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Background Multiple myeloma (MM) is a hematological cancer characterized by the proliferation of plasma cells (PC). Progressive disease (PD) criteria are primarily characterized by an increase in (1) serum M-protein (SPEP), (2) urinary M-protein (UPEP), (3) serum free light chains (sFLC), or (4) PC percentage in the bone marrow. An essential aspect of the PD criteria is the requirement for confirmatory results from blood or urine analyses. However, in many cases, patients may be taken off treatment in favor of a new therapy after only one assessment. This can introduce clinical trial endpoint inaccuracies when confirmatory values are missing, resulting in increased censoring and underestimation of the progression rate (if the second assessment is not done). Moreover, in subjects with high tumor burden and rapidly progressing disease, it is often urgent to confirm PD before starting a subsequent line of treatment. This study assessed the necessity of repeating biomarkers to confirm PD if two biomarkers meet the progression criteria. Methods In this retrospective study, between 2002 and 2020, we assessed all consecutive patients with relapsed MM enrolled in any therapeutic clinical trial at Mayo Clinic. We included patients with measurable disease if SPEP and sFLC were available at the time of assessment. Patients required at least two consecutive assessments (by the same method) to be included in our study. PD was based on the IMWG criteria. Relapse characterized by the development of new bone lesions or extramedullary relapse without meeting biochemical progression criteria was an exclusion criterion. Patients starting a new line of treatment after non-confirmed PD were also excluded. Furthermore, patients who did not meet simultaneous progression criteria and had unconfirmed PD (did not meet IMWG criteria at the second assessment) were excluded from the analysis (n=15). The probability of meeting the progression threshold with 1 and 2 consecutive assessments was calculated. We then examined the proportions of patients meeting PD by 2 biomarkers simultaneously at the first assessment. Results We evaluated episodes of treatment among 434 patients enrolled in clinical trials, including 601 episodes: 593 episodes of biochemical confirmed PD and 8 episodes when patients did not progress but who met simultaneous progression criteria at the same time with two different biomarkers. Per measurable disease criteria, MM isotypes were 71.0% IgG, 16.8% IgA, 0.9% IgM, 1.2% IgD, 0.3% biclonal, and 9.8% light chain. According to the standard IMWG response criteria (sequential criteria), 78% and 9% of episodes reached the progression threshold with serum and urine M-components, respectively, with two consecutive assessments. For the FLC, 21% of episodes met the progression criteria. No patients had a second bone marrow examination for confirmation of PD. At the first available assessment, 82%, 19%, and 2% of episodes met progression threshold, respectively, with serum M-protein, urine M-protein, and both (serum + urine M-protein). Additionally, 58%, and 19%, episodes met the progression threshold at the first assessment with FLC and bone marrow examination, respectively. Moreover, 67% of patients with IgA myeloma meet the progression threshold with the total quantitative IgA. With the simultaneous criteria at time of first assessment, 403 episodes meet progression threshold: 45% with SPEP+sFLC (n=269/593), 4% with SPEP+UPEP (n=21/593), 9% with sFLC+UPEP (n=55/593), 11% with BME only (n=66/593), and 0.3% with qIgA +dFLC/UPEP (n=2/593). Thus, among these 413 episodes, which meet progression criteria by 2 simultaneous values at the time of the first assessment, 98% (n=413/(413+8)) of these patients (positive predictive value of the new criteria) subsequently had confirmed progression by sequential value. Conclusions In summary, we showed that the simultaneous criteria allow to identify patients with true progressive disease without overestimating the progression rate of myeloma. Therefore, confirmatory results of progression for relapsed MM patients having two biomarkers meeting criteria of progression at the same time is useless for determining disease progression.

Introduction: Multiple myeloma (MM) with t(11;14)(q13;q32), resulting in CCND1;IGH rearrangement, remains the only subset amenable to targeted therapy with the BCL2 inhibitor, Venetoclax. Nevertheless, not all patients with this translocation respond to treatment and some progress early after initial response. The mechanisms behind this clinical heterogeneity are largely unknown. Methods: To investigate mechanisms of heterogeneity in response to Venetoclax, we interrogated 44 whole genome sequencing (WGS) and 24 whole exome sequencing data from a training cohort including 33 patients with t(11;14) MM treated with Venetoclax at the Mayo Clinic. In a validation cohort, we extracted single nucleotide variants (SNV) and indels from 17 MM patients with t(11;14) with available RNAseq data treated with Venetoclax at Emory University. Results: In the training cohort the median number of lines of therapy was 4 and Venetoclax drug regimens included combinations with dexamethasone, proteasome inhibitors, anti-CD38 agents, and immunomodulatory agents. After a median follow up of 30 months, the median progression free survival (PFS) was 9 months, and 13 patients (39%) achieved a complete remission (CR) to therapy, while 9 (27%) were refractory (i.e. no response or progressed within the first 60 days). For our driver discovery analysis, we integrated SNV, indels, copy number variants (CNV) and structural variants (SV). Deletion 17p (del17p) or TP53 mutation, MCL1 gain/amp, and hyper-APOBEC did not correlate with PFS in our cohort (p=0.59, p=0.71, and p=0.05, respectively). In contrast, mutations involving the MAPK pathway (NRAS, KRAS, BRAF, FGFR3) displayed the strongest association with short PFS, which was retained after adjusting for del17p/TP53 mutations, and 1q gain/amp (HR: 4.76, CI: 1.8-12.9, p=0.002). The association between suboptimal response and resistance to Venetoclax in MM patients with MAPK pathway mutations (n=7) was also observed in our validation cohort from Emory University (p=0.046). This is in line with what has been reported in functional studies in acute myeloid leukemia, where RAS pathway promotes MCL1 under the selective pressure of Venetoclax (Zhang et al. Signal Transduct Target Ther 2022). While MAPK mutations explained most cases of refractory patients (86%), the mechanisms through which initially responding patients subsequently progressed remain unclear. To investigate this, we interrogated 10 patients with pre- and post-treatment samples collected at disease progression (PD). We identified genomic events acquired/selected at PD of genes involved in the anti-apoptotic MCL1/BCL2 and MAPK pathways in 6/10 cases. One patient was refractory to Venetoclax and had double minutes involving BCL2, which caused multiple focal amplifications (>30 copies), likely reflecting extrachromosomal DNA. Another patient progressed with an acquired, focal deletion on BCL2L11. In two patients, the progressing clones harbored mutations in MAPK pathways (one BRAF and the other WNK2). Two patients who relapsed after sustained CR had major branching evolution where a previously undetectable focal deletion of PMAIP1 (NOXA) was seen at PD. Importantly, one of these deletions was a biallelic event. Venetoclax sensitivity assays conducted on PMAIP1 knockout in the MM t(11;14) cell line, KMS12PE, demonstrated significantly reduced cell death across all drug concentrations (p<0.05), validating the loss of NOXA as a mechanism of resistance to Venetoclax in MM. Finally, similarly with what was previously reported in chronic lymphocyte leukemia treated with Venetoclax (e.g., Herling et al Nat Comm 2018), CDKN2A/B monoallelic and biallelic focal deletions were selected post-Venetoclax, at PD in 4 patients while TP53 SNV/deletions were selected from subclonal to clonal in 2 patients at PD. Conclusion: In this study, we demonstrate that mutations involving the MAPK pathway identify a population of MM patients with t(11;14)(CCND1;IGH) who are unlikely to respond to Venetoclax. We also show that genomic events involving the MAPK and BCL2/MCL1 pathways are primarily responsible for disease progression after initial response. These findings highlight the critical role of comprehensive genomic profiling through WGS in identifying mechanisms of resistance to targeted therapies in MM, aiding patient selection for Venetoclax, and guiding the development of novel drug combinations.

Background: 24-hour urine (24HRU) assessments are a key component of International Myeloma Working Group (IMWG) response criteria for multiple myeloma (MM) and are requested longitudinally in clinical trials even if not routinely used in real-world practice. Several analyses have suggested a limited role for 24HRU if serum immunofixation or free light chain (LC) testing is abnormal (Dejoie Blood 2016, Lahuerta Blood 2023). Adopting urine-free response criteria (with 24HRU removal from every level of IMWG response) may change responses in <5% of patients (Natsuhara et al, Leuk Lymph 2023) but not have been studied in terms of progression-free survival (PFS) in a larger population. Methods: This was a secondary analysis of data from the Phase 3 BMT CTN 0702 (STaMINA) trial (Stadtmauer et al, JCO 2019) studying post-autologous stem cell transplantation (ASCT) strategies. Only patients evaluable for Day +56 response following ASCT were included, and patients with progressive disease (PD, n=8) at this timepoint were excluded. Post-ASCT responses relative to diagnosis were calculated by two sets of criteria. First, traditional responses were calculated per IMWG criteria (Kumar et al, Lancet Onc 2016) as stable disease (SD), partial response (PR), very good PR (VGPR), or complete response (CR, including stringent CR). Second, urine-free response assessments were calculated as follows: negative 24HRU immunofixation no longer required for CR, 24HRU M-protein < 100 milligrams (mg) per day no longer required for VGPR, and no 24HRU M-protein thresholds (e.g., < 200 mg per day or > 90% reduction) required for PR. PFS was estimated (using PD by traditional IMWG criteria or death as events) using proportional hazard models. Results: Of 636 evaluable patients (84% of all BMT CTN 0702 participants), LC-only disease was noted in 18.9% (n=120). Pre-enrollment 24HRU testing at diagnosis was done in 28.9% of patients (n=184). IMWG responses were as follows: CR, 29.4% (n=187) traditional vs 29.7% (n=189) urine-free; VGPR, 37.0% (n=235) traditional vs 36.6% (n=233) urine-free; PR, 30.7% (n=195) for both; SD, 3.0% (n=19) for both. A total of 4 responses (0.6%) changed with urine-free criteria. Three patients were ‘upgraded’ (VGPR to CR, n=2, PR to VGPR, n=1) because 24HRU requirements for CR/VGPR were no longer required. One patient without a measurable serum paraprotein was ‘downgraded’ from a traditional VGPR (with negative 24HRU immunofixation) to a urine-free PR based on LC-only criteria. Among the subset of 184 patients with 24HRU electrophoresis results at diagnosis, there were no differences between traditional and urine-free IMWG responses. Median PFS and 95% confidence intervals (CI) were the same at each depth of both traditional and urine-free criteria: 63.0 months (50.8 - 71.4 months) for CR, 49.6 months (42.6 - 55.1 months) for VGPR, 44.9 months (37.2-54.8 months) for PR, and 34.4 months (14.0-51.5 months) for SD. Urine-free IMWG criteria were highly prognostic for PFS (p = 0.006, 95% CI 1.05-1.36). The achievement of urine-free CR vs urine-free ≤CR was also prognostic (p = 0.005, 95% CI 0.54 - 0.89). Urine-free VGPR vs urine-free ≤VGPR was of borderline significance (p = 0.102, 95% CI 0.66 - 1.04), with median PFS 53.8 months (48.0 - 57.6 months) vs 44.6 months (36.3 - 54.7 months) respectively. Discussion: In this secondary analysis of a Phase 3 trial, omitting 24HRU assessments from IMWG criteria changed <1% of responses and had no impact on PFS prediction at any response depth. 24HRU assessments had been performed for less than a third of patients as standard of care prior to study entry, underscoring its infrequent use outside of trials. Urine-free IMWG assessments would reduce time toxicity and discomfort for patients, particularly for those with limited dexterity. If implemented in trials, they would also simplify study logistics and lower operational costs. Outside of situations such as AL amyloidosis where 24HRU assessments remain critical or for patients with urine as their only measurable MM biomarker, our results support the removal of 24HRU assessments from future iterations of IMWG response criteria. Funding: Grants #U10HL069294 and #U24HL138660 to the BMT CTN from the NHLBI and the NCI; contributions by Celgene Corporation and Millennium Pharmaceuticals, Inc.; funding by The Alliance for Clinical Trials in Oncology, the ECOG-ACRIN Cancer Research Group and SWOG.

Background: Light chain (AL) amyloidosis is a rare and understudied plasma cell disorder. It is caused by abnormal misfolding of monoclonal immunoglobulin light chains, leading to extracellular deposition of amyloid, ultimately resulting in organ dysfunction and death. Since AL amyloidosis is uncommon, therapies are adapted from the more common plasma cell disorders like multiple myeloma. Daratumumab, a monoclonal antibody targeting CD38, was first tested in several prospective and retrospective studies in AL patients with relapsed refractory disease. The ANDROMEDA study (Kastritis E et al, NEJM 2021) established daratumumab in combination with CyBorD (Cyclophosphamide-Bortezomib-Dexamethasone) followed by daratumumab maintenance as the new standard of care compared to CyBorD alone for newly diagnosed patients. Consequently, daratumumab maintenance has become universally offered to all newly diagnosed AL amyloidosis patients. Notably, there was no second randomization before the initiation of maintenance therapy in ANDROMEDA. Hence, a critical question that remains is whether the superior efficacy observed in the investigational arm of the ANDROMEDA trial is solely a result of the addition of daratumumab during the induction phase or if the maintenance phase contributed to the favorable outcomes in the investigational arm. We hypothesize that a shorter duration of maintenance would prove non-inferior to the standard duration of daratumumab maintenance in patients with AL amyloidosis that have responded to Daratumumab-CyBorD induction. To answer this question we are conducting a pragmatic phase 2 randomized trial assessing the optimal duration of daratumumab maintenance in patients with AL amyloidosis who have responded to Daratumumab-CyBorD induction. Study Design, Patients and Methods: We use broadened eligibility criteria consistent with pragmatic trial design. The estimated total enrollment is up to 96 patients. The main eligibility criteria are patients with biopsy-proven AL Amyloidosis who have completed 6 cycles of Dara-CyBorD-based induction treatment and achieved an adequate response to induction (defined as hematological complete response (CR), hematological very good partial response (VGPR), or hematological partial response (PR) with at least one organ response). Patients in whom bortezomib and/or cyclophosphamide were omitted from induction due to toxicity concerns or adverse effects are allowed. The major exclusion criteria are patients who were concomitantly diagnosed with multiple myeloma based on International Myeloma Working Group (IMWG) criteria. This phase II, open-label, multicenter trial (NCT05898646) randomizes patients who have attained an adequate response to 6 cycles of Dara-CyBorD-based induction in a 1:1 ratio, using a dynamic allocation procedure that balances the marginal distributions of the stratification factors (age, cardiac stage, and t(11;14) status). Randomization is to either single-agent daratumumab maintenance of 6 cycles duration (experimental arm) or 18 cycles of daratumumab maintenance (control arm) (cycle = 28 days). Treatment is allowed to be given at a local facility After completing maintenance treatment, patients will follow up every 3 months for up to 36 months from registration. The primary endpoint of the trial is to evaluate the non-inferiority of a shorter (6 cycles) daratumumab maintenance measured by event-free survival (EFS) compared to the standard maintenance of 18 cycles in newly diagnosed AL amyloidosis who receive 6 cycles of daratumumab-CyBorD based induction. EFS in this study is defined as time from registration to any of the following events: hematological progression, organ progression, subsequent anti-plasma cell therapy in the absence of hematological progression but with persistent organ dysfunction or death, whichever occurs first. Secondary endpoints are depth of hematological and organ response, bone marrow measurable residual disease negativity rate, duration of response, quality of life, and survival. By investigating whether a shorter duration of maintenance is non-inferior to the standard duration, we aim to inform clinical practice and potentially reduce the toxicities associated with prolonged maintenance therapy, including hypogammaglobulinemia, recurrent infections, the need for intravenous gammaglobulin replacement, and financial toxicity.

Background:Patients (pts) with multiple myeloma (MM) often receive immunomodulatory drugs (IMiDs; eg, lenalidomide), proteasome inhibitors (eg, bortezomib), and anti-CD38 monoclonal antibodies (mAbs) as part of their initial treatment. However, most pts relapse following initial treatment, with increasing attrition rates observed at each subsequent line of therapy (LOT), creating an unmet need for treatment options for pts with relapsed/refractory MM (RRMM) who have prior exposure to these agents. Current regimens for pts who have prior exposure to lenalidomide and anti-CD38 mAbs include elotuzumab, pomalidomide, and dexamethasone (EPd; approved in Europe and the US) and pomalidomide, bortezomib, and dexamethasone (PVd; approved in Europe and Australia), but disease control, as well as depth and durability of response, could be improved. Talquetamab (tal) and teclistamab (tec), targeting CD3 and either G protein-coupled receptor family C group 5 member D (GPRC5D, tal) or B-cell maturation antigen (tec), are the first T-cell redirecting bispecific antibodies (BsAbs) approved as monotherapies in Europe and the US for the treatment of pts with triple-class exposed (TCE) RRMM. Use of these novel immunotherapies in earlier LOTs, either with a dual-antigen targeting approach or by enhancing the activity of the BsAb with an IMiD (pomalidomide), may provide deeper, more durable responses and improve pt quality of life. Preliminary data using these combination treatments have shown promising results: in the phase 1b MonumenTAL-2 study (NCT05050097), tal+pomalidomide (tal-P) demonstrated overall response rates (ORRs) of >84% in pts with RRMM with ≥2 prior LOTs and in the phase 1b/2 RedirecTT-1 study (NCT04586426), tal+tec (tal-tec) showed ORRs of >77% in pts with TCE RRMM. MonumenTAL-6 (NCT06208150) is a randomized, open-label, multicenter, phase 3 study comparing the efficacy of tal-P vs investigator's choice of EPd or PVd and tal-tec vs investigator's choice of EPd or PVd in pts with RRMM with 1-4 prior LOTs, including lenalidomide and an anti-CD38 mAb. Study Design and Methods: Eligibility includes pts aged ≥18 years with documented MM per International Myeloma Working Group (IMWG) criteria, with measurable disease at screening, an Eastern Cooperative Oncology Group performance status of 0-2, who have received 1-4 prior LOTs (including lenalidomide and an anti-CD38 mAb in any prior LOT), and have progressive disease on or after their last therapy. Pts with prior exposure to pomalidomide, tec, or GPRC5D-directed therapies are excluded. Approximately 795 pts are randomized 1:1:1 to receive tal-P, tal-tec, or investigator's choice of EPd or PVd. Tal is administered subcutaneously at 0.8 mg/kg every other week (Q2W), and tec is administered subcutaneously at 3.0 mg/kg every 4 weeks (Q4W) following step-up doses of each. Pts are permitted to switch from Q2W to Q4W tal dosing following a confirmed very good partial response or better (≥VGPR) at cycle 5. Pomalidomide, EPd, and PVd are administered per approved dosing schedules. Randomization is stratified by number of prior LOTs (1 vs ≥2), International Staging System stage at screening (I vs II/III), and investigator's choice of treatment (EPd vs PVd). Pts receive study treatment until disease progression, start of subsequent anti-MM treatment, death, intolerable toxicity, withdrawal of consent, or end of study, whichever occurs first. A finite dosing approach is employed for tal and tec. The primary endpoint is progression-free survival (PFS). Secondary endpoints include ORR assessed per IMWG 2016 criteria, ≥VGPR rate, complete response (CR) or better rate, rate of minimal residual disease-negative CR, overall survival, PFS on next LOT, time-to-next treatment, and pt-reported outcomes. This study is actively recruiting, with more than 160 locations in 26 countries. Results will provide insights into the efficacy of the combination of BsAbs in therapeutic strategies that exploit their different mechanisms of action to overcome treatment resistance and improve outcomes of pts with TCE RRMM in earlier LOTs.

Introduction. Platelet-activating anti-Platelet factor 4 (PF4) antibodies mediate a number of thrombotic thrombocytopenic syndromes, including heparin-induced thrombocytopenia (HIT), and more recently recognized conditions, vaccine-induced immune thrombotic thrombocytopenia (VITT), adenovirus-associated thrombotic thrombocytopenia, and monoclonal gammopathy of thrombotic significance (MGTS). In MGTS (Blood. 2023 Apr 6;141(14):1772-1776), a monoclonal IgG antibody to PF4 mediates a chronic prothrombotic state characterized by recurrent thrombosis. More recent evidence suggests that MGTS may present with negative results in HIT enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA), and positivity only in PF4-dependent functional assays such as the PF4-dependent P-selectin expression assay (PEA; submitted). The presence of MGUS in patients has long been associated with high risks of thrombotic disease (Blood. 2010 Jun 17;115(24):4991-8), but the mechanism by which they predispose to thrombosis has been relatively unexplored. In this study, we evaluated a cohort of MGUS patients who experience thrombosis for the presence of platelet-activating anti-PF4 antibodies. M ethods. An administrative database search was performed of Mayo Clinic records to identify patients who had a diagnosis of MGUS, and thrombosis. Research samples from 134 MGUS patients with IgG monoclonal protein and thrombosis were obtained from Mayo Clinic's dysproteinemia biobank and subjected to PF4 antigen-based and functional screening. Research studies were approved by the Institutional Review Board of Mayo Clinic. Results. Of the 134 patients, 17 (12.7%) activated platelets in the PEA (≥19%). To exclude non-specific activation (e.g. due to HLA antibodies), secondary PEA testing was performed to assess whether platelet activation was mediated by a HIT-like antibody (i.e., PF4- and IgG-dependent, and inhibitable with high concentrations of heparin). Four patients, hereafter referred to as patients 1-4, were identified as having a HIT-like antibody, as demonstrated by PF4-dependent platelet activation that could be inhibited with high concentrations of heparin (100 U/mL), and blockade of the platelet IgG receptor, FcγRIIa. Patients 1 and 2 had persistent, platelet-activating anti-PF4 antibodies based on PEA testing of multiple samples obtained several years apart. Patient 1 had a recurrent, multi-year history of deep venous thrombosis (DVT), while patient 2 had one DVT episode. Patient 3 demonstrated a HIT antibody-like profile in the PEA in two of three samples available for testing and had a history of thrombotic cerebrovascular accident (CVA). Patient 4 demonstrated a HIT-like platelet-activating antibody in only one of four samples tested, and consistent with this finding, had an MGUS identified on clinical testing at only one time point (“small” IgG lambda, not quantified), 3 months prior to the platelet-activating sample. This patient experienced multiple thrombotic events including DVTs, left atrial thrombus and embolic CVA, pulmonary embolism and acute limb ischemia. All patients tested negative in two FDA-approved antigen-based HIT assays tested (Immucor PF4 IgG and Zymutest HIA IgG), similar to a recent MGTS case (submitted). SRA was negative in both Patients 1 & 2, weakly positive in Patient 3 (33%, with high CV), and positive (72%) in Patient 4. Conclusions. Definitive diagnosis of patients with MGTS provides insight into a mechanism of “unexplained” thrombosis and may offer additional treatment pathways for these patients with recurrent thrombosis (New Eng J of Med, 2024 Aug 8). Serologic testing in two patients correlated strongly with observations of thrombosis, and the study confirmed recent findings that anti-PF4 antibodies may present with unusual serology that is neither HIT- nor VITT-like (with negative results in both ELISA and SRA). The incidence of confirmed/suspected MGTS was 3% (4/134) in this population of MGUS patients with thrombosis. Prospective studies are needed to confirm these observations.

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy with several recurrent primary cytogenetic abnormalities including t(11,14)(q13;q32), resulting in increased expression of CCND1. Although 40% of patients with t(11;14) MM respond favorably to the BCL-2 inhibitor venetoclax (ven), 60% fail to achieve an objective response. Previous studies have suggested upregulation of PMAIP1, encoding the pro-apoptotic NOXA protein, in association with ven sensitivity (Leblay, Blood, 2024). Here we sought to evaluate the role for NOXA in ven response in t(11;14) MM. Methods: We analyzed whole exome, whole genome and RNA sequencing data of CD138+ tumor cells from 1,359 patients with MM in the CoMMpass and Mayo Clinic cohorts. The histone mark H3K27ac was assessed in t(11;14)+ KMS12PE and U266B1 human myeloma cell lines using ChIP-seq. Gene expression and protein levels of PMAIP1 (NOXA) were also detected by qRT-PCR and Western blot, respectively. Live cell detection was evaluated by Annexin V and PI using flow cytometry and by YoYo3 dye using IncuCyte following treatment with ven. Results: The t(11;14) MM subtype was associated with a 1.6-5.5-fold increased expression of PMAIP1 compared to non-t(11,14) MM in the CoMMpass and Mayo Clinic cohorts (p<0.0001). These findings are consistent with a significant correlation between CCND1 and PMAIP1 expression in both cohorts (p<0.001). To evaluate whether cyclin D1 is directly associated with increasing expression of PMAIP1 in t(11;14) MM, we generated a cyclin D1 knock out (KO) version of the U266B1 line using CRISPR/cas9. Two independent U266B1 KO cell lines showed reduced expression of PMAIP1 and NOXA protein compared to the WT U266B1 consistent with a role for cyclin D1 in regulating PMAIP1 expression. In addition, the KO lines had a 4.8-fold reduced H3K27ac signal at the PMAIP1 locus in comparison to the WT control suggesting a role for cyclin D1 in promoting H3K27ac. We previously observed increased expression of CCND1 in association with the G allele compared to the A allele of rs9344 in both the CoMMpass and Mayo Clinic cohorts (Leukemia, in press). We next asked whether the G risk allele of rs9344 within CCND1 impacted gene expression and H3k27ac levels at the PMAIP1 locus. To study the allele specific effect of CCND1, we used the CRISPR/cas9 edited KMS12PE WT (rs9344 AA) and Knock In (KI) (rs9344 GG) cell lines. In the KI cells, the G allele was accompanied by an ~2-fold increased expression of PMAIP1 and NOXA protein detected by western blot (<0.05). Increased PMAIP1 expression in KI lines was also accompanied by a 2.2-fold increased H3K27ac further suggesting a role for the G allele of rs9344 in promoting H3K27ac and gene expression at the PMAIP1 locus. Finally, we evaluated the consequence of PMAIP1 expression levels in relation to ven response. Increased expression of PMAIP1 and elevated levels of NOXA protein in the KMS12PE KI lines was associated with increased sensitivity to ven resulting in ~50% increase in cell death of KI vs. WT lines (p<0.02). Conclusion: We demonstrate a role for cyclin D1 in regulating PMAIP1 expression through H3K27ac resulting in increased NOXA protein. Levels of NOXA are associated with the G allele of rs9344 and increased venetoclax response.

Introduction: In myeloma, accurate risk stratification remains a challenge due to substantial biological heterogeneity and highly variable survival outcomes. Cross-sectional imaging is commonly obtained at the time of diagnosis, but imaging findings are not currently incorporated into risk stratification efforts in a standardized manner, despite their prognostic significance. Here, we investigate the use of novel image-based prognostic biomarkers obtained by automated image segmentation and radiomics feature extraction using machine learning methods. Methods: We obtained the pre-treatment 18F-FDG PET/CT scans of 443 patients with newly diagnosed myeloma and complete demographic and cytogenetic data (seen at Mayo Clinic between 2005 and 2019). The native DICOM images were pre-processed and subjected to automated image segmentation using nnU-Net (v1.0, inference using a pre-trained hematologic malignancies model). Limitations of this automated segmentation approach include heterogeneity introduced by varying image quality, technical differences related to PET reconstruction, and potential inclusion of non-neoplastic FDG-avid lesions in segmented regions. The quality of the automated image segmentation was evaluated by comparing the radiomics features obtained from ground-truth segmentation (manual lesion segmentation) and model segmentation in a separate cohort of patients with hematologic malignancies (n=72). We selected 42 radiomics features that moderately (r≥0.30, p<0.005, n=23) or strongly (r≥0.50, p<0.001, n=19) correlated between ground-truth (manual) and model (automated) segmentation for further evaluation in myeloma patients. PyRadiomics was used to extract discrete radiomics features for clinical prediction modeling. We evaluated the prognostic significance of tumor size-, shape-, and texture-related radiomics features and compared their predictive power to established prognostic factors (International Staging System [ISS], Revised ISS [R-ISS] stage and high-risk FISH cytogenetics). The outcome of interest was overall survival and the predictive power of competing proportional hazards regression models was assessed using Harrell's C-statistic. For modeling purposes, patients without measurable tumor volume were categorized as having undetectable radiomics features (0 for all features). All radiomics features were median dichotomized for downstream modeling. Forward feature selection and testing for multicollinearity were used to remove redundant features. Select radiomics features were divided into quintiles to generate an additive radiomics risk score and associated risk strata. Results: Median age at diagnosis of the 443 myeloma patients was 63 years (32-91) and 270 patients (62%) were men. After a median follow-up of 2.4 years (95% CI 0.1-7.4), median overall survival was 6.5 years (95% CI 5.5-NR). Most patients had detectable metabolic tumor volume (n=309, 70%) on their pre-treatment 18F-FDG PET/CT scan. Several of the 42 radiomics features were intercorrelated and two features were selected for further investigation based on their independent prognostic significance (forward feature selection). Higher maximum axial tumor diameter (HR 1.90, 95% CI 1.31-2.76, p=0.001) and a lower concentration of low gray-level values in the tumor (HR 1.73, 95% CI 1.20-2.49, p=0.004) were independently associated with overall survival in newly diagnosed myeloma. These associations were independent of age at diagnosis, sex, and R-ISS stage. We devised a simple additive risk score representing the sum of quintiles of the two radiomics features and divided the patients into three risk categories akin to the R-ISS. The predictive power of this radiomics risk score (Harrell's C=0.60) was comparable to the ISS (C=0.59) and the R-ISS (C=0.57). Conclusions: Automated image segmentation and radiomics feature extraction from 18F-FDG PET/CT scans is feasible. This approach yields image-based biomarkers that correlate with results obtained by manual imaging segmentation. Several radiomics features such as tumor size and heterogeneity have independent prognostic significance in newly diagnosed myeloma. These novel biomarkers rival the predictive power of the currently established risk stratification methods and may be used to improve risk stratification in myeloma.

Introduction: Measurable residual disease (MRD) is a well-validated prognostic marker and interim outcomes measure in patients with multiple myeloma (MM). Despite its broad acceptance as a robust prognostic marker, the MRD status is only available down the treatment line, and there is limited information on the markers at diagnosis that can predict achievement of MRD negativity post-transplant. This study explored the relationship between baseline risk factors and post-autologous stem cell transplantation (post-ASCT) MRD status, as well as the combined impact of these baseline factors and post-ASCT MRD status on progression-free survival (PFS) and overall survival (OS). Patients and Methods: We conducted a retrospective, single-center study of patients achieving at least VGPR after ASCT between 01/01/2016 and 12/30/2023, majority (n=814, 98%) received ASCT in the frontline setting. MRD testing was performed between days 60-100 post-transplant using next-generation flow cytometry with a sensitivity of at least 1x10^5 nucleated cells (minimum 2x10^6 cells assessed). Treatment response and disease progression were determined by IMWG criteria. High-risk disease (HRD) was defined as presence of International Staging System ISS stage 3, high lactate dehydrogenase, 1q gain, 17p deletion, or t(4;14) chromosomal abnormalities. We calculated PFS/OS from transplant day 0 to first documented event (progression or death). Categorical variables were compared using chi-square and Fisher's exact tests. Non-parametric tests compared continuous variables, and logistic regression analyses tested associations between clinical/lab variables and MRD status post-ASCT. Time-to-event analyses were performed using Kaplan-Meier and compared using log-rank test. Multivariable analyses were conducted using the Cox regression model. Result: The study cohort included 831 newly diagnosed MM patients. The median follow-up for the cohort was 50 months (95% CI: 48-51). 48% (n=402) achieved MRD negativity (MRD-neg) post-ASCT. Univariate analysis demonstrated that R2-ISS stage, depth of response pre-ASCT, and presence of t(11;14) to be associated with worse MRD-negativity rates. Multivariate analysis revealed that t(11;14) significantly decreased the odds of MRD-negativity (p<0.0001, OR=0.4, 95% CI: 0.3-0.6) and a deeper pre-ASCT response was associated with increase odd of MRD-negativity OR-4.7 (95%CI 3.2-7.4) p<0.0001. Patients with MRD-positive disease post-transplant had an inferior estimated median PFS of 57.6 months (95% CI: 49-69) with a 5-year PFS of 49% (95% CI: 43-56), compared to an estimated median PFS of 83.3 months (95% CI: NA) and a 5-year PFS of 66% in MRD-negative patients (p<0.0001, HR=1.9, 95% CI: 1.5-2.5). Similarly, MRD positivity was associated with an inferior OS [estimated median of 92.6 months (95% CI NA) ,5-year OS 76% (95%CI 72-82) for MRD-pos vs. Median OS NR, 5-year OS 85%(95%CI NA) in MRD-neg; p=0.003, HR=1.69, 95% CI: 1.2-2.38)]. In MRD-neg patients, a baseline bone marrow plasma cells BMPC% >60% was significantly associated with worse PFS (HR=1.9, 95% CI: 1.26-2.99; p<0.01). High-risk disease was not associated with an inferior PFS or OS in patients with MRD-negativity. For MRD-neg patients, estimated median PFS was not reached for those without HRD, 5-year PFS was 66% (95%CI 51-85), compared to 83 months(95%CI 65-NA) for those with HRD, 5-year PFS was 65%(95%CI57-74), p=0.6441. The median OS was not reached in either group (p=0.07). Five-year OS was 94% (95%CI 86-100) in MRD-neg without HRD vs. 81%(95%CI 75-87) with HRD (p=0.07). In MRD-pos patients, median PFS was 81 months (95%CI 58-NA) for those without HRD, 5-year PFS of 59% (95%CI 46-77), compared to PFS of 41 months(95%CI 36-52) for those with HRD ,5-year PFS of 39% (95%CI 31.3-47) , p=0.003. Median OS was not reached in MRD-pos patients without HRD, but it was 59.8 months ( for those with HRD (p=0.0012). Five-year OS was 94% (95%CI 89-99) in MRD-pos without HRD vs. 67%(95%CI 60-74) with HRD (p<0.0001). For every 1 log increase in the residual cell % on MRD testing in MRD-pos patients, the hazard ratio for PFS increased by 42% (p=0.0002, HR=1.42, 95% CI: 1.2-1.7). Conclusion: Patients with t(11;14) have a reduced likelihood of achieving MRD negativity post-ASCT, whereas high-risk cytogenetics did not impact MRD-negativity rates. Achievement of MRD negativity post-ASCT mitigates the impact of high-risk characteristics.

Background: Multiple Myeloma (MM) is predominantly a disease of older adults. However, older patients vary widely in physiological status and treatment tolerance. The simplified frailty index (Facon 2020) incorporates age, ECOG performance status, and comorbidities (Charleston Comorbidity Index [CCI]) to classify patients as frail or non-frail. While frailty is associated with increased toxicity with conventional systemic therapies, its impact on outcomes of older patients treated with bispecific antibodies (BsAbs) has not been established. Thus, we conducted a retrospective study to evaluate the efficacy and toxicity of BsAbs in older patients based on frailty. Methods: This is a single institution study. We included patients with MM ≥65 years who initiated treatment with a BsAb at Mayo Clinic in Rochester, MN from August, 2018 to January, 2024. Patients who received a BsAb on trial or as standard of care were included. For patients who received 2 different BsAbs at different timepoints, we included the first treatment only. We grouped patients into frail and non-frail based on the simplified frailty index (score ≥2 = frail). We evaluated the overall response (ORR) and ≥very good partial response (VGPR) rates, progression-free (PFS) and overall (OS) survival, early mortality (within 90 days) including early non-relapse mortality (NRM), and toxicities including cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS) and healthcare utilization. P values were insignificant (>0.05) unless otherwise stated. PFS and OS were calculated from the day of BsAb start. Results: A total of 99 patients age 65-89 years were included. The products used were teclistamab (N=43), talquetamab (N=2), and investigational agents (N=54); 67% were ≥70 and 29% were ≥75 years; 39% were female, 95% were non-Hispanic White and 4% were Black/African American. Seventy patients (71%) were considered frail and 29 (29%) were non-frail. At the time of BsAb start, a higher proportion of frail patients had platelets <75 x 10^9/L (30% vs 8%, P=0.04). Otherwise, there was no significant difference between frail and non-frail patients in age (median: 72 vs 70), hemoglobin <8 g/dL (19% vs 12%), absolute neutrophil count <1 x 10^9/L (6% vs 8%), presence of high-risk FISH (70% vs 60%), ISS stage 3 (32% vs 41%), high LDH (58% vs 61%), presence of extramedullary disease (39% vs 25%), serum creatinine ≥2 mg/dL (15% vs 8%), bone marrow plasma cell% (20% in both), triple- (83% vs 90%) and penta-class refractoriness (63% vs 48%), number of prior lines of therapy (median: 6 in both), prior exposure to BCMA-directed therapy (24% vs 21%, P=0.80) and prior autologous stem cell transplant (73% vs 86%). Step-up dosing was used in 81% vs 48% of patients (P=0.001), and treatment was initiated in the outpatient setting in 46% vs 28% (P=0.12) in frail and non-frail patients, respectively. The median follow-up was 1.9 (95%CI: 1.0-2.3) years. There was no significant difference between frail and non-frail patients in ORR (52% vs 62%), ≥VGPR (46% vs 48%), 1-year PFS (36% vs 48%, P=0.26), or 1-year OS (57% vs 61%, P=0.30), respectively. Age and CCI alone were not associated with PFS or OS, but ECOG >1 was associated with decreased PFS (hazard ratio: 3.1, P=0.001) and OS (hazard ratio: 3.1, P<0.001). There was no significant difference between frail vs non-frail patients in grade 3/4 thrombocytopenia (39% vs 17%, P=0.06), grade 3/4 neutropenia (27% vs 35%), total days of hospitalization (median: 8 vs 5, P=0.35), number of unplanned admissions in 6 months (median: 1 in both), or total hospital days for unplanned admissions (1 vs 2 days). There was no significant difference between frail and non-frail patients in early mortality (23% vs 10%, P=0.17), including early NRM (4% in both), CRS (59% vs 66%), grade 3-4 CRS (3% vs 7%), or CRS recurrence (21% vs 10%). ICANS occurred in 5 (9%) frail patients and 1 non-frail patient (grade 2); among frail patients, grades were: 1 in 3 patients, 3 in 1 patient, and 4 in 1 patient. Similarly, there was no significant difference in CRS/ICANS between the 2 groups when patients with and without step-up dosing were analyzed separately. Conclusion: Frailty, defined using the simplified frailty index, was not significantly associated with inferior efficacy or increased toxicity with BsAb therapy among older patients with MM. Poor performance status predicted inferior PFS and OS, while age and comorbidity scores did not.