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Introduction: Light chain multiple myeloma (LCMM) is a distinct subtype of multiple myeloma (MM) characterized by the exclusive production of immunoglobulin light chains, unlike the common form of MM that produces the complete immunoglobulin. There is a paucity of literature on the specific tumor cell alterations leading to light chain-only disease as opposed to the typical presentation in MM. Additionally, data on treatment response and outcomes in this subgroup of patients is sparse. This study explores the differences in baseline characteristics, including cytogenetic abnormalities and treatment response between LCMM and multiple myeloma with heavy chain involvement. Materials and Methods: We reviewed patient charts to obtain clinical and laboratory information for individuals diagnosed with LCMM and those with heavy chain MM (comparison cohort) from 2004 to 2022. Patients were matched for age, sex, and year of diagnosis. Data collected included treatment assessment, overall survival, and progression-free survival (PFS). Associations between outcomes and categorical variables were analyzed using the chi-square test, while the Mann-Whitney U test was employed to compare medians. Progression-free survival following initiation of initial therapy and overall survival were assessed using Kaplan-Meier analysis. The Mayo Clinic Institutional Review Board (IRB) approved the study. Results: We identified 848 patients, representing 17.1% of newly diagnosed MM cases. The median age of the LCMM cohort was 63 years (range 16-92), with a female-to-male ratio of 1:5. The LCMM cohort had higher levels of lactate dehydrogenase (LDH), beta-2 microglobulin (p<0.0001), higher baseline creatinine (p<0.0001) and calcium (p<0.0001). The two cohorts had no significant difference in the International Staging System (ISS) or revised ISS (R2-ISS). Additionally, hemoglobin (Hb) and white blood cell count at presentation were higher in the LCMM cohort. Chromosome 1 abnormalities were lower in the LCMM cohort: 1q gain (OR-0.7; 95% CI: 0.6-0.9) and 1p deletion (OR-0.7; 95% CI: 0.6-0.9, p=0.02). Likewise, the frequencies of trisomies and hyperdiploidy were lower in the LCMM cohort, with odds ratios of 0.32 (p=0.012) and 0.37 (p<0.001), respectively. The frequencies of translocations t(4;14) (OR-0.35, p<0.001) and t(14;16) (OR-0.6, p=0.03) were lower in LCMM compared to heavy chain MM. In contrast, the frequencies of t(11;14) (OR-3.4) and t(6;14) (OR-4.9) and deletion of 13q(OR-1.3) was higher in the LCMM cohort. There was no significant difference in the occurrence of del(17p)/TP53 abnormalities. The median PFS after the first regimen was shorter in the LCMM cohort (23 months vs. 27 months, p=0.03), and this difference remained significant after adjusting for baseline adverse prognostic indices and treatment regimen. Median PFS in LCMM patients with monoclonal antibody (Mab) as part of their treatment was 37 months vs. 23 months (Proteasome Inhibitor or immunomodulator-based regimen without Mab), though this difference is not statistically significant p=0.87. No significant difference was found in overall survival between the two groups. Conclusion: This study highlights unique features of light chain multiple myeloma, including increased frequencies of specific cytogenetic abnormalities. Further evaluation of observed differences in cytogenetic abnormalities may enhance our understanding of LCMM and potentially guide baseline treatment choices to improve outcome.

Background: Proven salvage therapies for pts with triple-class refractory MM who progress on BCMA-targeted therapies are lacking.Fc receptor-homolog 5 (FcRH5) is a type I membrane protein that is expressed exclusively in the B-cell lineage. Cevostamab is a FcRH5xCD3 BsAb that facilitates T cell-mediated killing of MM cells. In an initial Phase I study (GO39775), cevostamab monotherapy was clinically active and had manageable toxicity in pts with heavily pretreated relapsed/refractory MM (Trudel et al. ASH 2021). In Cohort A1 of the subsequent Phase I/II CAMMA 2 study (CO43476; NCT05535244), cevostamab was highly active and had manageable safety at the 160mg target-dose (TD) level in pts with triple-class refractory MM who had received a prior BCMA-targeted antibody-drug conjugate (ADC; overall response rate [ORR]: 60%, 6/10 pts) or chimeric antigen receptor T-cell (CAR-T) therapy (ORR: 73%, 8/11 pts) but had not received a prior BCMA-targeted BsAb (Kumar et al. EHA 2024). We present initial results from Cohort A2, which enrolled pts with triple-class refractory MM who had received prior BCMA-targeted therapies, including ≥1 BCMA-targeted BsAb. An exploratory analysis of baseline patient and tumor-related factors is also presented. Methods: Cevostamab was initiated with step dosing (Cycle [C] 1 Day [D] 1: 0.3mg; C1D2, D3, or D4 depending on the emergence of cytokine release syndrome [CRS] after the initial administration: 3.3mg) and continued at the 160mg TD on C1D8 and on D1 of each subsequent 21-day cycle until disease progression (PD) or unacceptable toxicity. Cytokine, tumor antigen, T-cell function/activation, and inhibitory immune checkpoint expression were assessed in blood and tumor samples collected at baseline and during treatment. Results: As of February 23, 2024, 21 pts (median age: 64 years, range: 46-84) had been enrolled into Cohort A2. Seven pts (33%) had extramedullary disease and 5/12 pts (42%) with a conclusive assay result had high-risk cytogenetics (t(4;14), t(14;16), or del(17p)). All pts were heavily pretreated (median prior lines of therapy: 8, range: 5-14); 62% had received ≥2 prior BCMA-targeted therapies (median time since last: 122 days, range: 68-558) and 33% had received ≥2 prior BsAbs (median time since last: 139 days, range: 68-782). Most pts (86%) were refractory to their last line of therapy; 62% were penta-drug refractory. Median follow-up was 173 days (range: 20-360). Grade (Gr) 3-4 adverse events (AEs) occurred in 71% of pts (15/21). Gr 3-4 AEs in >3 pts were neutropenia, anemia, thrombocytopenia (38% each), and infections (29%). CRS occurred in 76% of pts (16/21) and was mainly Gr 1 (43%) or Gr 2 (29%); 1 patient had Gr 3 CRS. One treatment-related Gr 5 (fatal) AE of pneumonia was reported in the context of PD. No treatment-related AEs leading to cevostamab discontinuation occurred. Objective responses (partial response [PR] or better) were observed in 2/21 pts (ORR: 10%). Both pts achieved a very good PR and had ongoing responses at cut-off. Stable disease was observed in 10 pts (48%). Pts in the prior BsAb group had received more prior lines of therapy than those in the prior ADC and CAR-T groups (median 8 vs 5 and 6, respectively) and more prior BCMA-targeted therapies (median 2 vs 1 and 1). The percentage of pts with low levels (<4.5 ng/mL) of soluble BCMA in plasma was also higher in the prior BsAb group (47% vs 0% and 18%). At baseline, inhibitory immune checkpoint expression across T-cell subsets was more common in the prior BsAb group. Notably, the median frequency of PD1+CD8+Tnaive and TIGIT+CD8+Tnaive cells in blood was higher in the prior BsAb group (21% and 29%, respectively) than in the prior ADC (3% and 6%) and CAR-T groups (4% each). Peak median IL-6 and IFN-γ levels were delayed until the first TD in the prior BsAb group, suggesting a different pharmacodynamic profile. Conclusions: Cevostamab has manageable safety in pts with triple-class refractory MM who have received prior BCMA-targeted therapies, including ≥1 BCMA-targeted BsAb. Clinical responses were less frequent in the prior BsAb group than in the prior ADC and CAR-T groups, which could be explained by the higher number of prior lines of therapy and T-cell exhaustion due to multiple prior exposures to BsAbs. Notably, the high frequency of CD8+ T-cell subsets expressing inhibitory immune checkpoints in the prior BsAb group suggests a level of T-cell dysfunction that could prevent effective T-cell activation.

Background The American Society of Clinical Oncology (ASCO) launched the Diversity in Oncology initiative in 2017 to address the need for increased minority representation in the hematology-oncology workforce. A recent series of reports, titled “State of the Oncology Workforce in America,” published in JCO Oncology Practice in 2021 and 2022, focused on this issue. Notably, these reports did not include any data regarding the representation of Asian minorities in the field. Therefore, our objective was to assess the proportion of Asian professionals within the hematology-oncology workforce, encompassing both practitioners and trainees. Methods We conducted a comprehensive search for publicly available data regarding Asian representation among US hematology-oncology practitioners and trainees in both adult and pediatric fields. Our primary data sources included the 2020 US Census, the Association of American Medical Colleges, the Accreditation Council for Graduate Medical Education, and various professional societies. We defined practitioners to include hematologist-oncologists, gynecologic oncologists, radiation oncologists, surgical oncologists, hematopathologists, hematology-oncology nurse practitioners, hematology-oncology physician assistants, and nurses specializing in hematology-oncology. Trainees encompassed fellows and residents. Additionally, we gathered comparable data on the US population, medical students, adult internal medicine residents, gynecology residents, pediatric residents, pathology residents, surgery residents, nurses, nurse practitioners, and physician assistants. To provide a comprehensive overview of the hematology-oncology workforce, we utilized data from 2020 to 2022, and whenever possible, we collected longitudinal data on trainees over the years. Results As of 2020, Asians represented 6.2% of the US population. Among hematology-oncology physician practitioners, the percentage of Asian individuals ranges from 14.7% to 32.1%, including 32.1% of adult hematologist-oncologists, 20.4% of pediatric hematologist-oncologists, 25.9% of radiation oncologists, 14.7% of gynecologic oncologists, and 7.0% of hematology-oncology physician assistants. In the realm of physician trainees, Asian representation is between 17.9% and 35.3%, with 35.3% of adult hematology-oncology fellows, 20.0% of pediatric hematology-oncology fellows, 26.4% of radiation oncology residents, and 17.9% of gynecologic oncology fellows. Additionally, the proportion of Asian residents from which hematology-oncology fellows are drawn ranges from 14.9% to 35.9%. Specifically, 35.9% of internal medicine residents, 22.0% of pediatric residents, 14.9% of gynecology residents, 26.6% of pathology residents, and 19.2% of surgical residents are Asian. Furthermore, 26.7% of medical students identify as Asian. Notably, the proportions of Asian hematology-oncology trainees and medical students have remained relatively stable from 2005 to 2022. Data on hematopathologists, surgical oncologists, and non-physician practitioners are limited, although Asian representation among hematology-oncology physician assistants is 7.0% and among nurses is 7.2%. Conclusions Asians comprise a large segment of the hematology-oncology workforce in the US. While only 1 in 20 people in the US are Asians, between 1 in 3 and 1 in 5 hematology-oncology physician practitioners and trainees, are Asians. Data are limited for non-physician practitioners. Asian minority representation should be highlighted and considered as an example of success in diversity in hematology-oncology workforce. This framework should be embraced moving forward to continue advancing minority representation throughout the field of hematology-oncology.

Introduction MYD88 L265P mutations (MUT) are found in 90% of patients with Waldenstrom macroglobulinemia (WM) and lead to the activation of pro-survival signaling. Limited literature has demonstrated that patients with MYD88 wild-type (WT) WM have poorer responses to conventional therapies and a higher likelihood of histological transformation. Increasingly the role of non-coding genomics in lymphomagenesis has demonstrated importance and currently, the epigenetic landscape of MYD88 WT WM remains underexplored. This study utilized a multi-omics approach to assess DNA methylation in MYD88 WT WM compared to MYD88 MUT patients and controls. Methods Bone marrow samples were prospectively collected from 20 subjects [3 MYD88 WT/CXCR4 WT WM, 12 MYD88 MUT WM and 5 normal controls] and positive selection for CD19+ and/or CD138+ B cells was performed. The analysis focused on the comparison between MYD88 WT WM to normal controls and MYD88-WT to MUT WM. Genome-wide DNA methylation analysis was performed, and CpG methylation ratios were segmented into 200-bp regions. Differentially methylated regions (DMRs) were determined using a Q-value <0.05 and absolute methylation difference ≥10%. Next, RNA-seq data was interrogated and differentially expressed genes (log2fold change>0.5 or <-0.5, false discovery rate<0.05) with corresponding promoter hypo- or hypermethylation were assessed. Ingenuity Pathway Analysis was used to determine potentially epigenetically regulated pathways using a corrected p<0.05. Results Comparing MYD88 WT WM samples to controls, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (29129 vs 6300), 3'untranslated region (UTR) (2068 vs 1551), 5'UTR (10169 vs 735), intron (30344 vs 24511), exon (2224 vs 6855), CpG island (30372 vs 1240) and CpG shore (14477 vs 5872) (all p<0.001). Next, mRNA analysis demonstrated that 214 genes were differentially up-/downregulated with concordant promoter hypo-/hypermethylation. On the assessment of promoter-methylation-based pathways, multiple pathways involved in cell signaling, proliferation, gene regulation, cytokine and immune signaling and metabolism were found to be deregulated. Most pathways were found to be negatively enriched in MYD88 WT patients compared to controls including several members of interleukin (IL) signaling (IL-2, IL-3, IL,7) and regulators of cell proliferation and signaling including mTOR, PI3K/AKT, and p53. Positive enrichment of the PTEN, FGFR3 and ERK/MAPK pathways were observed. Underlying multiple pathways was promoter hypermethylation with corresponding mRNA downregulation of AKT3 and PIK3CA. Furthermore, promoter hypomethylation with transcript upregulation of PTEN was observed. Next, in comparison of MYD88 WT to MUT samples, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (24829 vs 2544), 3'UTR (3534 vs 378), 5'UTR (6111 vs 605), intron (52292 vs 5360), exon (21066 vs 2187), CpG island (18334 vs 1551) and CpG shore (16351 vs 1957) (all p<0.001). Assessing mRNA expression, 354 genes were up-/downregulated with concordant promoter hypo-/hypermethylation. Pathway analysis revealed positive enrichment of the mitotic G1 phase to G1/S transition pathway with upregulation of the CDK4, LYN, and PSMA6 transcripts in MYD88 WT compared to MUT samples. Further, negative enrichment of cell signaling regulators including the RHO GTPase family, IL pathways (IL-4, IL-8, IL-13, IL-17) and integrin signaling were found in MYD88 WT compared to MUT samples. Underlying multiple pathways were downregulation with promoter hypermethylation of BCL2, an important regulator of apoptosis. Conclusion Genome-wide methylation analysis of MYD88 WT WM revealed a unique DNA methylation profile, with potential epigenetic alteration of key oncogenic transcripts and pathways. The negative enrichment of cell signaling and cytokine/inflammatory pathways in MYD88 WT samples compared to controls and MYD88 MUT WM suggests an epigenetic mechanism for immune evasion and potentially underlies the reduced response to conventional WM-directed therapies. Overall, this study reveals that MYD88 WT WM is epigenetically distinct from MYD88 MUT WM and regulators such as methylation may underlie the unique clinical and phenotypic properties observed.

Introduction: The outcomes of patients diagnosed with multiple myeloma (MM) have improved significantly with the advent of immunomodulatory drugs, proteasome inhibitors, anti-CD38 monoclonal antibodies, and high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (HSCT). Further advancements include immune-based approaches such as bispecific antibodies and chimeric antigen T-cell receptor therapy. However, most of these therapies are predominantly available in developed countries, with limited access globally. Our study aimed to ascertain the landscape of interventions being used in developing countries for MM, and their efficacy. Methods: A comprehensive search was conducted on March 13, 2023, using Embase via Elsevier, PubMed, Scopus, CINAHL, and CENTRAL. We included retrospective and prospective studies that were evaluating interventions for patients with plasma cell dycrasias in the developing country setting. We defined developing country according to the United Nations. For comparative studies, we calculated the risk ratio (RR) for binary outcomes such as overall survival (OS) and progression-free survival (PFS), estimating 95% confidence intervals (CIs) and pooling data using the DerSimonian and Laird random effects model. For non-comparative studies, we calculated overall event rates and transformed them using the Freeman-Tukey double arcsine method, then pooled the data using the same random effects model. All statistical analyses were conducted using R version 4.4.0. Results: A total of 448 records were identified, of which 37 studies met the eligibility criteria. The overall risk of bias was moderate. The included studies evaluated induction treatments using doublet and triplet-based therapies, including TD (thalidomide and dexamethasone), VD (bortezomib and dexamethasone), MP (melphalan and prednisone), CTD (cyclophosphamide, thalidomide, and dexamethasone), VTD (bortezomib, thalidomide, and dexamethasone), and VAD (vincristine, doxorubicin, and dexamethasone). Lenalidomide was notably absent in the induction regimens apart from one non-comparative study reported on the use of RVD (lenalidomide, bortezomib, and dexamethasone). Studies were divided into comparative (n=11) and non-comparative groups (n=26). Comparative studies included: HSCT vs. no HSCT, induction with alkylating agents vs. thalidomide or bortezomib, and thalidomide vs. bortezomib or CTD vs. VTD. Lenalidomide use in comparative studies was reported only post-HSCT, compared against observation. High-dose chemotherapy followed by autologous HSCT was commonly utilized, with most studies reporting outcomes for patients who underwent autologous HSCT. Two comparative studies from 2 countries (Colombia and India) compared the overall survival of autologous HSCT in patients diagnosed with MM (N=130) vs. patients who never received ASCT (N=293). At one year of follow-up, there was no significant difference in overall survival (OS) between patients who received autologous HSCT and those who did not (relative risk [RR] = 1.11, 95% CI: 0.93-1.33). However, a significant improvement in OS was observed at five years post-autologous HSCT (RR = 1.59, 95% CI: 1.38-1.82). The pooled estimate for five-year OS in single-arm studies, which included nine cohorts from seven countries (Pakistan, India, Colombia, Algeria, Sri Lanka, Mexico, and Peru), was 62% (95% CI: 48-75). Progression-free survival (PFS) at five years post-HSCT, reported in three cohorts from three countries (India, Algeria, and Mexico), was 44% (95% CI: 23-67). Conclusions: Most of the recent advancements in MM treatment, including immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, were not widely utilized globally, particularly in developing countries. This limited access is a major issue, hindering the potential improvement in patient outcomes. However, high-dose chemotherapy followed by autologous HSCT showed good outcomes, demonstrating a significant survival advantage at five years follow-up. The lack of survival benefits at one-year post-HSCT may be due to high toxicity and/or limited resources to manage side effects in developing countries. Addressing the disparity in treatment availability is crucial for improving MM management and patient survival globally.

Background: Recent advancements in multiple myeloma (MM) diagnosis and treatment significantly improved patients' survival and quality of life. Severe Renal Impairment (RI) remains a significant challenge in newly diagnosed MM (NDMM), complicating treatment and affecting outcomes. Prior studies reported improved overall renal recovery in the era of novel therapies. However, there is a lack of evidence documenting the outcomes of patients presenting with severe RI. We present an updated analysis of the characteristics and outcomes of patients with NDMM presenting with severe RI in an ethnically diverse, low socio-economic population. Methods: A single-center retrospective review of patients with NDMM presenting to Montefiore Medical Center (Bronx, New York) was conducted from 2011-2023. Severe RI was defined as an estimated glomerular filtration rate (eGFR) of less than 30 mL/min/1.73 m². MM risk was assessed using the mSMART 3.0 tool. Results: Out of 284 patients with NDMM, 58 (20%) patients presented with severe RI. Black patients (p = 0.018) and older adults (64 years vs 60 years, p value 0.032) were more likely to present with severe RI. Severe RI at diagnosis correlated with high-risk myeloma (p = 0.002), high R-ISS stage (p < 0.001), and a lower likelihood of receiving stem cell transplants (p < 0.001). Among those with severe RI, 23/58 required dialysis of which 6/23 were taken off dialysis at last follow-up after renal recovery. Median eGFR at diagnosis, 6 months and 1 year after diagnosis was 11, 23 and 23.5 ml/min/1.73 m2, respectively. 14 out of 58 patients (39%) achieved renal recovery to eGFR > 30 one year post-diagnosis, with no significant difference in race, sex, or ethnicity. The most common first-line treatment for patients with severe RI was CyBorD (62%). The median time from diagnosis to first treatment (including plasma exchange) was 10 days. By the last follow-up, 35/58 patients (60%) had a stem cell transplant, with a median time from diagnosis to transplant of 275 days. The median overall survival (OS) for patients with severe RI at diagnosis was 49 months, while it was not reached for those without severe RI (p < 0.001). Early death (≤ 2 months after diagnosis) occurred in 2 patients, both with severe RI. Among patients with severe RI, multivariate Cox regression survival analysis showed that achieving free light chain (FLC) below 500 mg/L (HR 0.212, p < 0.001) and receiving a stem cell transplant (HR 0.209, p 0.002) improved outcome. Conclusions: Despite improvements in the life expectancy of patients with MM, severe renal failure remains a challenge, particularly among minorities. Socioeconomic factors need to be explored as a contributor to outcomes in patients with severe RI. Achieving FLC clearance and undergoing stem cell transplant may play a role in survival in patients with NDMM and severe RI.

Introduction: Advances in AI have enabled the generation of synthetic individual patient data (SynthIPD) that mimics the statistical properties of the real IPD, which can help generate subgroup-specific insights in the absence of subgroup-specific IPD. We apply this technology in evaluating Daratumumab treatment benefit in high-risk cytogenetic multiple myeloma subgroup. The addition of daratumumab to standard multiple myeloma (MM) regimens has been associated with improved response rates and clinical outcomes. A systematic review (meta-analysis) enables further evaluation of the effectiveness of daratumumab for MM patients with high-risk cytogenetic factors. However, concerns have been raised about meta-analyses due to the statistical model assumptions they impose. This study aims to address these limitations by employing SynthIPD to provide a more robust analysis. Methods: This study focuses on newly diagnosed multiple myeloma (NDMM) and relapsed or refractory multiple myeloma (RRMM) patients with high-risk cytogenetic profiles. We included six published studies: ALCYONE, CASSIOPEIA, and MAIA for NDMM, and CANDOR, CASTOR, and POLLUX for RRMM. Large language models were used to extract up-to-date clinical evidence from the literature. SynthIPD including cytogenetic risk profile were generated via digitization and an innovative optimization algorithm. Meta-analyses based on random effects models were then conducted, and the results were compared with those from pooled analyses using synthetic data. Results: SynthIPD for cytogenetic high-risk patients were generated for three studies for NDMM: ALCYONE (treatment vs control: n=53 vs n=45), CASSIOPEIA (n=82 vs n=86), MAIA (n=48 vs n=44); and three studies for RRMM: CANDOR(n=48 vs n=26), CASTOR (n=40 vs n=35), POLLUX (n=35 vs n=35). The generated hazard ratios (HRs) for each study closely aligned with the reported HRs for high-risk patients, with a maximum relative error of less than 2%. By pooling these datasets, we obtained SynthIPD datasets for NDMM and RRMM patients. Stratified Cox regression analyses yielded HRs of 0.764 (95% CI 0.529-1.104) for NDMM and 0.431 (95% CI 0.294-0.613) for RRMM. Random-effects meta-analyses showed HRs of 0.764 (95% CI 0.529-1.104) for NDMM and 0.425 (95% CI 0.287-0.621) for RRMM. Compared with traditional meta-analysis techniques, the synthetic data approach provides additional clinical insights that were not available in the original publications. For instance, the clinical results for the six studies within high-risk group (e.g., KM curves in high-risk subgroup) can be reproduced, which were not reported in the original studies. Conclusion: AI-generated synthetic data enhances the robustness of conventional meta-analysis outcomes. The identification of HRs and corresponding confidence intervals through SynthIPD enhances the credibility of our proposed method, offering a reliable approach to evaluating the efficacy of daratumumab in high-risk patients. Since SynthIPD includes individual covariate information, any statistical results derived from the original IPD can be effectively approximated using SynthIPD. Using the SynthIPD, we show that the addition of daratumumab to standard regimens improves PFS for RRMM patients with high-risk cytogenetic factors, although its benefit for NDMM patients is not statistically significant. This analysis can be extended to other multiple myeloma high risk subgroups.

We previously showed that both reactive oxygen species-inducing chemotherapy and some genetic subtypes of B precursor acute lymphoblastic leukaemia (ALL) can induce bone marrow (BM) mesenchymal stromal cells (MSC) to become cancer associated fibroblasts (CAFs). Exposure of MSC to ALL-cell conditioned media (CM) is sufficient to generate CAFs - direct cell-cell contact is not required (Burt, Dey et al, 2024). We showed that CAFs can lead to chemoresistance via transfer of mitochondria to ALL cells through tunnelling nanotubes (Burt, Dey et al 2019). We noted that CAFs in the ALL BM had features of senescence, prompting the current study in which we aimed to investigate the link between senescence and CAFs in ALL. We used 3 genetic subtypes of ALL; BCR::ABL1 (SD1 cells, representing “adult” ALL) , ETV6::RUNX1 (REH cells, representing “childhood” ALL) and KMT2A::AFF1 (SEM cells, known from our previous work NOT to induce CAFs). We exposed HS27a stromal cells to the ALL CM at a number of timepoints over a period of three weeks and characterised senescence using qPCR, cytochemical B-galactosidase assays, flow cytometry and immunofluorescence (IF). Cytokine arrays were performed to quantify the senescence associated secretory phenotype (SASP) components and assess the complexity of SASP components in all three ALL subtypes. A more prolonged exposure (> 10 days) to CM was required for senescence to develop in contrast to CAF formation which occurs early (2-3 days) in HS27a MSC cells. Senescence marker expression peaked at around day 11 to day 14 of CM exposure before subsiding to lower levels around day 21. On day 14, 70% of HS27a cells treated with SD1 CM were positive for beta galactosidase compared to below 25% positive for REH and SEM cells. Cell cycle arrest was demonstrated by P16 and Ki67 IF. 95% of SD1 CM treated HS27a cells were positive for P16 compared to less than 10% in RPMI control. Likewise, only 29% of HS27a exposed to SD1 CM were Ki67 positive compared to 66% in the RPMI control. Expression levels of other senescence-associated markers such as SERPINE1, SERPINB2, SOD2, NFKB1 were quantified by qPCR and were all significantly (p<0.001) upregulated in SD1 CM treated HS27a (2 to 40 fold) compared to SEM CM (-2 to 5 fold) and REH CM (-0.5 to 1.0) treated HS27a. SASP associated cytokines IL6 (1000-5000pg/ml), IL8 (2500-5000pg/ml), CXCL10 (500-1500pg/ml), CCL5 (1500-2250pg/ml), IL10 (25-50pg/ml), were significantly (p<0.0001) upregulated in the SD1 CM treated HS27a compared to controls and other ALL cell line exposed HS27a. Finally, HMGB2 and LMNB1 genes, loss of which is associated with senescence were significantly (p<0.001) downregulated in the SD1 CM (-2.5 to 0.5 fold) treated HS27a compared to controls. In view of the prominence of senescence in SD1-exposed HS27a cells, we tested the effects of two different senolytic drugs venetoclax (BCL2i) and navitoclax (BCL2i/BCLXLi) in vitro. Briefly, HS27a cells were exposed to SD1 CM for 11 days, and after washing, increasing concentrations of the drugs were added for up to 72 hrs followed by washing, fixing and DAPI-staining and counting. HS27a exposed to SD1 CM was highly sensitive to low dose treatment of navitoclax (0.5uM) with 80% cell death 48 hrs post treatment. HS27a cells exposed to SD1 CM were less sensitive to venetoclax with only 50% cell death observed at higher doses (5uM) even after 72 hrs post treatment. Further senolytic drug screening experiments are ongoing. Our data suggest that BCR::ABL1 but not ETV6::RUNX1 or KMT2A::AFF1 ALL cells, can induce paracrine senescence in HS27a MSC cells. We suggest that paracrine senescence in the stromal microenvironment may be a component of treatment response in B-ALL. ^^ and ** contributed equally

Abstract Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma associated with an increase in serum immunoglobulin M (IgM) monoclonal protein. The underlying causes of the disease are still not fully understood, but cells in the bone marrow (BM) microenvironment (TME) are anticipated to play a significant role in disease progression. We previously reported that myeloid-derived suppressor cells (MDSCs), a phenotypically heterogeneous group of cells known for their ability to suppress immune responses, are expanded in the bone marrow of patients with WM and that the expansion of MDSCs is particularly notable for increased numbers of polymorphonuclear MDSCs (PMN-MDSCs). In the present study, we evaluated the ability of WM cells to attract MDSCs to the tumor microenvironment (TME). We also evaluated whether PMN-MDSCs suppress the cytotoxic function of T-cells and thereby promote the survival of WM cells. Our study confirms the expansion of MDSCs, notably PMN-MDSCs in BM of WM patients as compared to normals. We also found that PMN-MDSCs expressing CD66b was a predominant population in WM patients. To determine the phenotype and transcriptome of both PMN-MDSCs and monocytic MDSCs (M-MDSCS) in WM, we compared BM aspirates from WM patients to normal bone marrow (NBM) and performed cellular indexing of transcriptomes and epitopes by sequencing (CITEseq) analysis (WM=3, NBM=2) followed by qPCR validation. We found that MDSCs in WM express significantly higher levels of HIF1-α overall (p<0.02) compared to NBM. Furthermore, PMN-MDSCs are more abundant in WM bone marrow than M-MDSCs and express significantly higher levels of CXCR4 (p<0.001), myeloperoxidase (MPO; p<0.0003), and azuricidin (AZU1; p<0.0004), suggesting an activated phenotype. To determine whether WM cells attract MDSCs to the BM, we performed a transwell assay and showed that WM cells (BCWM.1 cell line) attracted MDSCs, particularly PMN-MDSCs, to a greater extent than M-MDSCs (n=3; p<0.001). We next evaluated whether MDSCs prevent cytotoxic T-cells from lysing WM cells. In-vitro co-culture studies with BCWM.1 cells and T-cytotoxic cells (B:CD8-T) showed that T-cytotoxic cells were able to lyse the WM cell line by 46.4% , (median range 46.4 - 47.8%, p=0.001). However, when BCWM.1 cells and T-cytotoxic cells were co-cultured in the presence of MDSCs (i.e B:CD8-T:M), the specific lysis of BCWM.1 cells was reduced by 12.6% respectively (range 2.2- 12.6%, p<0.001). Furthermore, we observed that B:CD8-T:M co-culture significantly reduced CD8-T cell activation as compared to activated T cells alone (17.9%) (median range 17.8- 18%, p<0.001). In contrast, no affect was observed with B: CD8-T coculture by 91% (range 90.6- 91.4% p<.001) confirming that MDSCs suppress CD8-T cell activation. Our study therefore provides evidence that MDSCs in WM have an activated phenotype and that PMN-MDSCs in WM are attracted by malignant cells in the bone marrow. Furthermore, we find that the increased presence of PMN-MDSCs in the WM bone marrow environment suppresses T-cell lysis of malignant cells and may thereby contribute to disease progression. These findings provide a strong rationale for targeting MDSCs as a potential therapeutic strategy in WM.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and severe hematologic disorder caused by somatic mutation(s) in hematopoietic stem cells, which results in dysregulation of complement activation and complement-mediated hemolysis. Several complement inhibitors are approved for PNH, including the intravenous or subcutaneous C5 inhibitors (C5is) eculizumab, ravulizumab, and crovalimab; the subcutaneous self-administered C3 inhibitor pegcetacoplan; and the oral factor B inhibitor iptacopan and factor D inhibitor danicopan (as add-on therapy to a C5i). Although oral self-administered medications would be expected to lighten treatment burden over injectable medications in patients with PNH, adherence to anticomplement therapies is essential to maintain stable hematologic response while avoiding adverse events of breakthrough hemolysis and thrombosis. Real-world adherence to pegcetacoplan in patients with PNH was evaluated and compared with reported real-world adherence to long-term oral therapies for chronic conditions. Methods: A descriptive review of recent literature was conducted to summarize real-word oral medication adherence rates in chronic conditions. The Medical Subject Headings “compliance, medication” and “administration, oral” were applied in PubMed, and yielded reports on oral antidiabetic medications (OADs) in type 2 diabetes, oral anticoagulants (OACs) in atrial fibrillation (AF), and oral oncolytics in hematologic cancers; observational, real-world studies reporting medication adherence rates were reviewed and summarized. Adherence to pegcetacoplan therapy among patients with PNH in the US postmarketing setting was calculated as the percentage of patients who take pegcetacoplan as prescribed (adherence definition) based on the number of days patients had pegcetacoplan vials in their possession (dispensed) divided by the total number of days in a dispensing period (proportion of days covered) using central pharmacy prescription refill data. Results: In general, patients who take ≥80% of prescribed doses are considered adherent; using this definition, adherence rates among patients receiving long-term therapies for chronic conditions are typically 50% to 60% but vary greatly [Kleinsinger F. Perm J. 2018;22:18-033]. For example, reported OAD adherence rates in type 2 diabetes vary across countries (42% in Switzerland [Huber CA et al. Medicine (Baltimore). 2016;95:e3994] to 67% in Canada [Simard P et al. Acta Diabetol. 2015;52:547]) and by medication types (36.7% with thiazolidinediones to 47.3% with dipeptidyl peptidase 4-inhibitors [Farr AM et al. Adv Ther. 2014;31:1287]). OAC adherence rates in AF range widely (~40% to ~90%), differing across countries, patient populations (incident AF vs. post cardiovascular event), OAC types (warfarin, direct OACs), and follow-up period lengths. OAC adherence is also influenced by patient demographic and clinical characteristics and regional determinants in US Medicare beneficiaries [Hernandez I et al. J Am Heart Assoc. 2019;8:e011427]. Interestingly, an inverse relationship between oral medication adherence and dosing frequency is observed in chronic diseases in general (progressively decreasing adherence rates from once-daily to twice, 3-times, and 4-times daily dosing regimens [Coleman CI et al. J Manag Care Pharm. 2012;18:527]), and with OACs in AF [Vrijens B, Heidbuchel H. Europace. 2015;17:514]. As with other chronic conditions, nonadherence to oral oncolytics in hematologic cancers is more prevalent than suspected (32.7%) and associated with poorer clinical response [Noens L et al. Blood. 2009;113:5401], which can further increase the economic burden on patients [Dashputre AA et al. J Manag Care Spec Pharm. 2020;26:186]. Pegcetacoplan adherence for PNH in the US postmarketing setting from launch (2021) to date (2024) was estimated at 97%, well above the 80% threshold defining medication adherence in the literature. Conclusions: Real-world patients with PNH who self-administer pegcetacoplan subcutaneously have adherence rates exceeding the reported real-world adherence rates for oral medications in chronic conditions, especially those with high dosing frequencies. Retaining a stable hematologic response and preventing potentially life-threatening complications in PNH relies on continuous patient adherence to lifelong anticomplement therapies.