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Aim: The definition of high-risk (HR) in Multiple Myeloma (MM) is ever evolving with the discovery of new genomic abnormalities and increasing therapeutic options. There are various stratification models incorporating laboratory and genetic features with different definitions of high-risk and varied emphasis on individual risk factors. Recognized high-risk cytogenetic abnormalities (HRCA) include t(4;14), t(14;16), t(14;20), del(17p) with recent inclusion of del(1p) and gain/amplification (amp) of 1q (+1q) . In addition to del(17p), the presence of biallelic del(1p) and ≥2 HRCAs (“double-” or “triple-hit”) appear to confer poorer outcomes. A new definition proposed by the International Myeloma society (IMS) defines HR as the presence of del(17p) and/or TP53 mutation; an IgH translocation - t(4;14), t(4;16) or t(14;20) along with +1q and/or del(1p); monoallelic del(1p32) along with +1q; bi-allelic del(1p32); or β2 microglobulin ≥5.5 with normal creatinine (<1.2mg/dL). Herein, we report real-world outcomes on these HRCAs and the new IMS consensus definition of HR MM. Method: We retrospectively reviewed newly diagnosed MM patients from the Australian and New Zealand Myeloma and Related Diseases Registry across 4 Australian states. Patients with FISH and/or SNP microarray testing for IgH translocations, 1q21 (CKS1B), del(1p32) (CDKN2C) and del(17p) (TP53) were identified. Positive threshold was 30%. We used Kaplan-Meier methods to compare progression free survival (PFS) and overall survival (OS) between groups and first performed univariate analysis (UA) for each HRCA. We next analyzed patients with gain1q vs amp1q and monoallelic vs biallelic del(1p). In the final analysis, two risk groups were formed: cases meeting the new IMS-defined HR criteria were considered IMS-HR and the rest were considered standard risk (IMS-SR). Indeterminate cases with missing variables that could influence risk classification were excluded. Results: Of 839 newly diagnosed patients (pts) included, 31% had ISS (International staging system) stage 3 disease. High-risk IgH translocations t(4;14) and t(14;16) were present in 8% and 4%. Secondary HRCAs del(17p), del(1p), gain1q and amp1q were present in 9%, 14%, 30% and 11% respectively. In this cohort, 41% received ASCT. Of the transplant eligible pts, 41% received triplet therapy (PI, IMiD and corticosteroid) and 57% received PI-based induction. Of the transplant ineligible pts, 31% received triplet therapy, 47% PI-based and 19% IMiD-based therapy. Frontline Anti-CD38 antibody use was <1%. Median follow up was 35 months[m] (IQR 18-60). On UA, the risk of progression and/or death was increased with ISS III vs I, amp1q, del(17p), t(14;16), del(1p) and gain1q in descending order by hazard ratio. The risk remained significant in ISS III, del(17p), amp1q and gain1q after multi-variate analysis (MVA). The risk of death was increased with ISS III vs I, del(17p), amp1q, del(1p) and t(14;16), ISS II vs I in descending order by hazard ratio. After MVA, the risk remained significant for ISS stage III, del(17p), amp1q and t(14;16). Of the 117 pts with del(1p32), 108 had monoallelic and 9 had biallelic loss. Pts with biallelic del(1p32) had an inferior PFS (median 16.3 vs 23.2m, p<0.001) and OS (median 24.8m vs NR, p<0.001) compared to those with monoallelic del(1p32). In pts with 1q+, pts with amp1q had an inferior PFS (median 16.3 vs 27.5m, p<0.001) and OS (median 37.0m vs NR, p<0.001) vs pts with gain1q. Based on the new definition of HR myeloma - 385 pts met the criteria for IMS-HR and 216 for IMS-SR. There were 238 indeterminate cases due to missing data. The IMS-HR group had an inferior PFS (median 20.2 vs 39.7m, p<0.001) and OS (median 44.2m vs NR, p<0.001) when compared to IMS-SR. Noting that the IMS-HR group had a higher median age, poorer performance status and lower rates of ASCT use, adjusted analysis for these variables still showed an inferior PFS (aHR 1.68; 95% CI 1.30-2.16, p<0.001) and OS [aHR 1.72; 95% CI 1.21-2.44, p=0.002). Conclusion: ISS III, del(17p), amp1q are the strongest predictor of OS and PFS. Differentiating between monoallelic vs biallelic del(132p) and gain vs amp1q21 has important prognostic value. The IMS- HR definition which takes into account del(1p), “double-” and “triple-hit” disease predicts for a group of patients with a PFS of ~20 months and inferior OS, and provides a standardized approach to risk stratification in MM.

Background The detection, isotyping and quantitation of over-expressed immunoglobulin(s) (Igs) (M-protein) is key for diagnosing and monitoring patients with monoclonal gammopathies. Traditionally, our lab has used a combination of agarose gel protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) for these purposes. In 2018, a mass spectrometric method (Mass-Fix) was implemented to replace IFE for M-protein isotyping. This work documents the validation of a quantitative version (Q-Mass-Fix) to replace both SPEP and IFE. Methods 24,645 samples submitted for M-protein testing between 7/2018 and 9/2023 with quantitative IgG, IgA and IgM measurements, Mass-Fix analysis and SPEP results were utilized for this study. A separate cohort of 100,000 M-protein negative samples were utilized to mathematically model the spectral shapes IgG, IgA and IgM polyclonal background. To detect M-proteins, software was developed that fits the polyclonal Ig portion of the spectra and M-proteins are detected as peaks above the polyclonal background. M-proteins isotypes are determined by matching similar peaks in spectra in 5 sperate immuno enrichments (IgG, IgA, IgM, kappa and lambda). M-protein quantitation was obtained by multiplying total M-protein isotype quantity by the fraction area of the M-protein in the spectra. M-protein dilutional studies were used to examine the linearity and level of detection of Q-Mass-Fix. Results SPEP and Q-Mass-Fix M-protein quantitation had the best agreement between the range of 10 to 40 g/L. Below 10 g/L, Q-Mass Fix had on average, lower M-protein values. Above 40 g/dL, Q-Mass-Fix values were on average, higher than SPEP. The automated isotyping assignment was 98.8 percent consistent with the isotype assigned by visual inspection. Dilutional studies revealed that Q-Mass-Fix quantitation was linear over a range of 70 to 0.04 g/L (slope 1.0, r2 0.99) with an analytical limit of quantitation of 0.1 g/L and a limit of detection of 0.04 g/L. Conclusion Q-Mass-Fix represents a significant improvement in comparison to our current SPEP method. Q-Mass-Fix is more capable of removing the interference of polyclonal Igs below 10 g/L and is not subjective to the dye saturation effect at high M-protein levels. The increased analytical measuring range and improved limit of detection of detection could be beneficial in tracking patient's response. Q-Mass Fix is more automated than SPEP and the workflow in our laboratory.

Background: Financial toxicity (FT), or the negative impact of medical expenses experienced by patients, is a known adverse effect of cancer treatment that result in worse health outcomes for patients. However, there is limited literature investigating FT in lymphoma with even less data on predictors such as age, gender, and socioeconomic factors. As lymphoma treatment options expand, it is vital to understand the impact of these treatments on patients' financial well-being. We investigate FT through patient reported outcomes from the Lymphoma Epidemiology of Outcomes (LEO), a large multi-institution prospective cohort study of non-Hodgkin lymphoma (NHL). Methods: Patients with newly diagnosed NHL were enrolled into the LEO cohort from 2015-2020. Clinical, pathology and treatment data were abstracted from medical records, and participants were offered self-administered surveys at enrollment and selected follow-up time points. Participants were included if they completed at least four of six survey questions on FT adapted from the Medical Expenditure Panel Survey (MEPS) within the first year after diagnosis. Questions focused on either material hardship (unable to cover costs; file for bankruptcy; borrow money; work extra job or hours) or psychological stress (worried about paying large medical bills). We evaluated FT as answering positively to any question (any vs none). Associations with FT were evaluated using chi-squared tests. All analyses were performed using R (v4.2.2). Results: 3202 patients completed at least four survey questions relating to FT. Mean age at diagnosis was 62 years (18-94), 56% were male, and majority identified as white (90%). Characteristics associated with FT are reported (p<0.001). FT was more likely in younger patients: 18-25 years (64%, 30/47), 26-44 years (73%, 299/313), and 45-64 years (58%, 799/1375) as compared to those 65-79 years (36%, 459/1272) and ≥80 years (35%, 69/195). FT was highest in patients without a high school diploma (54%, 45/84), followed by patients with a high school diploma (52%, 209/402), and an associate's degree (57%, 358/627). It was lowest in patients with a bachelor's (45%, 224/296) or post-graduate degree (39.6%, 216/545). Higher FT was seen in those receiving disability income (65%, 47/72); whereas less FT was seen in retired patients (36%, 299/828). FT rates inversely correlated with income: 65% for income < $21,000 (184/283), 61% for $21,000- $39,999 (224/370) and 60% for $40,000-$65,999 (293/491). Lower FT noted for income range of $66,000-$106,000 (47%, 323/681) or > $106,000 (37%, 324/872). Patients separated from a partner (82.9%, 19/23) had the highest FT, followed by patients living with a partner (64%, 48/75), divorced (56%, 103/184), and never married (53.4%, 79/148). Patients who were married (47%, 793/1672) or widowed (41%, 60/146) reported the lowest FT. Higher FT was reported in DLBCL (55%, 631/1146), MCL (55%, 172/315), and TCL (53%, 119/226) when compared to FL (44%, 327/746), MZL (44%, 137/316), and other NHL (44%, 200/453). This is likely related to treatment as more FT was seen in patients who received an anthracycline and/or alkylator containing therapy (54%, 1066/1963) versus non-systemic (43%, 120/278) and observation (35%, 168/483). In multivariable analyses, FT strongly correlated with age at diagnosis, household income, and treatment type. The odds ratio and 95% confidence intervals for the most significant category within each variable are as follows: age per 1 year (0.96, [0.95-0.97]), income > $106,000 vs < $21,000 (0.28, [0.21-0.38]), and observation vs anthracycline and/or alkylator (0.56, [0.44-0.71]). Conclusion: Nearly 50% of patients with lymphoma experience FT, seen as material hardship and psychological stress, within the first year of diagnosis. Patients with a younger age (18-44), less education, and lower household income reported higher levels of FT; whereas those who were retired or married/widowed reported lower FT. Lymphoma subtypes that require more anthracycline and/or alkylator containing therapy imposed higher FT. Understanding these risk factors can help clinicians identify patients who may benefit from additional resources during lymphoma management.

Introduction: Progression-free survival (PFS), a common endpoint used in multiple myeloma (MM) trials, is defined as the time to the earliest occurrence of disease progression or death. Disease progression in MM is determined by the International Myeloma Working Group (IMWG) Uniform Response Criteria based on imaging and biomarkers from blood, urine, and bone marrow biopsy testing. In clinical trials, IMWG-recommended assessments are protocol-based. However, in routine care, they are often performed and recorded differently based on a multitude of factors. Algorithms to ascertain progression events using routinely collected biomarker data are important to classify disease progression and align with trial-defined endpoints. The aim of this study was to construct and evaluate an optimized real-world Progression (rwP) algorithm using real-world data (RWD) and demonstrate how biomarker data are captured and used to derive endpoints in routine practice. Method: A cohort of 270 transplant ineligible newly diagnosed MM (NDMM) patients starting on lenalidomide and dexamethasone (Rd) and meeting select MAIA (NCT02252172) trial eligibility criteria were included from the nationwide Flatiron Health electronic health record-derived deidentified database from 2015 to 2023. A rwP algorithm was developed using key IMWG-recommended biomarkers, including serum protein electrophoresis (SPEP), urine protein electrophoresis (UPEP), and serum free light chains (FLCs). The initial baseline value for each biomarker was identified, if available, during either the pre-treatment period (45 days prior through 7 days after first line treatment (1L) start) or the on-treatment period (8 days after 1L start through 1L end). Available biomarkers were followed for disease assessment. If any subsequent lab value during 1L treatment was less than the initial baseline value, the subsequent “on-treatment baseline” value was adopted as the new lowest value; otherwise, the initial value was used as the “on-treatment baseline” value for assessing progression events. Three hierarchies, which describe the order of utilizing key IMWG-recommended biomarkers to define rwP events, were explored, including: i) strict hierarchy (SH) [SPEP > UPEP > FLCs], ii) partial hierarchy (PH) [SPEP = UPEP > FLCs], and iii) no hierarchy (NH) [SPEP = UPEP = FLCs]. The PH option closely resembles the IMWG criteria, with FLCs only used for rwP assessments when no measurable SPEP/UPEP are identified. The NH approach considers all available biomarkers equally and resembles how they are utilized in real-world clinical practice. The feasibility of requiring confirmatory events following the first suggestion of progression was also explored. Assays following the progression event up to the end of 1L were considered as confirmatory progression events if they met IMWG criteria. Results: We applied the rwP algorithm with each hierarchy to all eligible NDMM patients (N = 270). The SH, PH, and NH approaches respectively identified 106 (39.3%), 106 (39.3%), and 123 (45.6%) patients with progression events. Results from the SH and PH approach are consistent likely due to the rarity of UPEP usage in clinical practice. The NH approach captured more progression events because FLCs are used in the presence of measurable SPEP or UPEP, resulting in 73 progression events captured by FLCs in comparison to 32 from other approaches, potentially increasing false-positive capture due to the volatility of FLC results. Among the 106 PH-identified patients who progressed, 84 (79.2%) had IMWG-recommended tests following their progression event, and 74 (69.8%) had their progression confirmed before the end of 1L, with 35 (33.0%) receiving their confirmatory reading within 2 months after progression. These results suggest that requiring confirmatory testing as part of the rwP is feasible, though it may introduce false negatives for patients receiving clinical progression confirmation instead of getting confirmatory labs in routine clinical practice. Conclusion: We present a comprehensive assessment of important design choices for an rwP algorithm for NDMM, including a hierarchy of key assays and the inclusion of confirmatory testing. Further validation of the algorithm with health care provider assessments and the inclusion of bone marrow biopsy and imaging data is important for evaluating the performance of the algorithm in RWD.

INTRODUCTION: Non-Hodgkin's Lymphoma (NHL) survival is influenced by various factors, including marital status and sex. This study explores how these intersecting factors affect cancer-specific (CSS) and overall survival (OS) in NHL patients. OBJECTIVE: To examine the intersection of sex and marital status on survival outcomes among individuals with NHL. METHODOLOGY: We conducted a retrospective cohort study using SEER registry data for individuals aged 18-85 diagnosed with NHL. Cox regression models assessed the influence of marital status and sex on CSS and overall survival OS, adjusting for age, race, household income, disease stage, primary site, and treatment (chemotherapy, radiotherapy, surgery). Kaplan-Meier curves and log-rank tests compared survival distributions. RESULT: The study analyzed 319,013 individuals with NHL from 2000 to 2020. Participants were predominantly white (73.0%) and female (54.7%), with 54.6% married and a median age of 66 years (IQR 55-76). Females exhibited a 33% higher hazard of death for cancer-specific survival (HR = 1.34, 95% CI: 1.30-1.38) and a 40% higher hazard for overall survival (HR = 1.40, 95% CI: 1.37-1.44). The interaction of marital status and sex significantly impacts both CSS and OS among individuals with NHL. Widowed individuals, regardless of sex, showed the highest risk of death; males had a cancer-specific hazard ratio (HR) of 2.50 (95% CI: 2.35-2.65) and overall HR of 2.31 (95% CI: 2.21-2.41), while females had a cancer-specific HR of 3.09 (95% CI: 2.87-3.31) and overall HR of 2.97 (95% CI: 2.82-3.12). Divorced and separated individuals also exhibited elevated risk. Notably, married males had the lowest hazard ratios, with cancer-specific HR of 1.07 (95% CI: 1.03-1.12) and overall HR of 0.87 (95% CI: 0.84-0.89). CONCLUSION: This study highlights that marital status and sex significantly influence survival outcomes in NHL patients. Widowed individuals have the highest mortality risk, while being married is associated with better survival, especially for males. These findings emphasize the role of social support and gender-specific factors in NHL prognosis.

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has shown substantial promise in treatment of hematological malignancies, including non-Hodgkin Lymphoma (NHL). A subset of patients develop prolonged cytopenia, comprising quality of life, increasing healthcare utilization, and imparting considerable morbidity and mortality. Therapy-related myeloid neoplasms (t-MN), typically seen in patients who receive DNA-damaging agents such as radiotherapy and alkylators, have also been observed in CAR-T recipients. CAR-Hematotox score predicts the development of hematotoxicity in CAR-T recipients using laboratory parameters obtained at lymphodepleting (LD) chemotherapy, a timepoint when the patient has already gone through leukapheresis thus committed to the CAR-T process. Therefore, it is critical to identify patients at the highest risk of prolonged cytopenia and/or t-MN prior to apheresis, making it possible for a better triage of those who will proceed with CAR-T and those who might benefit from other strategies. Methods: A retrospective analysis was conducted in NHL patients who received commercial CAR-T products between January 2018 and March 2024 at Mayo Clinic Rochester, Arizona and Florida. Clinical and laboratory data including age, hemoglobin (Hb), absolute neutrophil count (ANC), platelet (PLT), ferritin, and LDH, obtained at the initial CAR-T evaluation, typically within 1 week of leukapheresis, were used for analysis. Prior lines of therapies including autologous stem cell transplant (ASCT) were collected. Prolonged cytopenias were defined as Hb < 8 g/dL, ANC < 0.5 x 109/L, and/or PLT < 50 x 109/L at 3 months after CAR-T. t-MN was diagnosed per WHO 2016. For prolonged cytopenia, the optimal cutoff points for continuous variables were determined by maximizing the sum of sensitivity and specificity. Logistic regression was then used to determine the factors associated with cytopenia at 3 months post-CAR-T. For t-MN, maximally selected log-rank statistics was used to determine the optimal cutoffs. We used competing risk analysis to determine the factors associated with development of t-MN. Death without t-MN was considered a competing risk. For both analyses, the factors with P < 0.1 in univariate analysis (UVA) were included in multivariate analysis (MVA). Results: Among the 387 NHL patients, with a median follow-up of 11 months (95% CI 8.7 - 12.2 months), 56 (14.4%) developed (41, 10.5% with 1 cytopenia; 14, 3.6% with 2 and 1, 0.2% with cytopenia in all 3 lineages), 20 (5.2%) developed and 315 (81.2%) did not have either. Factors associated with prolonged cytopenia: were age >, PLT ≤ 150, ferritin ≥ 600, and LDH ≥ 250. Univariate analysis identified baseline Hb, ANC, PLT and ferritin to be significantly associated with prolonged cytopenia. MVA identified Hb ≤ 11 (HR = 1.98; 95% CI 1.03-3.85, P = 0.042) and PLT ≤ (HR = 2.56; 95% CI 1.39-4.78; P = 0.003) to be associated with the development of prolonged cytopenia. Factors associated with t-MN:were age > 65 years, Hb ≤ 9 g/dl, ANC < 3.8, PLT ≤ 80, Univariate analysis identified age, Hb, ANC, PLT, ferritin, and ≥ 3 prior lines of treatment to be significantly associated with t-MN. On MVA, age ≥ 65 (HR = 3.91; 95% CI 1.52-10.03; P = 0.004), Hb ≤ 9 (HR = 3.49; 95% CI 1.38-8.85; P = 0.008), ferritin ≥ 300 (HR = 3.06 ; 95% CI 1.08-8.67; P = 0.035) and ≥ 3 prior lines of therapy (HR = 3.25; 95% CI 1.33-7.94 ; P = 0.010) were associated with t-MN development. Association of prolonged cytopenia with t-MN development: Prolonged cytopenia was noted in 45% of patients who developed t-MN and 15% of those who did not (P = 0.002). Cumulative incidence of t-MN at 1 year was 3.3% and the presence of prolonged cytopenia was associated with a higher 1of t-MN (HR 8.93, 95% CI 2.15 - 37.1, P = 0.003). Conclusions: Readily accessible clinical and lab data such as blood count and inflammatory markers at the time of CAR-T evaluation were associated with the development of prolonged cytopenias. Similarly, advanced age, anemia, high ferritin levels and history of ≥3 lines of therapy were associated with the development of t-MN. These findings, when validated in larger dataset, can be used for patient counseling and therapy selection by identifying patients at high-risk of developing hematotoxicity and/or t-MN development.

Background: Daratumumab (DARA) has demonstrated clinical benefit in multiple myeloma (MM) and is approved for the treatment of both newly diagnosed MM (NDMM) and relapsed refractory MM (RRMM). While single-center studies have shown the benefit of DARA retreatment in patients (pts) who are refractory to DARA (D-Ref), there is a lack of real-world data on the characteristics of pts who are retreated with DARA across multiple lines of treatment (LOTs). In this study, we examine DARA retreatment patterns and outcomes among pts who are D-Ref and those who are sensitive to DARA (D-Sens) after the first DARA exposure in the US real-world clinical practice. Methods: This study used the Flatiron MM Core Registry data to identify pts with MM treated with a DARA-containing regimen in ≥2 separate LOTs between November 1, 2015, and March 31, 2024. The first DARA-based LOT was defined as D1, and the second DARA-based LOT was defined as the retreatment LOT (D2). The following criteria, developed by Janssen Scientific Affairs, were used to classify pts as Dara-retreated: 1) pts treated with the same DARA-containing regimens in consecutive LOTs were classified as Dara-retreated if there was a gap of >90 days between D1 and D2, 2) pts treated with different DARA-containing regimens in consecutive LOTs were classified as Dara-retreated, 3) pts treated with DARA-containing regimens had ≥1 LOTs between D1 and D2 they were classified as Dara-retreated, regardless of whether the regimens were the same or different. Derived disease progression reported in the Flatiron data was used for this analysis. If disease progression was reported during D1, or within 60 days of discontinuing D1, the pt was considered as D-Ref, otherwise, the pt was considered as D-Sens. Kaplan-Meier (KM) analyses were conducted to evaluate duration of D2, proportion of pts not receiving subsequent treatment after D2, and progression-free survival (PFS) rates during D2. Results: Data for 201 pts retreated with DARA were identified in this analysis, 150 were D-Sens and 51 were D-Ref. In the D-Sens group, the median age at D2 initiation was 71 years, 44.0% of pts were female, 27.3% were ISS stage II and 16.0% were African American. In the D-Ref group, the median age at D2 initiation was 67 years, 45.1% of pts were female, 21.6% were ISS stage II, and 25.5% were African American. At D2 initiation, 64.7% (D-Sens) and 60.8% (D-Ref) of pts were frail, and 56.0% (D-Sens) and 52.9% (D-Ref) of pts had a Quan-Charlson Comorbidity Index of ≥3, indicating a high burden of comorbidities. About 31% of pts in both groups had high cytogenetic risk abnormalities, and about 20% had a 1q21 gain or amplification. The median time from MM diagnosis to start of D1 and to start of D2 was shorter in the D-Sens group (10.8 and 34.9 months) than in the D-Ref group (18.4 and 44.4 months). D-Sens pts were less likely to receive D1 in the third LOT or later compared to D-Ref pts (30.0% vs 45.1%). The median number of LOTs before D2 was three in both groups. The median gap between D1 and D2 was 9.3 months in D-Sens pts and 9.0 months in D-Ref pts, however, more D-Sens pts received D1 and D2 LOTs consecutively compared to D-Ref pts (62.0% vs 51.0%). The most common regimen used in D1 was DARA + lenalidomide + dexamethasone (dex; 18.7%) and DARA + bortezomib + dex (18.7%) among D-Sens pts, and DARA + pomalidomide + dex (23.5%) among D-Ref pts. The most common regimen used in D2 was DARA mono (31.3%) in D-Sens pts, and DARA mono (17.6%) and DARA + carfilzomib + dex (17.6%) in D-Ref pts. Overall, 32.0% of D-Sens and 35.3% of D-Ref pts received agents from the same drug class in D1 and D2. The KM median duration of D2 was 7.4 months (95% CI: 5.5-9.5) among D-Sens pts and 6.4 months (95% CI: 3.8-23.5) among D-Ref pts. The proportion of pts that had not received subsequent treatment after D2 was 38.7% and 29.5% at 12 months in the D-Sens and D-Ref groups, respectively. PFS rates at 12 months after D2 initiation were 77.1% in the D-Sens group and 49.4% in the D-Ref group. Conclusion: DARA retreatment is utilized frequently and is effective in the real-world, suggesting the ability to use this approach in subsequent LOTs among pts with RRMM. In the absence of other feasible alternatives, this may be a treatment strategy for patient benefit, regardless of whether they are D-Sens or D-Ref. Future studies are needed to definitively investigate the benefit of DARA retreatment in combination with drugs with novel mechanisms of action.

Introduction: Nodal lymphomas of T-follicular helper cells (n-TFHLs) are considered neoplasms of mature TFH cells and are classified by the World Health Organization (WHO) under one umbrella with three subtypes: angioimmunoblastic-type (n-TFHL-AI), nodal TFHL, follicular type (n-TFHL-F), and nodal TFHL, not otherwise specified (n-TFHL-NOS). TET2 mutations in n-TFHL-AI subtype frequently co-occur with IDH2R172 mutations. Methods: To elucidate the cooperative role of TET2 and IDH2R172K mutation in n-TFHL-AI pathogenesis, we generated a murine model with Tet2 loss and IDH2R172K knock-in mutation in CD4+ T cells (Tet2-/-/IDH2R172K) by crossing Tet2Flox/Flox/IDH2R172K mice with Cd4-Cre transgenic mice. This resulted in Tet2 exon 3 deletion and IDH2R172K knock-in mutation specifically in CD4+ T cells. Murine tumors were comprehensively evaluated through immunohistochemistry and flow cytometry. Transcriptomic (RNA-seq), 5-hydroxymethylcytosine (5hmC), and 5-methylcytosine (5mC) profiling, and genomic (whole exome sequencing [WES]) analyses were conducted on both earlier time points (preneoplastic) and neoplastic cells. Functional validation involved proliferation, T-cell polarization, and Western blotting assays. Results: The transcriptomic analysis (p < 0.05) in n-TFHL-AI (double-mutant) cases showed enrichment of TFH-like signature, IL-12-signaling, NF-ΚB, and downregulation of TH1 cell differentiation (including IFNγ signaling). Evaluation of double-mutant mice revealed an earlier tumor onset than Tet2 mutant mice, with a median survival of 1.3 years compared to 1.7 years (p=0.006), respectively. With chronic stimulation by T cell-dependent antigens (sheep red blood cells [SRBCs]), these mice showed B-cell expansion with a 100% incidence of lymphoid neoplasms, in contrast to PBS-treated mice, where myeloid and lymphoid diseases occurred at an equal ratio. Genomic analysis of murine tumors revealed a unique mutation spectrum in metabolic genes (Ata3d3a, Atp1a3, Ppp4r3a, and Nemf), T- and B-cell interaction (Gnai3), Stat pathway (Jak1), and DNA repair (Zfp451). Transcriptomic analysis of tumors showed downregulation of Tbx21, increased TFH-related genes, and increased expression of Bcl6 with downregulation of its known suppressed genes. Transcriptomic analysis of murine CD4+ T cells isolated at 12 weeks revealed downregulation of genes associated with TH1-cell differentiation and Trp53 signaling, concurrent activation of T-cell receptor (TCR) and MAPK signaling, and API-complex. We demonstrated elevated levels of 2-hydroxyglutarate (2-HG) in double-mutant cells, and enasidenib (IDH2R172 inhibitor) treatment significantly decreased 2-HG levels and increased Tbx21 expression. Mutant cells showed a proliferative advantage in IL-2 and IL-21 in vitro conditions, resulting in enhanced TCR, AKT, ERK1/2, p-65, and inactivation of p-FOXO1 protein. While these cells showed a growth advantage in in vitro culturing with TFH conditions, their growth and polarization were impaired in TH1 conditions, indicating a differentiation block. Tet2-/-/IDH2R172K mice (12-week) with SRBCs treatment revealed a significant induction of B and CD4+ T- cell populations compared to wild-type (WT) mice (p=0.01). In vitro co-culturing experiments demonstrated that Tet2-/-/IDH2R172K mutant CD4+ T-cells confer a substantial proliferative advantage to B-cells (p=0.021). Notably, these double-mutant CD4+ T cells exhibited superior proliferation compared to WT CD4+ T-cells when co-cultured with B-cells isolated from WT or double mutant mice (p=0.004). When Tet2-/-/IDH2R172K neoplastic cells with whole tumor were allografted into NOD scid gamma (NSG) mice, lymphoma developed within three months, but also showed numerous B-cells in the TME. This suggests that the propagation of neoplastic T-cells likely require B cell help. Overall, the TFH program remained intact in transplanted cells, and a global decrease in 5hmC levels and an increase in 5mC levels were observed. Conclusion: Tet2-/-/IDH2R172K murine CD4+ T cells exhibited hyperactive TCR signaling, impaired DNA damage response, and NF-ΚB activation as key oncogenic transitions. Both in vitro co-culturing and the allografting of neoplastic Tet2-/-/IDH2R172K cells showed dependence on B-cells, suggesting that interrupting T- and B-cell interaction could be a therapeutic approach.

Introduction The major primary genetic events in multiple myeloma (MM) include translocations of immunoglobulin heavy chain (IgH) and a hyperdiploidy (HRD) karyotype (Kuehl, 2002). The main drivers of pathogenesis in HRD MM are unknown (Fonseca, 2003), therefore further research is needed in this subgroup. Translational research in HRD MM has relied on analysis of genomic and transcriptomic alterations, however changes at the genome and transcriptome level do not always correlate directionally with protein abundance. Because proteins are considered the main and final effectors of most cellular processes, proteogenomics can improve our understanding of the drivers of HRD MM. We employed it here to identify potential biomarkers in HRD MM and determine whether any given trisomy may lead to a selective advantage. Methods: Protein and mRNA were extracted from CD138+ cells from 47 HRD MM and 14 t(11;14) MM samples. RNA-seq data was processed using MAPR-Seq pipeline using default parameters and gene-wise raw counts were extracted for each sample. Proteomics data was processed using Specranaut and protein-wise intensities were extracted for each sample. A generalized linear model configured with negative binomial distribution was used to analyze gene-wise count data in edgeR software. Genes with an adjusted p-value of <=0.05 were considered significantly differentially expressed. Protein intensity data were normalized using trimmed mean of M-values and log2 transformed. A generalized linear model configured with Gaussian distribution was used to compare protein intensities between the groups. Proteins with an adjusted p-value <= 0.05 were considered significantly differentially expressed. Two separate rank lists for protein and mRNA data were generated for pathway analysis. Results When comparing our small cohort of HRD MM patient samples to those with t(11;14) MM, out of approximately 16,600 total transcripts and 9,700 proteins evaluated we identified approximately 2,100 significantly variant transcripts and 500 significantly variant proteins in HRD MM. GSEA pathway analysis of the transcriptome and proteome revealed significantly upregulated translational and ribosomal processing pathways in HRD MM. Transcriptional MYC target pathways were also significantly upregulated. RNAseq revealed significantly higher expression of MYC (Rank 24.5, p=5.3E-6) in HRD MM. It is known that patients with t(11;14) MM have increased expression of CCND1 (Kuehl and Bergsagel, 2012). As expected, we found that the HRD MM subgroup had significantly lower expression of CCND1 compared to the t(11;14) MM subgroup (Rank -79.9, p=2.8E-21) and this was also true at the protein level (Rank -21.7, p=2.2E-5). The most significantly abundant protein in the HRD MM subgroup compared to t(11;14) MM was BAHD1, which is a protein that targets H3K27me3 and recruits transcriptional co-repressors (Fan, 2020). BAHD1 has been shown to repress erythroid genes and may recruit HDAC5, which plays a role in osteoblast differentiation (Rank 72.3, p=2.9E-20; Xu, 2021). The second most abundant protein in HRD MM was CKS2, which binds to the catalytic subunit of cyclin-dependent kinases (CDKs) and therefore positively regulates cell proliferation, invasion, and migration and has been shown to be implicated in tumor progression in several solid tumors (Rank 72.3, p=2.9E-20; You, 2015). TRIM proteins promote tumorigenesis and cancer development. TRIM14 mediates cancer development through mediating the JAK/STAT, PI3K/AKT, NFkB, and p53 pathways and was found to be significantly higher in HRD MM (Rank 70.6, p=9.0E-20; Huang, 2022). We also found increased protein abundance of LAMP5 in HRD MM (Rank 20.1, p=6.0E-5). LAMP5 may play an important role in MM progression and has been shown to be highly expressed in patients with mixed-lineage leukemia and is associated with a poor prognosis (Wang, 2023; Gracia-Maldonado, 2022). We identified many proteins (not all discussed here) that have not been well described in HRD MM and thus may be biomarkers/targets of disease pathogenesis of this subgroup. Conclusion Proteins are the final effectors of most cellular processes. Proteogenomics can improve our understanding of the drivers of HRD MM. Using a proteogenomic approach we identified proteins that may lead to selective advantage and/or pathogenesis of trisomies in HRD MM.

Background: Continuous single-agent ibrutinib (Ibr) treatment has been associated with extended progression-free survival in patients with Waldenström macroglobulinemia (WM). Previous clinical studies have shown that Ibr dose reductions (DRs) improved or resolved adverse events (AEs) in most patients, while hematologic responses were sustained. However, real-world data on long-term outcomes for patients with WM undergoing Ibr DR in first-line (1L) settings remain limited. Methods: Medicare Fee-for-Service (FFS) medical and pharmacy records were used to identify patients diagnosed with WM who initiated 1L single-agent Ibr (420 mg/day) or 1L Ibr + rituximab from January 1, 2015, to September 30, 2022. Observed patients were enrolled in Medicare FFS ≥12 months before and after 1L treatment initiation and had ≥1 qualifying post-treatment initiation AE of special interest (AESI; both prevalent and incident AEs), as identified via ICD-9-CM or ICD-10-CM diagnosis codes. Ibr DR was defined as a reduction of the starting dose (420 mg/day) following the first AESI during 1L treatment. Patients without an Ibr DR were defined as those who had received Ibr at 420 mg/day during 1L treatment. Patients without an AESI post-Ibr initiation and prior to DR were excluded from the analysis. Demographics, clinical characteristics, time to AESI, duration of therapy (DOT), and time to treatment discontinuation (TTD) were assessed in cohorts with and without DRs. DOT was measured at the date of Ibr treatment initiation through treatment discontinuation (end of 1L treatment). Patients were considered to have discontinued treatment if they received a subsequent systemic therapy after the 1L regimen, had a gap of ≥120 days with no systemic therapy following the last administration, or died while on the 1L regimen. Patients without a discontinuation were censored at the earliest date of end of follow-up or disenrollment. TTD was analyzed using the Kaplan-Meier method. A Cox proportional hazards model adjusted for patient demographics and clinical characteristics was used to identify factors associated with treatment discontinuation. Results: A total of 407 patients with WM were included in the analysis (DR, n=55 [14%]; no DR, n=352 [86%]). Patients in the DR and no DR cohorts, respectively, had similar a median age (78 years vs 77 years), Charlson Comorbidity Index score (3 vs 4), and prevalence of the most common baseline comorbidities: hypertension (67% vs 68%) and cardiovascular conditions (31% vs 28%). The median time between treatment initiation and the occurrence of the first AESI was 89 days versus 76 days, and total Ibr DOT mean ± SD [median] was 786.1 ± 692.6 [661] days versus 454.8 ± 510.4 [279] days (P=0.001) for DR cohort versus no DR cohort. Following DR, patients remained on Ibr for mean ± SD [median] 336.0 ± 422.8 [179] days until the end of 1L therapy. After adjusting for variations in baseline characteristics, patients with a DR had significantly longer TTD post-treatment initiation than those without a DR (median TTD, DR vs no DR: 22.5 months vs 9.3 months; adjusted HR [95% CI], 0.52 [0.37-0.72]). Conclusion: This real-world evidence study suggests that implementing DRs to manage AEs can be a valuable strategy in supporting patients with WM to remain on 1L Ibr longer, thus maintaining beneficial clinical outcomes. These findings are consistent with previous studies, demonstrating that Ibr DRs help to improve tolerability while maintaining treatment efficacy.