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INTRODUCTION Medicaid expansion under the Affordable Care Act (ACA) has aimed to improve healthcare access and outcomes for low-income individuals across the United States. Acute Lymphoblastic Leukemia (ALL), a rapidly progressing hematological malignancy, demands timely and comprehensive medical intervention to optimize patient survival rates. Despite advancements in treatment, disparities in cancer-specific survival persist (CSS), particularly among socioeconomically disadvantaged populations. OBJECTIVE This study investigates the impact of Medicaid expansion on CSS among individuals diagnosed with ALL, aiming to explore the extent to which increased healthcare access through Medicaid expansion may influence survival outcomes in this vulnerable patient population. METHODOLOGY We utilized the Surveillance, Epidemiology, and End Results (SEER) cancer registry to examine the impact of Medicaid expansion on CSS among individuals diagnosed with ALL. A difference-in-difference analysis (DID) was conducted to evaluate the effect of the policy on survival outcomes. Texas, a state that has not implemented Medicaid expansion, was compared with California, which adopted the policy in 2014. The analysis adjusted for significant covariates including age, sex, race/ethnicity, socioeconomic status, and treatment modalities. By comparing pre- and post-expansion periods in these two states, we aimed to isolate the impact of Medicaid expansion on CSS in ALL patients. RESULT Among 22,303 patients, 60.0% were in California (13,384), while 40.0% were in Texas (8,919). The pre-ACA period included 14,129 patients (62.0% in California and 38.0% in Texas), and 8,174 patients were in the post-ACA period (56.6% in California and 43.4% in Texas). In the post-ACA implementation period, there was a 23.5% reduction in the hazard of deaths (HR=0.77, 95% CI 0.70-0.85). Medicaid expansion significantly improved cancer-specific survival among individuals with ALL across different race/ethnicity and income levels (DID=-0.69, 95% CI -1.18 to -0.19). The most significant improvements were observed among minority groups: Blacks (DID=-0.80, 95% CI -1.39 to -0.21) and Hispanics (DID=-0.72, 95% CI -1.35 to -0.22), as well as individuals with lower income levels (DID=-0.66, 95% CI -1.14 to -0.18). CONCLUSION The implementation of the ACA and its Medicaid expansion component led to a substantial improvement in cancer-specific survival among individuals with acute lymphoblastic leukemia in California compared to non-expansion Texas.

Background: Indolent non-Hodgkin lymphomas (iNHL) are incurable and disproportionately affect adults age >65 in whom frailty and functional impairment can impact disease management. The Vulnerable Elders Survey (VES-13) is a 13-item screening tool that incorporates age, self-rated health, physical function limitations, and functional disabilities to identify vulnerable older community-dwelling adults. Adults with a VES-13 score of >3 have a fourfold increased risk of death or functional decline over 2 years as compared to adults with a score <3 (Saliba et al, JAGS 2001). Our team previously showed that VES-13 >3 was associated with a higher risk of 1-year mortality in NHL (OR 3.26, 95% CI 2.15-4.96, Fama et al, ASH 2019), including in adults age <65. In this study, we determined whether VES-13 scores >3 were associated with quality of life (QOL) changes in adults with iNHL across the age spectrum. Methods: We used the Lymphoma Epidemiology of Outcomes (LEO) Cohort Study to identify adults enrolled from 2015-2020 with grade 1-3A follicular and marginal zone lymphoma (FL, MZL) who completed the VES-13 at enrollment and QOL surveys at enrollment and follow-up. The LEO Cohort Study enrolls participants within 6 months of diagnosis and collects information on disease status, QOL, health behaviors, and patient-reported functional assessment. For this analysis we included QOL measured using the Functional Assessment of Cancer Therapy-General (FACT-G) at years 1 and 2 post-enrollment. The FACT-G total score was the sum of 4 subscales (range): physical (PWB, 0-28), social/family (SFWB, 0-28), emotional (EWB, 0-24), and functional (FWB, 0-28) well-being. A higher FACT-G total score (range 0-108) indicated better overall QOL. VES-13 scores ranged from 0 (lowest risk for death or decline) to 10+ (highest risk). Generalized linear mixed models evaluated changes in QOL from baseline based on VES-13 score >3 (vulnerable) vs <3 (non-vulnerable), within groups by age at enrollment >65 and <65. Results: Our study included 1,186 participants: 42% age >65, 54% female, 84% non-Hispanic White, 10% Hispanic, 3.3% Black, and 1.4% Asian/Pacific Islander. Most (68%) were diagnosed with FL. FLIPI was high risk for 20%, and MALT-IPI was high risk for 23%. In the VES-13 <3 group, the mean (SD) baseline FACT-G total score was 86.5 (12.0), PWB 21.0 (3.5), SFWB 24.3 (4.3), EWB 18.8 (3.7), and FWB 22.3 (4.9). In the VES-13 >3 group, the mean (SD) baseline FACT-G total score was 70.4 (16.5), PWB 15.4 (5.6), SFWB 22.3 (5.5), EWB 17.5 (4.5), and FWB 15.3 (6.4); all significantly lower than the VES-13 >3 group (all p<0.01). In adults <65, 63 (9%) had a VES-13 >3, which was associated with a significant improvement from baseline in PWB at years 1 and 2 when compared to VES-13 <3 (changes in baseline PWB were 2.1, 2.9 for VES-13 >3 vs 0.2, 0.1 for VES-13 <3, all p<0.05). Similarly, we observed improvement in FWB from baseline at years 1 and 2 in the VES-13 >3 vs <3 group (2.4, 2.9 vs 0.4, -0.1, respectively all p<0.05). The VES-13 >3 group had a decline in their EWB at 1 year vs the VES <3 group (-0.5 vs 1.2, respectively, p=0.003). At year 2, total FACT-G improved from baseline in the VES-13 >3 group (5.6 for VES-13 >3 vs -0.5 for VES-13 <3, p=0.002). SFWB changes from baseline did not differ between groups. In adults >65, 83 (17%) had a VES-13 >3, which was associated with a significant improvement from baseline PWB (1.3, 1.5 for VES-13 >3 vs. 0.1, -0.1 for VES-13 <3), FWB (3.3, 2.2 for VES-13 >3 vs. 0.5, 0.1 for VES-13 <3), and total FACT-G score (5.2, 3.4 for VES-13 >3 vs. 1.0, -0.4 for VES-13 <3) when compared to VES-13 <3 at years 1 and 2 (all p<0.05). SFWB and EWB changes from baseline did not differ between groups. While there was improvement from baseline PWB and FWB in the VES-13 >3 group, QOL scores across all subscales remained below the VES-13 <3 group in the study period for both age groups. Statistical significance was maintained in models adjusted for baseline characteristics, including initial treatment strategy (observation, systemic, vs local) for both age groups. Conclusion: Although the vulnerable group reported worse QOL throughout the study period compared to the non-vulnerable group, survivors in the vulnerable group saw greater improvement in their physical and functional well-being at 1- and 2-years post-diagnosis. Additional analyses are underway to identify drivers of improvement in physical and functional well-being in vulnerable adults diagnosed with iNHL.

Background: Epcoritamab (epcor) is a first-in-class CD3xCD20 bispecific antibody (bsAb) approved for the treatment of 3L+ LBCL and follicular lymphoma. In the single-arm, phase 1/2, EPCORE NHL-1 trial (NCT03625037), epcor given until disease progression or unacceptable toxicity, demonstrated deep and durable responses, a median overall survival (OS) of 18.5 months, and a manageable safety profile in R/R LBCL. A second CD3xCD20 bsAb given as fixed-treatment duration, glofitamab (glofit), also demonstrated durable responses, conferring a median OS of 11.5 months in the pivotal study. Understanding how long-term outcomes compare among bsAb agents remains an important knowledge gap. With over 2 years of follow-up, in the absence of a head-to-head study, it is now relevant to compare the efficacy of these agents via indirect comparison. This study evaluated OS, progression-free survival (PFS), and duration of complete response (DoCR) of epcor vs glofit in R/R LBCL using data from the respective pivotal trials. Methods: Two analyses were conducted to compare the efficacy of epcor and glofit. First, a matching-adjusted indirect treatment comparison (MAIC) was conducted using published OS and PFS data from the full cohort of the glofit pivotal trial (12.6 months of follow-up, Dickinson et al, 2022) and the epcor pivotal trial (EPCORE NHL-1). Piece-wise hazard ratios (HRs) were calculated when proportional hazards assumptions were not met. Baseline characteristic prognostic variables were selected based on literature, clinical judgement, and empirical statistical testing and were adjusted between epcor and glofit cohorts. Second, in sensitivity analyses, indirect comparisons were conducted using an updated analysis of glofit with longer follow-up that included DoCR, together with OS and PFS data starting at cycle (C) 3 with 32 months of follow-up (Hutchings et al, ASH 2023). Sensitivity analyses with longer follow-up could not be adjusted because glofit pt baseline characteristics were not presented. Aggregate OS and PFS curves for glofit were derived from subgroups of pts with complete response/partial response/no response (CR/PR/NR) at C3 (Hutchings et al, ASH 2023). Aggregate epcor OS and PFS curves were similarly derived for pts with CR/PR/NR at C3 without prior disease progression. Weighted Cox proportional-hazards models were used to estimate the OS and PFS HRs between the 2 comparators. The DoCR comparison included all glofit and epcor pts achieving CR. All comparisons used EPCORE NHL-1 data with a median of 37.1 months of follow-up. Results: Pts enrolled into the pivotal trials of epcor and glofit were comparable by several key baseline characteristics before adjustment: age ≥65 y; male gender; Ann Arbor disease stage III-IV; primary refractory disease; refractory to most recent anti-CD20 therapy; previous chimeric antigen receptor T-cell therapy (CAR T); refractory to CAR T; and previous autologous stem cell transplant (ASCT). MAIC analysis revealed no significant difference in OS benefit from C1 until month 6 (HR 1.08, 95% CI 0.71, 1.64; P=0.72), but a demonstrable OS advantage from 6 months onward (HR 0.60, 95% CI 0.37, 0.98; P=0.04) in favor of epcor. An OS comparison starting at C3 with longer glofit follow-up demonstrated a consistent survival advantage in favor of epcor (HR 0.68, 95% CI 0.46, 1.01; P=0.05). The proportion of pts who received CAR T or ASCT subsequent therapy were similar for epcor and glofit (CAR T: 7.0% vs 8.9%; ASCT: 6.4% vs 6.3%) (National Institute for Health and Care Excellence Glofit Single Technology Appraisal, 2023). The MAIC analysis showed comparable PFS between epcor and glofit (HR 1.06, 95% CI 0.79, 1.41; P=0.71), although there was substantial censoring in the glofit PFS curve due to immature follow-up. With longer follow-up in the analysis starting at C3, with less censoring, the glofit PFS curve remained similar to the epcor PFS curve from C3 until month 6 (HR 0.67, 95% CI 0.42, 1.07; P=0.10), where separation of the PFS curves were observed favoring epcor (HR 0.56, 95% CI 0.33, 0.97; P=0.04). Median DoCR was longer for epcor (36.1 months) vs glofit (26.9 months). Conclusion: These indirect treatment comparisons suggest that epcor may provide an overall survival benefit over glofit through longer PFS with more mature follow-up, potentially driven by longer DoCR. Survival data with longer follow-up in the full glofit cohort are needed to confirm these findings.

Background: Patients with relapsed/refractory multiple myeloma demonstrate an increasing incidence of extramedullary disease (EMD), noted in up to 30% of patients. Presence of EMD portends a markedly inferior prognosis with limited response to salvage therapies, including immune effector therapy. Understanding the molecular alterations in EMD is key to exploring novel therapeutic approaches but remains to be well-characterized. Here we describe the mutational landscape and biologic pathways with alterations in a large cohort of patients with EMD. Methods: We included 18 paraffin-embedded tumor tissues from true EMD biopsies (non-bone contiguous disease) for whole exome sequencing (WES); biopsies from paraskeletal/bone-contiguous soft tissue were not considered. Germline sequencing was performed utilizing cryopreserved peripheral blood mononuclear cells (PBMCs) at initial diagnosis from patients without evidence of circulating tumor cells. Additionally, we studied 12 CD138-sorted cryopreserved bone marrow (BM) aspirates collected at the time of development of EMD. Following standard DNA extraction and library construction practices, we performed WES on the EMD, BM and PBMC samples. Variant calling was performed using consensus calls observed in at least 2 out of the 3 callers (Mutect2, VarScan, Strelka2) and these were included for downstream analysis. Synonymous and low-impact variants were filtered. Pathway analysis was performed using the PanCancer driver gene sets and compared using Fisher's exact test. Results: The median age of patients with EMD was 65.6 years (range 46.2-86 years). Seven (39%) patients had de novo (previously untreated) EMD and 11 (61%) had secondary EMD (developed at relapse). The MAPK pathway was frequently mutated, most commonly involving NRAS (33%), KRAS (22%) and BRAF (22%) genes. These mutations were mutually exclusive. Alterations in TP53 were noted in 11% of EMD samples. Single nucleotide variants (SNVs) in epigenetic modulators were frequently noted in EMD, with the most prevalent alterations seen in ARID1A (33%), KMT2C (28%), EP300 (22%) and KMT2B (22%). Both ARID1A and EP300 are postulated to play a role in IRF4 regulation and plasma cell differentiation. Additionally, inhibition of EP300 can be a viable therapeutic strategy in MM through disruption of IRF4 signaling. We noted frequent mutations in the roundabout (ROBO) genes ROBO1 and ROBO2, observed in 22% and 28% of EMD samples, respectively. ROBO1 has been demonstrated to be necessary for myeloma cell adhesion to BM stromal and endothelial cells, with ROBO1 knockout MM cells infused in mice demonstrating a tendency for extramedullary dissemination. Similarly, variants in LRP1B, a frequently altered large putative tumor suppressor gene in the LDL receptor family, were noted in 33 % of EMD samples. Depletion of LRP1B has been demonstrated to promote anchorage-independent growth, cell migration and invasion in vitro. Notably, in the paired BM samples, no SNVs were noted in ARID1A, LRP1B, ROBO1 and ROBO2 genes (q-value not significant). In paired samples, among the shared mutations of interest, we noted expansion of the BRAF clone in 2 EMD samples [variant allele frequency (VAF) of 3% and 13% in BM and 58% and 29% in the paired samples, respectively]. An expansion of a shared KRAS mutation was noted in another EMD sample (VAF of 15% in BM and 29% in EMD). The frequently altered cellular pathways in EMD samples (n=18) included the MAPK pathway (89%), chromatin histone modifier pathway (83%), transcriptional regulation (83%), immune signaling (72%) and RNA abundance (72%). In the cohort of paired EMD and BM samples (n=24), notable enrichment was evident in EMD for MAPK pathway alterations [91% in EMD vs. 25% in BM q=0.02] and chromatin histone modifier pathway (83% in EMD vs. 42% in BM, q <0.01). Conclusions: Patients withEMD demonstrate a near universal presence of alterations in the MAPK pathway. Variants in chromatin modifiers regulating plasma cell differentiation and genes involved in BM stromal adhesion and homing of plasma cells were frequently noted, potentially facilitating the extramedullary spread of myeloma cells. Functional studies are needed to evaluate these hypotheses. Targeting EP300, ARID1A and downstream MEK/ERK inhibition as part of combination therapies may represent promising therapeutic approaches for further exploration in EMD.

Background Acute myeloid leukemia (AML) with TP53 mutation (m) has poor outcomes even in the contemporary era with median overall survival (OS) of 6-9 months. The only factor that has been shown to improve outcomes is allogeneic hematopoietic cell transplantation (allo-HCT) (Badar et al AJH 2022; Leukemia 2023). However, the role of allo-HCT for TP53-m AML continues to be a matter of debate given high rates of relapse after allo-HCT particularly in TP53-m AML with complex cytogenetics (CG). We used the COMMAND consortium database to investigate the factors that influenced physician's decision for allo-HCT in TP53-m AML. Method We conducted a multicenter observational study in collaboration with 7 U.S. academic centers under COMMAND consortium. We reviewed electronic medical records of 369 TP53-m AML patients to analyze reasons for not pursuing allo-HCT including refractory disease, donor availability, advanced age/co-morbidities, physician perspective on ineffectiveness of allo-HCT in improving outcome and patient's decision. Diagnostic criteria for TP53 allelic state were according to the International Consensus Classification (ICC) (Blood 2022; 140:1200). Next-generation sequencing and cytogenetic studies were performed according to institutional standard protocols. Results The median age at diagnosis was 68 years, with 222 (60.5%) patients being male. The majority of patients were diagnosed with de novo AML 179 (48.8%), followed by secondary AML (sAML) in 103 (28%) patients, and therapy-related (t)AML in 85 (23.2%) patients. Ninety-four percent of evaluable patients had complex cytogenetics (CG) and 192 (52%) had multi-hit TP53-m. One-hundred forty-seven (42%) patients received intensive chemotherapy (CPX 351 [66 (44%)], 7+3 [78 (53%)]), while 205 (58%) patients received low-intensity regimens including hypomethylating agents (HMA) plus venetoclax (137 [66%]) and HMA +/- investigational agents (68 [33%]). The rate of complete remission (CR)/CR with incomplete count recovery (CRi) was 34.4% and overall response rate (CR/CRi + partial remission [PR] + morphologic leukemia free state [MLFS]) was 52%. Forty-seven (13%) patients received allo-HCT in CR1 and 17 (5%) patients received allo-HCT in CR2. Of the 294 patients for whom data on allo-HCT decision-making was available, the most common reason for not proceeding to allo-HCT was refractory disease in 126 (43%) patients. Other significant reasons included advanced age/comorbidities (n=78 [30.6%]), physician recommendation (n=7 [2%]), patient's decision (n=24 [9.4%]), and patient relocation or changing healthcare providers (n= 9 [3.5%]). Two (0.4%) patients had no suitable donor available for allo-HCT. Amongst the 7 patients for whom allo-HCT was not pursued due to physician's decision, the median age of the patients was 69 years (R, 59-70), 3 (25%) patients had sAML, and all of them had multi-hit TP53-m associated with complex CG. Six out of seven (86%) patients received HMA plus venetoclax and 1 (14%) patient received HMA-based induction. Six patients achieved CR/CRi after induction and 1 patient had partial remission. The median survival of these patients was 11 months (R, 10.7-11.3), eventually all patients died from progressive AML. Conclusion Our survey suggests that the primary reason for not proceeding with allo-HCT was refractory disease, underlying the difficulty in achieving the disease control needed for allo-HCT. There is an urgent need for better frontline therapies tailored specifically for TP53-m AML, along with refined patient selection criteria to better identify candidates who would benefit most from allo-HCT to potentially improve their long-term outcomes.

Background: Hypomethylating agent (HMA) and venetoclax (VEN) therapy is a standard of care frontline treatment of acute myeloid leukemia (AML) amongst patients (pts) ineligible for intensive therapy. Despite its convenient flat dose, oral nature, and static excretion irrespective of body weight (wt), VEN has a large, variable volume of distribution and extensive protein binding (Yang, Molecules, 2022). Body mass index (BMI) is a more accurate, albeit imperfect, indicator of body fat that has not been evaluated as an independent predictor of differential VEN exposures and clinical outcomes. We sought to evaluate this possibility amongst pts treated with frontline HMA+VEN in a multi-center, real-world, retrospective study. Methods: We conducted a multi-center, retrospective study through four US academic centers in the Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND). 368 pts treated with frontline HMA+VEN from 6/2016-8/2023were evaluated based on BMI within 30 days of induction. Responses were defined by European LeukemiaNet (ELN) 2022 criteria. Pearson's chi-squared test and Wilcoxon rank-sum test were used for comparison between groups. The Kaplan-Meier method and multivariable Cox regression model were applied to overall survival (OS). Continuous variables were summarized as a median with interquartile range (IQR), and categorical variables were reported as a frequency (percentage). Results: The median age was 75 (IQR: 70-79) years, 58% were male, and 76% were white. 107 (29%) had a normal BMI of 18-25 kg/m2, 141 (38%) were overweight (BMI 25-30 kg/m2), and 120 (33%) were obese (BMI >30 kg/m2). There were no significant differences in baseline pt-, disease-, and treatment-specific factors when comparing pts with normal wt to BMI >25.The cohort was enriched for complex karyotype and TP53-mutated disease (27% and 32%, respectively), with 73% categorized as adverse risk by ELN 2022. There was no difference in 30-, 60-, and 90-day mortality rates (6-8%, 14-18%, 19-29%, respectively) by BMI group. Complete remission (CR/CRi) rate was 50% overall and was not impacted by BMI (41%, normal wt; 55%, overweight; 52%, obese) (p=0.10). Overall response rate (ORR) (CR/CRi/MLFS) was 59% overall and differed by BMI groups (50%, normal wt; 66%, overweight; 58%, obese) (p= 0.047). The median OS was 6.6 months for normal wt, 13.7 months for overweight, and 8.3 months for obese pts, and not significantly different when comparing normal to overweight (p=0.052) or obese pts (p=0.756). In multivariable analysis, the effect of BMI varied by the composite remission rate used. Overweight was borderline significantly associated with higher likelihood of achieving CR compared to normal wt (odds ratio [OR] 1.98, 95% confidence interval [CI] 0.99-4.03; p=.056), whereas obesity was not (OR 1.78, CI 0.86-3.74; p=.13). Other co-variates with a significant association with achieving CR included diagnosis of secondary AML (sAML) (OR 0.37, CI 0.18-0.74; p=.006) and presence of IDH (OR 2.81, CI 1.30-6.15; p=.009), ASXL (OR 0.39, CI 0.15-0.96; p=.046), TET2 (OR 2.43, CI 1.17-5.09; p=.02), and DNMT3A (OR 2.37, CI 1.19-4.78; p=.02) mutations. There was a trend towards higher likelihood of achieving CR/CRi for both overweight (OR 1.74, CI 0.92-3.32; p=.09) and obese pts (OR 1.88, CI 0.96-3.74; p=.07). Diagnosis of sAML and presence of TET2, ASXL, and DNMT3A mutations no longer had a significant association with achieving CR/CRi, while IDH mutations maintained an association (p=.01). None of these variables demonstrated a significant impact on ORR (inclusive of MLFS) except overweight (OR: 1.99, CI 1.06-3.80; p=.03) and sAML vs de novo (OR: 0.53, CI 0.29-0.99; p=.047). In multivariable logistic regression, higher ECOG score was associated with risk of early mortality within 60 (p=.01) and 90 (p=.01) days, but not 30 days. sAML vs de novo associated with higher 90-day mortality, presence of NPM and TP53 mutation associated with higher 30-day mortality, and cytogenetic risk associated with higher 60- and 90-day mortality. Conclusions: This multi-center, real-world study demonstrates that BMI may not have a linear effect on response to frontline HMA+VEN, with some indication of negative impact at both lower and higher BMI categories. However, additional dedicated studies are needed to determine if differences in BMI is associated with altered drug exposure as an explanation for these differences.

Background Follicular and marginal zone B cell lymphoma (FL; MZL) demonstrate an indolent course with heterogeneous outcomes and a potential for spontaneous remissions indicating immune system intervention. T cell-based therapeutic approaches are therefore an attractive proposition in FL and MZL. EO2463 peptide immunotherapy expands pre-existing memory CD8 T cells recognizing non-self-protein sequences from gut bacteria which cross-react with B cell antigens. The compound includes 4 HLA-A2 synthetically produced epitopes which exhibit molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268 (BAFF-receptor), as well as a the CD4 helper-epitope UCP2 derived from hTERT. EO2463 is being used to drive anti-tumor immunity against B cell malignancies, and has demonstrated long-lasting, specific CD8-memory T cell responses and clinical activity in relapsed/refractory FL and MZL [J Clinical Oncol 2024, 42 (S16), 7058]. Furthermore, CD8 T cells expanded from samples of patients (pts) treated with EO2463 can kill HLA-A2 expressing lymphoblastoid cell lines presenting the B cell targets [Hematological Oncol 2023, 41 (S2), 581-582]. Methods The current report describes testing of a possible biomarker in the phase 1 portion of the ongoing EONHL1-20/SIDNEY trial (NCT04669171).This part of the trial (“Cohort 1”) included pts (n=9) with relapsed (at least 1, no limit, prior treatments) FL and MZL, stage 1-3A, confirmed to be positive for HLA-A2. Pts received EO2463 (150 [n=3] or 300μg/peptide [n=6]) SC with adjuvant Montanide ISA 51 VG q2 weeks (w) x 4, then q4w, for a max of 12 months; at w7 lenalidomide (20 mg/day for 21/28 days up to 12 cycles) was added (EL), and if no complete remission (CR) at w19, rituximab (375 mg/m IV, q1w x 4, then q4w x 4) was also added (ER2). Immune responses and clinical outcomes of Cohort 1 were previously reported [J Clinical Oncol 2024, 42 (S16), 7058]. To assess a possible biomarker, tetramers were used to specifically detect CD8 T cells recognizing the 4 epitopes in the EO2463 compound (derived from gut bacterial proteins exhibiting molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268). The tetramers were all marked with phycoerythrin. Thawed samples of peripheral blood mononuclear cells taken at start of w5 (after 2 administrations of EO2463 monotherapy, each followed by 2w observation) were stained ex vivo (without any in vitro stimulation) with the tetramers and assayed by flow cytometry. Results are presented as percentage of EO2463 specific CD8 T cells among all CD8 T cells in the peripheral blood of the pt. Results Among the 9 treated pts, PET-CT at w6 (after 3 administrations of EO2463 monotherapy) suggested possible clinical activity in 4/9 pts (1 PR, 1 pt size reduction 15%, 1 pt 20% reduced tracer uptake, 1 pt 5/6 target lesions reduced metabolic activity). By PET-CT at w42 (after 12 administrations of EO2463 monotherapy, 11 cycles of lenalidomide, and rituximab treatment) the Lugano response assessment was complete response in 6/9 pts. A positive biomarker response was assessed in 5/9 pts (percentage of specific CD8 T cells ranged from 0.2-2.1%). EO2463 monotherapy PET-CT activity at w6 (n=4/9 pts): 4 of 4 pts with clinical activity were biomarker positive and 1/5 of the other pts was also positive; this pt had an isolated increase in FDG-uptake at w6, followed by a CR at w19. Four further other pts were biomarker negative. In this limited sample, the biomarker had sensitivity 100% and specificity 80%. EO2463 in combination with lenalidomide/rituximab PET-CT CR at w42 (n=6/9): 5 of 6 pts with CR were biomarker positive and 1 pt with CR was negative; the pt with a negative test had no objective tumor response until after rituximab. In this limited sample, the biomarker showed sensitivity 83% and specificity 100%. Conclusions The current preliminary assessment indicates that direct ex vivo tetramer staining for specific CD8 T cells after short term (5 w) treatment with EO2463 monotherapy could be a possible biomarker for clinical response, both for EO2463 monotherapy response, and for response to EO2463 in combination with lenalidomide/rituximab. We plan to validate these findings in the ongoing study cohorts of the trial; expansion phase 2 cohorts, exploring EO2463 monotherapy in the “watch & wait” setting, EO2463 plus rituximab for 1st line FL/MZL, and EO2463/lenalidomide +/- rituximab in relapsed FL/MZL.

Splenic marginal zone lymphoma (SMZL) is a B-cell malignancy with a typically indolent course. However, some patients have more aggressive disease and initial treatment can range from observation to chemoimmunotherapy. The reason for this spectrum of disease behavior is incompletely understood but the tumor microenvironment (TME) likely plays a role as recent studies have shown that an optimized antitumor immune response in SMZL patients would allow the immune system to better target malignant B cells and irradicate the disease. Regulatory CD4+T cells (Tregs) are crucial in maintaining TME homeostasis by suppressing effector T cells and malignant B cells and in this study, we therefore explored the key factors influencing the function and development of intratumoral Tregs in SMZL patients. We identified two distinct Treg subsets in the TME of SMZL patients: CD26+Tregs and CD161+Tregs, each with unique phenotypic profiles, functions, and prognostic significance. CD161+Tregs suppress CD8+T cells and malignant B cells more effectively than CD26+Tregs, which only inhibit CD8+T cells. Clinically, patients with inferior outcomes have significantly higher numbers of CD26+Tregs and fewer CD161+Tregs. To delineate differences between these subsets, we performed Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-seq) on biopsies from 7 SMZL patients. We found that cytokine-inducible SH2-containing protein (CISH) is the most highly expressed gene in CD26+Tregs compared to CD161+Tregs, aside from FOXP3. CISH has been reported to be a negative regulator of immune effector cell activation in tumor models. Although CISH deficiency leads to a loss of Foxp3 expression and reduced Treg suppressive function, the mechanism by which CISH modulates Treg function, especially immune suppression in hematologic malignancies, remains largely unknown. To further investigate the mechanism of CISH in Treg function in MZL patients, we conducted CITE-seq analysis on a larger cohort of MZL patients (n=12), divided into groups based on outcomes (EFS24 failed, n=4; EFS24 achieved, n=8). The CITE-seq analysis results showed significantly higher CISH expression in Tregs from patients with inferior outcomes (P=1.07E-06). We then explored factors influencing CISH expression and found that TCR engagement and treatment with cytokines (IL-15, IL-2, and IL-7) elevated CISH expression in Tregs. While we previously observed that lymphoma B cells could enhance Foxp3 expression in CD4+CD25-T cells, this study we found that B cells could also enhance CISH and CD26 expression in CD4+CD25-T cells in vitro, indicating that a high frequency of B cells in the environment may lead to increased CISH expression in Treg cells leading to an unfavorable outcome in patients. We further investigated the role of CISH in the function of Treg cells. Following CISH knockdown in Treg cells, we observed decreased CD26 expression and reduced suppressive function on CD8+ T cell proliferation. Using CRISPR-Cas9, we knocked out CISH in the Karpas 299 cell line, a human T cell lymphoma line that constitutively expresses CD25, Foxp3, and CISH. CISH-KO Karpas 299 cells showed significantly less apoptosis and Foxp3 expression compared to CISH-WT cells. Bulk RNA-seq analysis of CISH-KO and CISH-WT Karpas 299 cells revealed that CISH-KO cells showed enhanced DNA replication and cell cycle pathway activity. Taken together, our results suggest that high expression of CISH in Tregs may predict an unfavorable outcome in SMZL patients. Malignant B-cells in the TME of SMZL patients may promote expression of CD26 and CISH in Tregs, thereby modulating Treg function. Targeting CISH could decrease the immunosuppressive activity of Tregs in the TME, potentially optimizing antitumor immunity in SMZL patients.

Introduction: Ticagrelor is a widely used reversible platelet adenosine 5'-diphosphate (ADP) P2Y12 receptor inhibitor used in patients with acute coronary syndrome and coronary interventions. Ticagrelor treated patients may be concomitantly exposed to heparin treatment for prophylaxis/treatment of thrombosis, and heparin induced thrombocytopenia (HIT), a thrombotic thrombocytopenic adverse event may be suspected in such patients. Objective: Here we describe the serological profile of a patient on ticagrelor treatment and strong clinical suspicion of HIT who presented with negative serotonin release assay (SRA) results. A follow up systematic study on the impact of ticagrelor on this gold standard HIT assay is presented. Methods: Remnants from 23 patient samples submitted for clinical SRA testing (5- SRA negative; 6- “low positive” [20-50%]; 5- “moderate positive” [>50 to <90%]; 7- “strong positive” [>90%]) were spiked with ticagrelor in concentrations of 100, 250, 500, and 1000 ng/mL before SRA re-testing. Testing was performed using heat inactivated serum, with low (0.1 units/mL) or high (100 units/mL) heparin and washed platelets (Leger, THSNA 2022). A positive SRA result was defined as ≥20% serotonin release with the low heparin condition and >50% inhibition of serotonin release with high concentration of heparin. A subset of SRA positive patient samples that tested falsely negative upon ticagrelor addition were subsequently incubated with activated charcoal and retested in the SRA to determine if ticagrelor interference was reversible using this methodology. Results: The index patient on ticagrelor had a high 4Ts score, ELISA optical density >1.0 but tested negative in the SRA. Re-testing of this patient with an activated charcoal treated sample demonstrated a striking change in SRA to a strong positive result (91% serotonin release). Upon testing of ticagrelor-spiked samples in the SRA negative patient cohort, as expected, the drug had no effect on results, with all 5 samples persistently staying SRA negative. Of the 6 patients that had low positive SRA results, all converted to SRA negative with ticagrelor concentrations >500 ng/mL. One of these patients became SRA negative at 100 ng/mL and 3 at 250 ng/mL. Of the 5 patients with moderately positive SRA results, 2 converted to SRA negative with ticagrelor concentrations >100 ng/mL, and 4 of 5 patients became negative with spiking at 500 ng/mL. Finally, of the 7 patients with strong positive SRA results, 1 became falsely negative in the SRA at a ticagrelor concentration of 1000 ng/mL. Activated charcoal was incubated with 6 samples that demonstrated negative SRA results with drug concentration of 500 ng/mL. Two samples reverted to SRA positive, while 4 samples remained persistently negative. Conclusion: In preliminary studies, ticagrelor has been shown to interfere with the heparin induced platelet activation (HIPA) assay but its impact on the SRA has been relatively unexplored. Here, we demonstrate that patients on ticagrelor may present with spuriously false HIT functional assay testing by the SRA, particularly if the HIT antibody is not strongly platelet activating. Activated charcoal treatment was only able to reverse drug interference in a subset of samples, even at drug concentrations below peak levels recorded in patients (approx. 800ng/mL). Given these findings, we recommend de-emphasizing the utility of the SRA in ticagrelor treated patients with reliance more on criteria such as pre-test probability of HIT (e.g. 4Ts score), and ELISA results in order to facilitate an accurate diagnosis of HIT.

Background: Pneumocystis pneumonia (PCP) is an opportunistic fungal infection, which is frequently observed in immunocompromised patients. Although T-cell immunity is classically related to Pneumocystis defense, recent data support roles for B lymphocytes in the development of PCP in animals, and we have observed several cases of PCP in patients receiving rituximab. These observations prompted a systematic review and meta-analysis of our experience to see the efficacy of PCP prophylaxis (TMP-SMX) in lymphoma patients receiving rituximab. The efficacy of PCP prophylaxis in this population remains a subject of clinical interest. Research Question: To evaluate the effect of PCP prophylaxis compared to no prophylaxis on the risk of developing PCP in lymphoma patients receiving rituximab. Methods: We conducted meta-analysis including six retrospective cohort studies that compared the incidence of PCP in lymphoma patients receiving rituximab with and without prophylaxis. The cohort studies with incept up to January 2023 were retrieved from databases including PubMed, Google Scholar, EMBASE, PLOS ONE, Cochrane, and ClinicalTrials.gov. Data extraction was done using a spreadsheet, and data analysis was done using RevMan 5.4.1 for meta-analysis. Results: The analysis included 2304 patients, with 571 receiving PCP prophylaxis and 1733 did not receive prophylaxis. Studies reporting results of PCP prophylaxis were included. Enrolled studies were quality assessed with Newcastle-Ottawa Scale. The incidence of PCP was significantly lower in the prophylaxis group (3 out of 571) compared to the no prophylaxis group (67 out of 1733). The pooled risk ratio (RR) was 0.23 [95% CI, 0.09 to 0.58], indicating a 77% reduction in the risk of PCP with prophylaxis. Conclusion: The clinical course of cases of PCP in patients treated with rituximab can be quite fulminant, with significant mortality. Primary prophylaxis should be considered in patients at risk. PCP prophylaxis significantly decrease the risk of PCP in lymphoma patients receiving rituximab-based therapies, with a pooled risk ratio of 0.23.