore the urgent need for prospective studies to guide evidence-based management in this rare and understudied population.

Abstract Introduction: Anti-CD19 chimeric antigen receptor T-cell (CART) therapy is feasible with an acceptable safety profile in older patients (pts) with relapsed/refractory (RR) large B-cell lymphoma (LBCL) (Tun et al, AJH 2024). However, immune effector cell-associated neurotoxicity syndrome (ICANS) is a frequent and challenging toxicity in older pts, and its characteristics and associated risk factors are not well defined. Methods: Seven centers participated in this retrospective study. Pts aged ≥65 years (yrs) with RR LBCL treated with a commercial CART product were included. Baseline characteristics, treatment, CART toxicity, and outcomes were collected. ICANS was graded by the ASTCT criteria in most pts. Cumulative incidences of ICANS were estimated using deaths/progressions as competing risks, and associations between incidences and variables were assessed by Gray's test. A 30-day landmark was set after infusion to reduce immortal time bias. Progression-free survival (PFS; time from the landmark to disease progression or death) and overall survival (OS; time from the landmark to death) were analyzed with the Kaplan-Meier method. Statistical analyses were done in JMP v18. Results: A total of 224 pts were included. Median age at infusion was 71 yrs (range 65–89; ≥80 yrs, 12%. 142 (63%) were male and 27 (13%) had an ECOG PS scale of ≥2. The median HCT CI was 1 (range 0–6). LDH was elevated in 110 (51%) pts, 23 (19%) had bulky disease ≥7 cm, 176 (82%) had advanced-stage, and 145 (71%) had extranodal disease. Median number of prior treatments was 2 (range 1–8), and 44 (20%) had prior bendamustine. Prior stem cell therapy was done in 47 (21%) pts (autologous stem cell transplant [SCT], n = 45; allogeneic SCT, n=2). Axicabtagene ciloleucel (axi-cel) was most frequently used (58%; n=131) followed by lisocabtagene maraleucel (liso-cel) (16%, n=36) and tisagenlecleucel (tisa-cel) (25%, n=57). Median duration of ICANS was 6 days (interquartile range [IQR] 3–11; range 1–303). Median time to ICANS onset was 6 days (IQR 3.8–8.0; range 0–24). The 30-day cumulative incidence of all grade ICANS was 50.9% (95% confidence interval [CI] 44.7–57.9) and grade ≥3 ICANS was 29.8% (95% CI 24.0–37.0). There was no significant difference in incidence of all grade (P=0.39) and grade ≥3 (P=0.49) ICANS as well as duration of ICANS (≥14 vs <14 days; P=0.08) according to the age groups (≥65–69 vs ≥70–74 vs ≥75–79 vs ≥80–89). In contrast, pts with an ECOG PS scale of ≥2 were associated with a higher incidence of all grade (30-day estimate, 66.7% vs 47.0%; P=0.02) and grade ≥3 ICANS (30-day estimate, 53.8% vs 25.6%; P=0.002). In addition, pts with an elevated LDH level had a higher incidence of grade ≥3 ICANS than those without (30-day estimate, 37.5% vs 18.8%;P=0.004). The incidence of ICANS varied among various CART products, with 30-day estimates of 64.9% vs 33.3% vs 29.8% (P<0.0001) for all grades and 40.1% vs 13.2% vs 18.0% (P=0.004) for grade ≥3 ICANS in pts treated with axi-cel vs tisa-cel vs liso-cel, respectively. A trend toward higher grade ≥3 ICANS was seen in pts treated with ≥3 prior lines than those treated with 2 or 1 (30-day estimate, 35.7% vs 28.1% vs 10.5%; P = 0.07). ICANS was managed with steroids in 47 (41%) pts, levetiracetam +/- other agents in 46 (40%) pts, biologic agents +/- other drugs in 15 (13%) pts, and supportive care in 6 (5%) pts. Median follow-up from the 30-day landmark post-infusion was 17.2 months (95% CI 14.2–22.4). Median PFS was 10.5 months (95% CI 5.7–18.2) and OS was 18.2 months (95% CI 13.3 vs 27.5). There was no difference in PFS and OS in pts who experienced ICANS vs no ICANS (median PFS 14.7 vs 10.1 months; P=0.65 and median OS 18.2 vs 24.7 months; P=0.49). However, there was a trend toward inferior PFS and OS in pts who experienced high-grade ICANS compared to those who had grade 0-2 ICANS (median PFS 9.2 vs 21.2; P=0.14 and median OS 11.7 =vs 22.9; P=0.39). Conclusions: This large study of older pts with RR LBCL demonstrated acceptable risks of ICANS, comparable to that in pivotal registration trials, with a relatively higher rate of grade ≥3 ICANS. The risks of ICANS were similar regardless of patient age, including those aged ≥80 yrs, but different among CART products. Similar to younger pts, high tumor burden disease and poor performance status at CART infusion were associated with higher risks of ICANS. A comprehensive neurologic and frailty assessment is warranted to identify suitable CART candidates.

Abstract Introduction There is currently no standard-of-care for patients (pts) who cannot tolerate full dose anthracycline regimens in frontline large B-cell lymphoma (LBCL). R-miniCHOP is a commonly used regimen that provides curative intent treatment with manageable toxicity. Despite this, lymphoma and treatment-related mortality are the leading causes of death in these pts. Additionally, many pts may not be eligible to receive R-miniCHOP due to comorbidity or frailty. There are limited clinical trial and real-world data focused on this population. Methods Pts with LBCL age 18 years (yrs) and older were identified from the Lymphoma Epidemiology of Outcomes (LEO) cohort, a prospective study of newly diagnosed pts with non-Hodgkin lymphoma enrolled from 2015-2020 at 8 academic medical centers in the US. Pts with LBCL were included in this analysis if they had received R-miniCHOP or a regimen that did not include an anthracycline (nonAC). NonAC regimens included R-CVP, BR, rituximab, radiation and lenalidomide and rituximab. Pts completed the Vulnerable Elders Survey (VES-13) and were considered vulnerable if they had a score >3. Event-free survival (EFS) was defined as the time from start of treatment until progression/relapse, retreatment, or death due to any cause; EFS at 24 months (EFS24) was a secondary endpoint. Overall survival (OS) was defined as the time from start of treatment until death due to any cause. EFS and OS for R-miniCHOP and non-IC groups, as well as vulnerable and not vulnerable by VES-13 were estimated using Cox models. Results A total of 190 pts received R-miniCHOP (N=139) or nonAC (N=51). R-miniCHOP-treated pts were older (median 82 vs 72 yrs) and more commonly female (55% vs 35%). The majority in both groups (86% vs 78%) received their treatment at a LEO center and did not receive therapy on a clinical trial (89% vs 100%). Most pts receiving R-miniCHOP had high-intermediate (32%) or high (30%) international prognostic index (IPI) score, while both high-intermediate (22%) and high (20%) IPI were lower in the nonAC-treated pts. Cell of origin was determined by immunohistochemistry as available clinically, with GCB 42% vs 31%, non-GCB 32% vs 29%, and not done/unknown 26% vs 39% in R-miniCHOP vs nonAC pts. Double-hit status by FISH was known in 115 pts and was positive in 13% of R-miniCHOP pts and 10% of nonAC pts. The diagnosis-to-treatment interval (DTI) was ≤14 days in 30% of R-miniCHOP pts and 24% of nonAC pts. Of pts who had a VES-13 score (N=119), vulnerable status trended higher in the R-miniCHOP (66%) vs the non-IC group (50%; p=0.15). With a median follow-up of 50.5 months, the median EFS was 37.1 months and median OS was 50.0 months. R-miniCHOP, compared with non-IC, was associated with improved EFS (unadjusted HR=0.54, 95% CI 0.36-0.81; p=0.003) and OS (unadjusted HR=0.58, 95% CI 0.38-0.88; p=0.011). When adjusted for age, IPI, LBCL subtype (diffuse large B-cell lymphoma, not otherwise specified vs other), DTI and VES-13, R-miniCHOP was strongly associated with improved EFS (HR=0.26, 95% CI 0.14-0.50; p<0.001) and OS (adjusted HR=0.29, 95% CI 0.15-0.57; p<0.001). EFS24 rate was 56% in all pts and was superior in pts treated with R-miniCHOP vs non-IC (62.7% vs 34.1%; p=0.001). In the R-miniCHOP group, vulnerable status (adjusted for IPI) was associated with inferior OS (HR=2.22, 95% CI 1.09-4.50; p=0.028) and trended towards inferior EFS (HR=1.76, 95% CI 0.93-3.32; p=0.083). In a competing-risk cumulative-incidence analysis, lymphoma was the leading cause of death for pts receiving R-miniCHOP at 2 (0.16, 95% CI 0.11-0.24) and 5 (0.20, 95% CI 0.14-0.28) yrs, as well as for those receiving non-IC at 2 (0.35, 95% CI 0.24-0.53) and 5 (0.38, 95% CI 0.26-0.56) yrs. Treatment-related mortality was similar in both groups. In the R-miniCHOP group, the 2-yr (0.20 vs 0.03) and 5-yr (0.20 vs 0.11) lymphoma-related mortality was higher in pts who were vulnerable vs not by VES-13, while there were no differences for mortality due to therapy or other causes by vulnerable status. Conclusions These data support R-miniCHOP as a curative regimen in older and frail pts with the majority achieving EFS24. R-miniCHOP compared to non-IC use is strongly associated with improved EFS and OS when adjusted for disease and patient related variables. Within the R-miniCHOP group, vulnerable status is associated with inferior OS. Additional research efforts are needed to optimize outcomes for this group of pts.

Abstract Background: Rural/nonmetropolitan (nonmetro) counties face known barriers to comprehensive cancer care compared with metropolitan (metro) counties, which may impact the outcomes for lymphoma patients. We evaluated the impact metro vs nonmetro status on outcomes after a diagnosis of non-Hodgkin lymphoma (NHL) in the context of clinical, demographic, and lifestyle factors. Methods: Of 7724 participants enrolled in the prospective LEO cohort study from 2015-2020, 7610 (98.5%) had their county of residence at diagnosis assigned a RUCC (2013) code. We compared outcomes of participants from metro (defined as RUCC 1-3, which are central counties with ≥1 urban areas of ≥50,000) vs nonmetro counties (RUCC 4-9, which are counties with smaller urban areas as well as completely rural). We estimated hazard ratios (HRs) and 95% confidence intervals (CI) from univariable and multivariable Cox models for associations of metro status with overall survival (OS). Cause of death (lymphoma [including treatment related], non-lymphoma, unknown) was assessed using a competing risk approach. Results: Compared with metro counties (N=6096), LEO participants from nonmetro counties (N=1514) were older (median age 64 vs 62 years; p<0.001), more likely to be non-Hispanic white (94% vs 75%; p<0.001) and married (84% vs 75%; p<0.001), and less likely to have completed graduate or professional school education (16% vs 27%; p<0.001). There were no notable differences (>5%) by metro status for sex, employment status, NHL subtype, stage, performance status, abnormal lactate dehydrogenase (LDH), international prognostic index (IPI), number of comorbidities, time from diagnosis to treatment interval (DTI) or participation in a clinical trial. Compared with LEO participants from metro counties, those from nonmetro counties had inferior OS at 5 years (74.5% vs 78.4%; HR=1.23, CI 1.10-1.37; p<0.001). The OS association only slightly attenuated after adjustment for age, sex, race/ethnicity and NHL subtype (HR=1.20, CI 1.07-1.34; p=0.002), and held in models that adjusted for clinical prognostic factors (stage, NHL subtype, IPI, DTI), socioeconomic status (SES; education, marital status), health status (comorbidity, frailty), or lifestyle factors (smoking, body mass index). Using a competing risk model, the cumulative incidence of death at 5 years due to lymphoma (16.9% vs 14.1%; p=0.002) and non-lymphoma (5.9% vs 3.9%; p=0.002) was higher for participants from nonmetro vs metro counties, respectively. For participants <65 years, nonmetro vs metro status had inferior OS (HR=1.40, CI 1.17-1.67; p<0.001). This association remained after adjustment for age, sex, race/ethnicity and NHL subtype (HR=1.36, CI 1.14-1.63; p<0.001), as well as after adjustment for clinical prognostic, SES, health status, or lifestyle factors. The cumulative incidence of death at 5 years due to lymphoma was higher in participants from nonmetro vs metro counties (15.1% vs 11.5%; p=0.002), while there was no difference by metro status for non-lymphoma related deaths (2.1% vs 1.7%; p=0.47). In contrast, for participants age 65+ years, there was no differences by metro status for OS (HR=1.04, CI 0.91-1.20; p=0.56). After adjustment for age, sex, race/ethnicity and NHL subtype, participants from nonmetro areas had inferior OS (HR=1.12, 0.97-1.29) although this was not statistically significant (p=0.13), and was attenuated in models adjusting for clinical, health status, or lifestyle factors. The cumulative incidence of death at 5 years due to lymphoma was no different in participants from nonmetro vs metro counties (18.9% vs 18.0%; p=0.37), while non-lymphoma related deaths were higher in nonmetro counties (9.9% vs 6.8%; p=0.017). Conclusion: LEO participants from nonmetro vs metro counties had inferior OS overall, although this was predominately observed for younger (<65 years) participants. These associations did not appear to be explained by demographic, NHL subtype, clinical, SES, health status, or lifestyle factors. Of particular note, the risk of death due to lymphoma was higher in nonmetro areas for participants <65 years but not those 65+ years, while the risk of death due to other causes was higher in nonmetro areas for participants 65+ years but not those <65 years. These findings require further study, particularly related to barriers to care, and suggest the potential need for age-specific strategies to optimize outcomes for NHL patients from nonmetro/rural areas.

Abstract Background: Isocitrate dehydrogenase (IDH) mutations are frequently implicated in the development of acute myeloid leukemia (AML). In a recent study (Salman MY, Blood Adv 2025), it was reported that IDH2 mutation subtypes, R140 and R172, may respond differently to intensive chemotherapy. In this study, we evaluated mid-cycle and end-of-cycle bone marrow blast responses in AML patients with IDH2 R140, IDH2 R172, and IDH1 R132 mutations, aiming to determine whether these subtypes exhibit different response dynamics. Methods: We reviewed the charts of AML patients diagnosed at Mayo Clinic, Rochester, between the years 2016 - 2023 that had available next generation sequencing (NGS) data. Institutional review board approval was obtained. All data were recorded at the time of AML diagnosis. Patients without molecular data or not treated with intensive chemotherapy (7+3 or CPX-351) were excluded from this study. Bone marrow blast percentages were assessed at mid-induction (days 14-21) and end of cycle (counts recovery). Blast clearance dynamics and complete remission rates were compared across IDH mutation subgroups and correlated with outcomes. GraphPad Prism 10.0.0 was used for statistical analysis. Results: Out of 381 AML patients, 31 had IDH2 mutations (23 with R140 and 8 with R172), and 8 had IDH1 mutations (all R132). Among patients with IDH2 mutations, those with R172 had numerically higher median mid-cycle bone marrow blast percentage compared to R140 (10% vs. 4%, p = 0.37). There was no significant difference in mid-cycle blast percentages between patients with IDH1 and IDH2 mutations (4.5% vs. 4%, p = 0.8). At mid-cycle, 68% of patients with IDH2 R140 had blast counts <5%, compared to 38% of those with IDH2 R172, suggesting a trend for faster mid-cycle clearance in the R140 group, though not statistically significant (OR 3.57, p = 0.2). In a broader comparison, 60% of IDH2-mutated patients and 50% of IDH1-mutated patients achieved blast clearance (blasts <5%) at mid-cycle, though not statistically significant (p = 0.7). At the end of the induction, patients with IDH2 R140 had significantly lower median bone marrow blast percentages compared to those with R172mutations (2% vs. 4%, p = 0.01). Similarly, patients with IDH1 mutations showed low blast counts compared to the IDH2 group (1.5% vs. 3%, p = 0.1). A higher proportion of IDH2 R140 patients achieved blast clearance compared to those with R172 mutations (100% vs. 75%, p = 0.06), while proportion of patients who achieved blast clearance were similar between IDH1 and IDH2 patients (87.5% vs 93.1%, P = 0.5). Analysis of overall complete remission rates (including both complete remission [CR] and CR with incomplete hematologic recovery [CRi]) within the IDH2-mutated group showed a higher rate in patients with the R140 mutation compared to those with R172 (87% vs. 75%), though the difference was not statistically significant (p = 0.5). When comparing IDH1- and IDH2-mutated patients, overall complete remission rates were 75% and 84%, respectively, with no significant difference observed (p = 0.6). Kaplan–Meier analysis of time to end-of-induction blast clearance revealed no statistically significant difference between IDH2 R140 and R172 patients (median time to clearance: 31 vs. 37 days, p = 0.3). Similarly, no difference was observed between IDH1 and IDH2-mutated patients (33 vs. 31 days, p = 0.9). Overall survival analyses showed no significant difference between IDH2 R140 and R172 groups (median 66.8 months vs not reached, P = 0.4). Likewise, no difference was observed between IDH1 and IDH2 patients (44.4 months vs not reached, P = 0.7), indicating similar overall survival across mutation subtypes. Conclusion: IDH2-R140 was associated with more effective blast cytoreduction following intensive induction compared to IDH2-R172. Although not statistically significant, IDH2 R172consistently showed trends toward higher blast percentages, delayed response, and lower overall complete remission rates. These findings align with the recent study reporting significantly slower blast reduction and delayed response in R172-mutated AML. Also, no significant differences were found between IDH1- and IDH2-mutated patients in terms of response kinetics or overall survival. Together, these findings highlight potential biological differences among IDH mutation subtypes that may impact treatment response and require further investigation in larger prospective cohorts.

Abstract Background: Pyroptosis is a form of programmed cell death characterized by cleavage of gasdermin (GSDM) family proteins that form pores in the plasma membrane, cell rupture, and release of pro-inflammatory cytokines. Its pro-inflammatory potential has garnered significant research attention in recent years but has not been studied in lymphoma. Patients and Methods: We performed immunohistochemistry for cleaved GSDMD (n-terminal) in two large cohorts of diffuse large B-cell lymphoma (DLBCL) with multiplex fluorescent IHC (mfIHC) data, and analyzed for prognostic and immune impact. Results: Cleaved GSDMD was frequently expressed in DLBCL, and only the cytoplasmic (versus nuclear) form of expression correlated with significantly better patient survival in two DLBCL cohorts. To understand the expression at the single-cell level, we examined mfIHC results, which revealed that GSDMD+cells were tumor or immune cells, and varied in Ki-67, BCL2, and MYC expression. Cases with cytoplasmic cleaved GSDMD expression had higher mean and median levels of CD38+ (activated) M1 macrophages in two cohorts and lower PD-1/PD-L1 expression, as well as phenotypes of cognate interactions between live activated M1 macrophages, DLBCL cells, and T cells in spatially resolved immune landscape by mfIHC. In contrast, phagocytosis of pyroptotic lymphoma cells by M2 macrophages and cell death of T cells were observed in cases with predominantly nuclear form of cleaved GSDMD expression. Bulk gene expression profiling analysis identified a large proportion of RNA genes upregulated in DLBCLs with cytoplasmic cleaved GSDMD expression, and deconvolution analysis found decreased frequency of the “Inflammatory” lymphoma microenvironment (LME) subtype, increased frequency of cell states and ecotypes with favorable prognostic associations, and decreased frequency of T cell exhaustion states. Summary: Analysis of pyroptosis-inducing cleaved GSDMD (n-terminal) expression in a large number of DLBCL patients revealed dinstinctive cytoplasmic and nuclear expression patterns in live and pyroptotic cells associated with different prognostic and immune effects. Cytoplasmic cleaved GSDMD expression is a marker of cognate interactions between live cells (e.g., activated M1 macrophages) associated with adaptive responses and favorable prognostic effects, whereas nuclear cleaved GSDMD expression is associated with immunosuppressive mechanisms, including T cell death and phagocytosis by M2 macrophages.

Abstract Introduction: The ZRSR2 gene, located at Xp22.2, encodes a protein component of the RNA spliceosome. Alterations in ZRSR2 have been reported in various myeloid neoplasms (MN) and their precursors. They are frequently accompanied by other co-mutations, most commonly TET2. Isolated ZRSR2 mutations, however, are less commonly encountered and their clinicopathologic features have not been well documented. Aim: To describe the clinicopathologic features of MN and their precursors with isolated ZRSR2 mutations. Methods: Next-generation sequencing (NGS) was performed on peripheral blood (PB) or bone marrow (BM) samples from patients with confirmed/suspected MN using a 35-47 gene MN-targeting NGS panel. MN and clonal cytopenia of undetermined significance (CCUS) cases with isolated likely pathogenic/pathogenic (LP/P) ZRSR2 mutations were identified. Pathology diagnoses were rendered following the 2022 WHO/ICC classification and the slides were reviewed by three pathologists. Electronic medical records were reviewed for collection of relevant laboratory data. This study was approved by our institution's Institutional Review Board. Results: Of 164 ZRSR2-mutated cases, 10 cases with isolated LP/P mutations in ZRSR2 were identified. Patients were all male, with a median age 69.5 years. Three patients were diagnosed with CCUS, five with myelodysplastic syndrome (MDS), one with chronic myelomonocytic leukemia (CMML, MDS/MPN) and one with mixed phenotype acute leukemia (MPAL). Six patients had additional variants of undetermined significance (VUS) detected in ASXL1, CALR, EZH2, IDH2 and TET2. The other four patients had no additional variants identified by NGS but had cytogenetic abnormalities. One patient with MPAL had MECOM and IGH gene rearrangements detected on cytogenetic analysis. All patients had macrocytic anemia. One CMML patient with accompanying VUS in TET2 also had absolute monocytosis. BM cellularity and erythroid and granulocytic morphology were variable, ranging from unremarkable to left-shifted and dysplastic morphology. Nine (90%) cases showed atypical megakaryocyte morphology, characterized by hypolobated forms and osteoclast-like forms with separated and eccentrically located nuclear lobes. Interestingly, this megakaryocytic morphology was also observed in CCUS cases, albeit with lower frequency, below the 10% threshold necessary for the diagnosis of MDS. Megakaryocyte distribution was normal. Conclusions: Isolated LP/P ZSRS2 mutations are seen in a spectrum of myeloid neoplasms, including CCUS. They show a strong association with macrocytic anemia and megakaryocytic atypia/dysplasia. The presence of additional cytogenetic alterations and variants of undetermined significance is a confounding factor in determining the role of ZRSR2 mutations in driving the pathogenesis and morphologic features of these diseases.