Subgroup Of Multiple Myeloma Patients Research Articles

An automated network-based tool to search for metabolic vulnerabilities in cancer

The development of computational tools for the systematic prediction of metabolic vulnerabilities of cancer cells constitutes a central question in systems biology. Here, we present gmctool, a freely accessible online tool that allows us to accomplish this task in a simple, efficient and intuitive environment. gmctool exploits the concept of genetic Minimal Cut Sets (gMCSs), a theoretical approach to synthetic lethality based on genome-scale metabolic networks, including a unique database of synthetic lethals computed from Human1, the most recent metabolic reconstruction of human cells. gmctool introduces qualitative and quantitative improvements over our previously developed algorithms to predict, visualize and analyze metabolic vulnerabilities in cancer, demonstrating a superior performance than competing algorithms. A detailed illustration of gmctool is presented for multiple myeloma (MM), an incurable hematological malignancy. We provide in vitro experimental evidence for the essentiality of CTPS1 (CTPS synthase) and UAP1 (UDP-N-Acetylglucosamine Pyrophosphorylase 1) in specific MM patient subgroups.

IGF2BP1 promotes multiple myeloma with chromosome 1q gain via increasing CDC5L expression in an m6A-dependent manner

Multiple myeloma (MM) patients with chromosome 1q gain (1q+) are clinically and biologically heterogeneous. The underlying molecular mechanisms are still under investigation, while the identification of targets for effective therapy of this subgroup of MM patients is urgently needed. We aimed to investigate the clinical significance and the regulatory mechanisms of insulin-like growth factor 2 messenger RNA (mRNA) binding protein 1 (IGF2BP1), a N6-methyladenosine (m6A) reader, in MM patients with 1q+. We found that MM patients with 1q+ exhibit a significantly higher level of IGF2BP1 mRNA than controls, while higher IGF2BP1 expression predicted a worse prognosis in MM patients with 1q+. IGF2BP1 overexpression promoted cell proliferation and G1-to-S phase transition of the cell cycle in NCI-H929 cells. Through comprehensive in silico analyses of existing public datasets and in-house generated high-throughput sequencing datasets, along with in vitro experiments, we identified CDC5L as a target of IGFBP1, which can bind to the m6A sites of CDC5L mRNA to up-regulate its protein abundance. Higher CDC5L expression also predicted a worse prognosis of MM patients with 1q+. Moreover, both knockdown and mutation of CDC5L attenuated the pro-proliferative effect of IGF2BP1. Furthermore, IGF2BP1 inhibitor BTYNB effectively inhibited CDC5L expression in MM cells with 1q+ and suppressed the proliferation of these cells in vitro and in vivo. Therefore, IGF2BP1 acts as a post-transcriptional enhancer of CDC5L in an m6A-dependent manner to promote the proliferation of MM cells with 1q+. Our work identified a novel IGF2BP1-CDC5L axis and provided new insight into developing targeted therapeutics for MM patients with 1q+.

IGF2BP1 Promotes Multiple Myeloma with Chromosome 1q Gain Via Increasing CDC5L Expression in an m6A-Dependent Manner

Background: Multiple myeloma (MM) patients with chromosome 1q gain (1q+) are clinically and biologically heterogeneous. The underlying molecular mechanisms are still under investigation, while identification of targets for effective therapy of this subgroup of MM patients is urgently needed. We aimed to investigate the clinical significance and the regulatory mechanisms of insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 1 (IGF2BP1), a N6-methyladenosine (m6A) reader, in MM patients with 1q+. Methods: Comprehensive in silico analyses of existed public datasets (MMRF CoMMpass study) and in-house generated high-throughput sequencing datasets (RNA-sequencing, methylated RNA immunoprecipitation sequencing, and crosslinking immunoprecipitation sequencing), along with routine molecular and cellular in vitro assays, as well as in vivo animal model studies, were conducted. Results: IGF2BP1 displays a high expression level in MM patients with 1q+ and its high expression predicts poor prognosis in these patients. IGF2BP1 overexpression promoted cell proliferation and G1-to-S phase transition of the cell cycle in H929 cells. Combinatory analyses of MeRIP-seq, RNA-seq, CLIP-seq and patients' survival data identified CDC5L as a target of IGF2BP1, which can bind the m6A sites of CDC5L mRNA to upregulate its protein abundance. Higher CDC5L expression also predicted worse prognosis of MM patients with 1q+. Moreover, both knockdown and mutation of CDC5L attenuated the pro-proliferative effect of IGF2BP1. Furthermore, IGF2BP1 inhibitor BTYNB effectively inhibited CDC5L expression in MM cells with 1q+, and suppressed the proliferation of these cells in vitro and in vivo. Conclusions: IGF2BP1 acts as a post-transcriptional enhancer of CDC5L in an m6A manner to promote the proliferation of MM cells with 1q+. Our work identified a novel IGF2BP1-CDC5L axis and provided new insight in developing targeted therapeutics for MM patients with 1q+.

Confirmed Pathogenic Germline Variants in Cancer Predisposition Genes Incidentally Detected in Somatic Genomic Profiling of Multiple Myeloma Patients

Introduction: We previously showed that ~10% of patients with multiple myeloma (MM) carry pathogenic or likely-pathogenic (P/LP) germline variants (PGVs) in known cancer predisposition genes, and that moderate/high-penetrance PGVs in DNA repair genes may be associated with MM risk and favorable response to alkylating chemotherapy. Increasingly applied in MM, genomic profiling by next-generation sequencing (NGS) can identify prognostically-relevant and therapeutically-actionable somatic mutations, but it may also incidentally unveil PGVs, which can have significant implications for patients and their families. We here test the diagnostic accuracy of a novel referral tool designed to flag likely PGVs warranting confirmatory germline testing among mutations identified in somatic NGS studies of 1161 MM patients from our institution. Methods: We retrospectively reviewed 1715 commercial NGS reports of 1161 MM patients treated at the Icahn School of Medicine at Mount Sinai (ISMMS) between 2015 & 2022 (targeted 448-gene somatic profiling panels performed by a single genomic testing company for clinical purposes on bone marrow aspirate samples). In collaboration with certified genetic counselors at our institution, we applied evidence-based criteria to develop a tool to flag P/LP variants of likely germline origin warranting a referral for confirmatory germline testing (“Likely PGVs”). The resulting referral tool flags variants detected in 35 well-established cancer predisposition genes at a variant allele frequency (VAF) of >10%, as well as 24 cancer predisposition founder variants at any VAF. Next, we tested the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of our approach for the detection of true PGVs by analyzing paired germline whole-exome sequencing (WES) in 382/1161 (33%) MM patients in the study cohort. Germline WES data were generated for research purposes using a clinically-validated pipeline for PGV detection and annotation. Results: 385 P/LP variants were reported in somatic NGS studies of 382 MM patients who had paired somatic/germline sequencing data. Of these, 38/385 (9.9%) were found to be true PGVs in confirmatory germline testing. True PGVs detected in more than one gene included nine APC, seven CHEK2, five BRCA1, three BRCA2, three ATM and three MUTYH variants. The referral tool flagged 41/385 (10.6%) of the cohort's somatic variants as likely PGVs, identifying 32/38 (84.2%) true PGVs. The six PGVs missed by the referral tool were detected in the following genes: one APC, one CHEK2, one MITF, one MUTYH, one NF1 and one SHDA variant. In contrast, the nine variants that were mislabeled as likely PGVs but were in fact somatic were: two ATM, two BRCA2, two TSC1, one CHEK2, one BRIP1, and one RET. With a variant allele threshold of >10%, the tool had a PPV of 78.0% and a NPV of 98.3% for the detection of true PGVs. The sensitivity and specificity were 84.2% and 97.5%, respectively. Among the 692/1161 MM patients who did not have confirmatory germline testing (67%), the proportion of variants flagged as likely PGVs was very similar to that of the paired somatic/germline group (77/696, 11.1%), and variants were detected in similar genes. Assuming a PPV of 78%, 60 of these variants could represent true PGVs. Conclusion: A novel tool designed to flag likely PGVs among mutations identified in somatic genomic profiling of MM patients had high sensitivity and specificity for the detection of true PGVs. 9.9% of all MM patients with paired somatic/germline testing were found to have a true PGV, validating the results of our prior study and once again highlighting the need to implement screening strategies in high-risk subgroups of MM patients, such as those with strong family history of cancer or prior personal history of cancer.

Cellular Mechanisms Associated with Suboptimal Immune Responses to Sars-Cov-2 Bivalent Booster Vaccination in Patients with Multiple Myeloma

Background: In response to the ever-evolving challenges posed by the antigenically diverse Omicron variants, the deployment of bivalent mRNA booster vaccines encompassing both the ancestral (WA1) and Omicron BA.5 spike components was initiated in fall of 2022. The real-world impact of these bivalent vaccines is largely unknown in patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster implementing cutting edge methodologies to identify patterns associated with continuing vulnerability in the current post-pandemic era. Methods: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assay. We measured spike specific T cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow cytometry-based T cell assays quantifying interferon-gamma (IFNg) production. In a subset of 38 patients, immune profiling was performed using high-dimensional flow cytometry. The frequencies of dendritic cells (DCs), B cells, Natural Killer (NK) cells and T follicular helper cells (TFH) were compared between responders and non-responders. Results: The serological effect of the bivalent vaccination in MM patients is nuanced with the overall cohort not demonstrating a significant increase post bivalent vaccination (median 196 AU/mL prior to bivalent to median 276 post bivalent p=0.11). The lack of antibody response was driven by patients undergoing anti-CD38 and anti-BCMA antibody therapy as compared to MM patients undergoing other therapies who demonstrated a significant increase in antibody levels post vaccination (p=0.021). Patients on anti-CD38 and anti-BCMA antibody therapy also did not significantly increase neutralizing capacity to WA1 (p=0.42) or BA.5 omicron strain (p=0.48) while MM patients on other therapies benefitted significantly, as illustrated by the increased neutralizing capacity of WA1 (p=0.024) and BA.5 (p=0.0055 Figure 1A). In addition to quantifying antibody responses, we profiled T cell responses using our published flow cytometry method using WA1 anti-S peptides as well as the QuantiFERON SARS-CoV-2 RUO assay (Qiagen). Spike reactive T cell responses significantly correlated with anti-Spike IgG levels in MM patients (r 2=0.49, p<0.001), WA1 neutralizing ID50 (R 2 = 0.6, p<0.01) and BA.5 neutralizing ID50 (R 2= 0.54, p=0.01) reinforcing the fact that T cell compensation is lacking in MM patients. We separated the lowest quantile (patients with Anti-S IgG <156 AU/mL) to delineate suboptimal producing patients compared to the remaining MM anti-S producing patients. Non-responders exhibited deficiencies in immune populations involved in robust antibody production including lower frequency of CD1c+ DCs (p=0.02 Figure 1B), B Cells (p<0.01 Figure 1B) and circulating CXCR3-CCR4+TFH cells (p<0.01 Figure 1B). Furthermore, immature NK cells were the predominant population in non-responders, while responders had more cytotoxic mature NK phenotype (p<0.01 Figure 1B) after bivalent vaccination. Conclusions: Our study highlights the varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairments in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate antibody and T cell responses to vaccinations. Our results indicate that the QuantiFERON SARS-CoV-2 assay could be deployed for clinical use bridging the need for suitable laboratory tests to measure SARS-CoV-2 specific T cell responses. Ongoing research will further investigate whether the compromised immune machinery and diminished T cell activity identified in this study can serve as discriminators for identifying MM patients at high risk of infectious complications stemming from anti-BCMA therapies. Such insights will aid in developing targeted strategies to address these challenges and improve patient outcomes.

Updated Analysis of a Prospective, Multicenter, Observational Study on the Prevalence of Type 1 Gaucher Disease in Patients with Multiple Myeloma

Introduction. Type I Gaucher disease (GD1) has been associated with increased cancer risk in the International Collaborative Gaucher Group (ICGG) Registry. Particularly, it is well recognized that the risk for multiple myeloma (MM) in affected individuals may be about 9 times higher than expected in the general US populations (Rosenbloom et al, 2022). Pathogenetic relationship between GD1 and MM is still unclear. It was hypothesized that accumulation of glucosylceramide in the macrophages leads to their activation and chronic immune stimulation favouring polyclonal gammopathies. In this scenario plasma cell clone selection could be promoted and subsequent MGUS/MM could derive from it. Diagnosis of GD1 is often inaccurate or delayed since symptoms and signs of disease are nonspecific or underhand. We designed a prospective study to investigate the prevalence of GD1 in a large MM patient population to provide substantial evidences to consider GD1 in the pathogenesis and management of MM. Patients and Methods. This is an observational, prospective, cross-sectional, multicentre study. Twenty-five Italian hematologic centers participated in this study. Due to the lack of data on the effective prevalence of GD1 in MM, the sample size has been determined considering clinically relevant a prevalence of the condition > 0.5% for defining as “high risk” the selected population. Considering an alpha error of 5% and a statistical power of 95%, approximately 1000 patients should beenrolled.. All MM patients are screened by Dried Blood Spots (DBS) sampling technique, that was centralized at the Istituto per la Ricerca e l'Innovazione Biomedica CNR-Palermo. All patients with a positive DBS test undergo genetic test to confirm the diagnosis of GD1. Primary end-point of the study is the prevalence of DBS test positivity in MM patients, which measures glucocerebrosidase activity in peripheral blood leukocytes (normal range 0.2-2.5 nMol/h/m). If the observed prevalence had been significantly higher than predicted, we would have tried to identify the population at higher potential risk of associated GD1. Results. We enrolled 902 patients with a median age of 68 years (range 37-90), 60% of which were male. No patients were Jews, 2 were Asian and 10 were Black, all others were Caucasian. Newly diagnosed and relapsed-refractory MM were 65% and 35%, respectively, whereas SMM and MM were 15% and 85%, respectively. Monoclonal component was IgG in 60%, IgA in 25%, light chain in 15%. Median Ferritin was 310 ng/ml (range 17-1236) and median Alkaline Phosphatase was 75 U/L (range 29-355). Descriptive data of DBS test, enzymatic activity and genetic tests are reported in the Table. In summary, DBS test was found positive in 12/902 (1.33%). Genetic test confirms GBA gene mutations, single heterozygous in 11 patients and double heterozygous in 1 patient. N370S and L444P loci accounted for 50% of GBA gene mutations found. Double-heterozygous patient started replacement therapy for GD1 with imiglucerase. Conclusions. After the enrolment of more than three quarter of the planned patients, the prevalence of DBS test positivity was higher than 0.5% (1.3%). Genetic test confirmed involvement of GBA genes, all but one in heterozygosity fashion. This study allowed to identify one patient with MM and GD1 requiring therapy for both diseases at the same time. The results of this study support further investigations on the implication of the GD1 in the pathogenesis and management of MM. More data on MM patient subgroups at higher risk of associated GD1 may be provided.

Cellular mechanisms associated with sub-optimal immune responses to SARS-CoV-2 bivalent booster vaccination in patients with Multiple Myeloma

The real-world impact of bivalent vaccines for wild type (WA.1) and Omicron variant (BA.5) is largely unknown in immunocompromised patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster, including neutralizing assays to identify patterns associated with continuing vulnerability to current variants (XBB1.16, EG5) in the current post-pandemic era. We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assays to assess inhibition of live virus. We measured spike specific T-cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow-cytometry based T-cell. In a subset of 38 patients, high-dimensional flow cytometry was performed to identify immune cell subsets associated with lack of humoral antibodies. We find that bivalent vaccination provides significant boost in protection to the omicron variant in our MM patients, in a treatment specific manner. MM patients remain vulnerable to newer variants with mutations in the spike portion. Anti-CD38 and anti-BCMA therapies affect the immune machinery needed to produce antibodies. Our study highlights varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairment in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate humoral and cellular responses to vaccination. National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), NCI Serological Sciences Network for COVID-19 (SeroNet) and The Icahn School of Medicine at Mount Sinai.

Co-Expression of GPRC5D, FcRH5 and BCMA Suggests That Targeting More Than One Cell Surface Marker May be a Viable Strategy in Relapsed/Refractory Multiple Myeloma (RRMM): Biomarker Results from the Phase I Study of Forimtamig, a GPRC5DxCD3 Bispecific Antibody

Background: Forimtamig is a GPRC5DxCD3 T-cell engaging bispecific antibody that induces T-cell mediated multiple myeloma (MM) cell killing and has shown promising clinical activity in a Phase I study (NCT04557150) in patients with RRMM (Carlo-Stella et al. ASH 2022). GPRC5D is known to be overexpressed in MM plasma cells (MMPCs), but little is known about its prevalence in relation to risk status and other identified MM targets like FcRH5 and BCMA. We developed a unique exploratory multiparameter flow cytometry panel to characterize GPRC5D, FcRH5 and BCMA expression on MMPCs (Lelios et al. Cyto 2023). We present data on the prevalence and co-expression of these targets, and their association with baseline patient characteristics and response to forimtamig. Methods: Baseline bone marrow aspirate samples were collected from patients who received intravenous or subcutaneous forimtamig during dose-escalation, and were analyzed with flow cytometry. Target expression was depicted as frequency of positive MMPCs and as receptor density (molecules of equivalent soluble fluorochrome). Patients were grouped by number of prior lines of therapy, ISS score, cytogenetic risk (high risk defined as presence of one or more of the following abnormalities: del(17p), t(4;14), t(14;16), and response to forimtamig (responder [≥PR] or non-responder [<PR]). All patients provided informed consent. Results: At data cut-off (June 15, 2023), 48/163 patients were biomarker evaluable (samples passing pre-defined quality standards and containing ≥100 acquired MMPCs). GPRC5D was detected at baseline in 46/48 (96%) patients, with most MMPCs expressing the target (median 95.6%; min-max: 0-100%). GPRC5D expression was more prevalent on MMPCs than FcRH5 (median 78.6%; min-max: 3.7-100%) and BCMA (median 40.3%; min-max: 0-100%; Figure). Interestingly, GPRC5D receptor density (median 20,612; min-max: 6495-208,072) was 47-100-fold higher and more homogenous than FcRH5 (median 436; min-max: 8-3921) and BCMA (median 206; min-max: 14-6657). Clinical response to forimtamig was not correlated with GPRC5D+ MMPC frequency or GPRC5D receptor density measured by flow cytometry ( Table). Percentages of GPRC5D+, FcRH5+ and BCMA+ MMPCs and receptor densities did not significantly differ between patient groups defined by ISS score, cytogenetic risk and number of prior lines of therapy. Patients previously exposed to BCMA-targeted agents (n=7) tended to have lower BCMA expression levels. GPRC5D and FcRH5 were co-expressed on 75.4% of MMPCs (min-max: 0-100%) followed by MMPCs co-expressing GPRC5D and BCMA (median 31.9%; min-max: 0-100%) or BCMA and FcRH5 (median 25.3%; min- max: 0-94.3%). In the majority of patients, double and triple negative MMPCs were rarely observed (0.5-6% of MMPCs expressed only one or none of the three targets across all analyzed patients). Conclusions: GPRC5D, FcRH5 and BCMA are highly prevalent across MM patient subgroups, including high-risk patients who may warrant targeted therapy approaches. Based on our data and previously published work for FcRH5- and BCMA-targeted T-cell bispecifics (Sumiyoshi et al. EHA 2021; Cortes-Selva et al. ASH 2022), target expression on MMPCs, as measured by flow cytometry, has not been associated with clinical response to the corresponding CD3 bispecifics, likely due to the high efficacy of T-cell mediated tumor cell killing. Co-expression of MM targets suggests that targeting more than one MM surface marker may result in double/triple hits on individual tumor cells. Our data support further testing of dual/triple MMPC targeting as a promising concept to promote fast and deep tumor clearance, and to achieve durable responses, while potentially avoiding clonal selection, in patients with RRMM. Further investigation is needed to understand the relevance of target expression differences measured by flow cytometry compared to other methods like immunohistochemistry, as well as to understand T-cell bispecific resistance mechanisms beyond target expression. Updated data will be presented.

A PREDICTIVE MODEL FOR ULTRA‐HIGH RISK IN PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA: A SINGLE‐CENTER STUDY IN CHINA

Introduction: Although novel therapies have significantly improved the prognosis of multiple myeloma (MM) patients, there are still a subgroup of MM patients experienced early relapse or disease progression. To early identify the patients with compromised prognosis in our center, the study aimed to establish a predictive model for ultra-high risk (UHR) in MM. Methods: A study from a tertiary hospital in western China analyzed the patients with newly diagnosed MM (NDMM) receiving bortezomib and/or lenalidomide-included regimens from January 2015 to June 2021. The last follow-up date was December 31, 2022. The patients were randomly divided (2:1) into training and validation cohorts, and the patients with progression-free survival (PFS) less than 12 months were defined as UHR. Univariate and multivariate Cox regression analyses were performed for clinical feature selection. Independent baseline factors were incorporated and scored to develop the predictive model. The UHR group was then defined beyond the optimal cut-off value of total scores. Results: A total of 314 MM patients were enrolled and 102 (32.5%) patients underwent autologous hemopoietic stem cell transplantation (ASCT). The median time of follow-up was 36.6 months. Disease progression and death occurred in 178 (56.7%) and 69 (22.0%) patients. The median PFS (mPFS) was 36.5 months, and the median overall survival (mOS) was not reached. The mPFS of patients stratified into R-ISS III, R2-ISS III, R2-ISS IV, IMWG high-risk and mSMART double/triple hit was 23.8, 25.6, 10.0, 17.2 and 10.5 months, respectively. Among 54 patients at UHR, less than half of them could be identified by the aboved stratification systems. In the training cohort, elevated LDH, hypercalcemia, 1q21 gain/amplification (1q+) and the presence of extramedullary disease (EMD) showed significant association with UHR in NDMM. Referring to the results, the UHR predictive model was developed as 5 points for elevated LDH, 6 points for hypercalcemia, 7 points for 1q+ and 10 points for EMD. Patients with total scores of more than 12 points can be predicated as UHR. This model identified UHR and non-UHR groups with significantly different mPFS (9.8 v.s. 37.5 months, HR = 3.24, 95% CI:2.09-5.03, P < 0.001) and 5-year OS (63.3% vs. 83.8%, HR = 2.92, 95% CI: 1.47-5.81, P = 0.002). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.77, and in the validation cohort, the UHR model kept good discrimination (AUC: 0.79) at the latest follow-up. Conclusions: In this study, elevated LDH, hypercalcemia, 1q+ and EMD were selected and scored to develop the UHR model for NDMM patients. At present, the extension of follow-up time and expansion of sample size in the validation cohort are still ongoing. External validity is also designed to verify the generalizability of the model. The research was funded by: Natural Science Foundation of Sichuan Province, China, Grant/Award Number: 2022NSFSC1299 Keywords: Risk Models, Multiple Myeloma No conflicts of interests pertinent to the abstract.

IL-32 is induced by activation of toll-like receptors in multiple myeloma cells.

Multiple myeloma (MM) is a hematological cancer characterized by accumulation of malignant plasma cells in the bone marrow. The patients are immune suppressed and suffer from recurrent and chronic infections. Interleukin-32 is a non-conventional, pro-inflammatory cytokine expressed in a subgroup of MM patients with a poor prognosis. IL-32 has also been shown to promote proliferation and survival of the cancer cells. Here we show that activation of toll-like receptors (TLRs) promotes expression of IL-32 in MM cells through NFκB activation. In patient-derived primary MM cells, IL-32 expression is positively associated with expression of TLRs. Furthermore, we found that several TLR genes are upregulated from diagnosis to relapse in individual patients, predominantly TLRs sensing bacterial components. Interestingly, upregulation of these TLRs coincides with an increase in IL-32. Taken together, these results support a role for IL-32 in microbial sensing in MM cells and suggest that infections can induce expression of this pro-tumorigenic cytokine in MM patients.

P-054: TRIM33 loss in Multiple Myeloma is associated with defective DNA repair and sensitivity to PARP inhibition

<h3>Background</h3> Chromosomal instability is a hallmark of Multiple Myeloma (MM) with most patients displaying cytogenetic abnormalities which can often act as prognostic indicators. Such abnormalities can arise due to defects in the DNA Damage Response (DDR). TRIM33 is an E3 ligase and transcription co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 is involved in PARP-dependent DDR and regulation of chromosomal stability. Here, we investigated the influence of TRIM33 loss in MM, focusing on its role in the DDR and whether this could be exploited therapeutically. <h3>Methods</h3> The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with survival, structural variants and common cytogenetic abnormalities. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells for in vitro studies. Protein expression and interactions were assessed by co-immunoprecipitation, western blotting and/or immunofluorescence. Clonogenic survival assays were used to assess response to Olaparib. <h3>Results</h3> Previously we identified a subset of MM patients with TRIM33 loss and identified that these patients exhibit significantly more chromosomal structural variants (deletions, inversions, duplications and translocations) (p<0.0001) and a significantly poorer overall survival (p<0.0001). Additionally, we have determined the frequency of common recurrent primary and secondary cytogenetic abnormalities for these patients. No recurrent primary cytogenetic abnormalities were associated with TRIM33 loss. However, high-risk secondary chromosome 1 aberrations were associated with loss of TRIM33, both del(1p) and gain(1q) (p<0.0001 and p=0.0474 respectively). In vitro, TRIM33 knockdown resulted in increased formation of 53BP1 foci and increased γH2AX expression (p<0.001) indicating unrepaired DNA damage typical of a DDR defect. Following induced DNA damage using 2Gy irradiation (IR), TRIM33 is recruited to chromatin within 5 minutes, with levels returning to basal by 30 minutes. The chromatin remodelling enzyme ALC1 is known to regulate sensitivity to PARP inhibition and TRIM33 is required for its timely removal from sites of damage. TRIM33 transiently interacts with ALC1 within 15 minutes of 2Gy IR. TRIM33 knockdown did not affect ALC1 expression. However, knockdown did sensitize MM cells to the PARP inhibitor Olaparib reducing the IC50 from 1.7µM to 780nM. <h3>Conclusion</h3> Here, we show that a subgroup of MM patients have TRIM33 loss and these patients have high-risk disease and poor outcome. We show that TRIM33 is recruited to damaged chromatin where it regulates ALC1 activity. Therefore, TRIM33 loss results in a DDR defect leading to chromosomal abnormalities. However, TRIM33 loss-associated DDR defects can be exploited therapeutically using Olaparib which is currently approved for the treatment of BRCA1/2 mutated breast and ovarian cancers.

Alterations of NK Cell Phenotype in the Disease Course of Multiple Myeloma.

Simple SummaryMultiple myeloma (MM) is a deadly cancer localized in the bone marrow, where changes can support progression and therapy resistance. This study examined the expression of numerous biological markers on natural killer (NK) cells in blood and bone marrow of patients with MM. NK cells play key roles in the innate immunosurveillance of MM, so we sought to identify biomarkers on NK cells that may be prognostic for patient outcomes and identify new therapeutic targets in these patients. Biomarker expression was compared on NK cells between MM disease stages and healthy donors, between blood and bone marrow, and associations with disease progression. The study shows that loss of certain biomarkers on NK cells may limit their anti-tumor function in MM patients, that several drug-targetable biomarkers are upregulated on NK cells, and that high expression of the biomarker, SLAMF7, may have prognostic potential to identify patients more likely to show rapid disease progression.Accumulating evidence demonstrates important roles for natural killer (NK) cells in controlling multiple myeloma (MM). A prospective flow cytometry-based analysis of NK cells in the blood and bone marrow (BM) of MM patient subgroups was performed (smoldering (SMM), newly diagnosed (ND), relapsed/refractory, (RR) and post-stem cell transplantation (pSCT)). Assessments included the biomarker expression and function of NK cells, correlations between the expression of receptors on NK cells with their ligands on myeloma cells, and comparisons between MM patient subgroups and healthy controls. The most striking differences from healthy controls were found in RR and pSCT patients, in which NK cells were less mature and expressed reduced levels of the activating receptors DNAM-1, NKG2D, and CD16. These differences were more pronounced in the BM than in blood, including upregulation of the therapeutic targets TIM3, TIGIT, ICOS, and GITR. Their expression suggests NK cells became exhausted upon chronic encounters with the tumor. A high expression of SLAMF7 on blood NK cells correlated with shorter progression-free survival. This correlation was particularly evident in ND patients, including on mature CD56dim NK cells in the BM. Thus, our NK cell analysis identified possible therapeutic targets in MM and a biomarker with prognostic potential for disease progression.

Activation of the ERK Pathway Drives Acquired Resistance to Venetoclax in MM Cell Models

Multiple myeloma (MM) is a hematological malignancy characterized by various genetic abnormalities including translocations involving the IgH gene at 14q32. Amongst these, t(11;14) is one of the most common translocations. Recent clinical data suggests a significant impact of Venetoclax, a small molecule inhibitor of BCL2, in this subgroup of MM patients, representing the first example of personalized medicine in MM and opening a wide range of research aiming at elucidating its mechanism of action. However, despite the initial positive response to the drug, a significant proportion of patients eventually develop resistance and relapse. To delineate the mechanisms that contribute to the development of an acquired drug-tolerant/resistance phenotype, we modeled the response to Venetoclax in 2 MM cell lines (KMS27 and KMS-12PE with IC50 of 35.47nM and 3.64nM, respectively). Whereas the vast majority of cells plated into 96-well plates were killed within a few days of exposure to a high dose of drug concentration, we detected a small fraction of viable, largely quiescent cells, which were expanded by culturing them in high doses of Venetoclax. We successfully generated 4 independent clones from each cell line, that were single cell-cloned with continued growth in the presence of high doses of Venetoclax. These clones labelled as drug-tolerant expanded persisters (DTEP) were investigated for the mechanisms driving drug tolerance and resistance against Venetoclax. First, we observed that altered expression of apoptotic regulators were associated with Venetoclax resistance in DTEP cells. We indeed observe a significant increase in the anti-apoptotic proteins MCL1 and BCL-XL in DTEP clones, which translated in our observation of improved sensitivity to MCL1 and BCL-xL inhibitors (S63845 and A-1155463 respectively). We performed both whole genome sequencing (WGS) and RNA-seq to evaluate if DTEP cells undergo transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprograming during development of acquired drug resistance. While, WGS analysis didn't show any significant differences between parental and resistant clones, transcriptomic analysis showed both shared and unique transcriptome signatures in the DTEP clones. Gene set enrichment analysis (GSEA) of the common significantly modulated genes in the resistant clones revealed that the genes belonging to the PKA-ERK-CREB pathway were significantly upregulated in resistant clones, while apoptotic genes were downregulated compared to parental cells. Western blot analysis confirmed activation of ERK and the downstream target cAMP response element-binding (CREB) gene in resistant clones; and importantly treatment with the ERK inhibitor U0126 rescued the resistance to Venetoclax, providing a synergistic activity in resistant clones but not in parental cells, with decreased cell viability and increased apoptotic cell death. To evaluate if the ERK pathway was also associated with intrinsic resistance to Venetoclax, we assessed a panel of 24 MM cell lines and then calculated Pearson correlation coefficients between the measured drug activity and individual gene expression levels (by RNA-seq) across all cell lines and subjected the resulting rank-ordered gene list to GSEA. This analysis showed that mechanisms driving the DTEP phenotype are different from those associated with the intrinsic resistance to Venetoclax. RNA processing and splicing pathways were strongly enriched, with high expression of these genes correlating with increased sensitivity. Moreover, among the genes correlated with a resistant phenotype, we observed that the gene G0S2 was significantly downregulated in the resistant cell lines. G0S2 is a tumor suppressor gene that binds and inhibits BCL2. Interestingly, we observed that while G0S2 is downregulated in MM compared to normal plasma cells, t(11:14) patients have a higher expression. We are now in the process of validating G0S2 in MM and its contribution to Venetoclax sensitivity in MM. In conclusion, we here provide evidences of molecular mechanisms of acquired resistance to Venetoclax with activation of the ERK pathway as one of the prime targets. Combining Venetoclax with ERK inhibitor may therefore prevent or overcome the acquired resistance to Venetoclax observed in MM patients. Disclosures Fulciniti: NIH: Research Funding. Munshi:C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BMS: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Long-Term Survival for Myeloma after Autologous Stem Cell Transplantation

Long-Term Survival for Myeloma after Autologous Stem Cell Transplantation

Outcomes of Multiple Myeloma Patients with Del 17p Undergoing Autologous Stem Cell Transplantation

Outcomes of Multiple Myeloma Patients with Del 17p Undergoing Autologous Stem Cell Transplantation

MM-203: Outcomes of Multiple Myeloma Patients with Del 17p Undergoing Autologous Stem Cell Transplantation

Context: High-risk (HR) multiple myeloma (MM) is a subgroup of MM patients that is variably defined. Patients with 17p deletion are considered HR and their optimal treatment approach has not been determined. Various strategies have been suggested to improve outcomes in MM patients harboring del 17p, including tandem transplantation. Objective: Evaluate the outcomes of multiple myeloma patients harboring 17p deletion that underwent autologous stem cell transplants (ASCT). Design: Retrospective study. Setting: Referral center in the US (Mayo Clinic). Patients or other participants: 116 MM patients with 17p deletion that underwent ASCT at Mayo Clinic. Patients were defined as 17p-deleted if they met the following criteria: If 50 cells in the bone marrow sample and 10 abnormal cells with the deletion were identified (20%) or if the bone marrow sample had between 20-50 total cells and 20% abnormal cells with the deletion were identified. We excluded patients that relapsed prior to ASCT, patients that underwent ASCT more than 12 months from the diagnosis, and patients that underwent tandem ASCT. Main outcome measures: PFS, OS. Results: There was no difference in the OS or PFS between patients that received VRd (bortezomib, lenalidomide, and dexamethasone) versus VCd (bortezomib, cyclophosphamide, and dexamethasone) induction. There was no OS or PFS difference between immunomodulatory drug (IMiD)-based and proteasome inhibitor (PI)-based maintenance, but there was a PFS advantage to patients that received combined maintenance (includes two novel agents). The OS and PFS of the entire cohort were NR and 29 months, respectively, similar to the outcomes in the single transplant arm in 17p-deleted patients that were recently published (EMNO2/HO95). Conclusions: The outcomes of our patients were similar to that of the single-arm ASCT in the EMN02 trial, and no difference in outcomes were found between patients that received VRd and VCd inductions, suggesting that tandem transplants may be considered for 17p-deleted MM patients treated with VRd induction. Dual novel agent maintenance therapy is important in improving outcome.

The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis.

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

IGLL5-BCL2L1 Rearrangement with Loss of BCL2 Dependency As Mechanism of Venetoclax Resistance in Multiple Myeloma (MM)

Background: Targeting the anti-apoptotic BCL2 protein in haematological malignancies has demonstrated significant anti-tumoral activity in a subset of multiple myeloma patients harbouring rearrangements involving the CCND1 and the immunoglobulin heavy chain enhancers (Eμ and α1/2). The mechanisms underlying the dependency of this subgroup of MM patients on BCL2 remains to be elucidated as well as the mechanisms of resistance to BCL2 inhibition with BH3 mimetic venetoclax. Methods and Results: Sorted bone marrow plasma cells from a cohort of t(11;14) myeloma patients treated with venetoclax were profiled through multi-omics single cell mRNA expression (scRNAseq), copy number profiling (scCNVseq) as well as chromatin accessibility with single cell ATAC-seq. Sequenced reads were aligned to hg38 reference genome. Samples were processed with CellRanger suite v3.0 and downstream analyses were realized with Seurat, Monocle, Signac, and Cicero R packages. Single plasma cells exhibited differential chromatin accessibility landscapes within and across individual patients as well as pre- and post-venetoclax with enrichment of MYC:MAX, RELA, IRF family, RUNX1/3 and ETS motifs. Integration of mRNA and ATAC data revealed a dynamic change of regulatory motifs across individual cell clusters with evidence of selective pressures driven by venetoclax treatment. Similarly mRNA profiling of the apoptotic genes pre- and post-venetoclax exposure showed loss of BCL2 and upregulation of MCL1 and/or BCL2L1 as well as loss of the BH3-only pro-apoptotic genes PMAIP1 and BCC3 in single cell clusters. mRNA levels mirrored open chromatin at the gene bodies and their respective promoter loci consistent with a direct transcriptional regulation. In a patient with several fold upregulation of the BCL2L1 transcript in the post-venetoclax sample (Figure A-B), scATACseq identified a gain in the chromatin accessibility mapping to a genomic region centromeric to BCL2L1 locus on chromosome 20 (chr20:31,617,200-31,619,900). Single cell CNV analysis identified a 5q loss (chr5:142,400,001-156,240,000) mapping to NR3C1 locus explaining with the clinical resistance to dexamethasone. Importantly scCNV also revealed a copy number gain mapping to the same locus with the newly acquired chromatin accessibility on chromosome 20. Mate-pair analysis of the sequencing reads identified the potent IGLL5 B-cell enhancer on chromosome 22 (chr22:22,960,001-22,980,000) as the mate partner juxtaposed the BCL2L1 locus (Figure C). This finding explains the robust upregulation of BCL2L1 mRNA observed in this patient and the shift in BCL2 dependency detected by ex vivo BH3 sensitivity profiling. Of note, while scCNV analysis also depicted a gain in 1q21 (chr1:149,940,001-169,980,001) MCL1 locus at the time of venetoclax resistance the acquisition of t(20,22) shifted the plasma cells dependency to BCL-xL rather than MCL1. This finding was corroborated by the plasma cells ex vivo resistance to dual BCL2 and MCL1 inhibition. Conclusion: Dynamic single cell epigenome and transcriptome profiling of pre- and post-venetoclax of primary plasma cells identified a de novo translocation driving BCL-xL transcription with the IGLL5 B-cell enhancer. This demonstrates that in addition to canonical TF-promoter regulation, restructuring of immunoglobulin regulatory sequences (i.e., enhancers) can also drive aberrant malignant circuitry endowing resistance to anti-BCL2 agents. Figure. Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Bahlis:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Lack of a Spleen Signal on Diffusion Weighted MRI Is Associated with High Tumor Burden and Poor Prognosis in Multiple Myeloma

▪Background: Whole body medical imaging is an integral component of Multiple Myeloma (MM) evaluation as it reveals bone disease and/or focal lesions. While established imaging techniques, such as PET-CT, are powerful in delineating focal pathologies, they suffer from low specificity when it comes to assessment of diffuse bone marrow (BM) signals. Especially in patients treated with G-CSF, discrimination between malignant and non-malignant BM signals is barely possible. Diffusion-weighted MRI with background suppression (DWIBS) is a novel functional MRI technique which measures water diffusion in vivo. Basically, diffusion of water is more restricted in tissues with high cellularity, making DWIBS a promising tool to investigate malignant diffuse signals. Initially, we searched for an internal reference to classify the level of BM infiltration. We selected the spleen, since it is the abdominal organ with the highest restriction on DWIBS. Unexpectedly, we observed significant heterogeneity in the spleen signal itself including a subgroup of MM patients with total loss of the signal on DWIBS. Perplexingly, these patients suffered from high tumor burden and poor outcome. Here, we report on our strategy to elucidate this phenomenon and to evaluate its clinical value.Methods: We investigated 295 transplant-eligible newly diagnosed MM patients and 72 cases with monoclonal gammopathy of undetermined significance (MGUS). This study was approved by the institutional review board (#205415). DWIBS was performed on a 1.5 Tesla Philips Achieva scanner. The spleen signal was assessed on DWI and ADC maps by two experienced investigators in consensus read. The Kaplan-Meier method was used for survival analyses. Molecular characterization included fluorescence in situ hybridization, and gene expression profiling of CD138-enriched plasma cells (PCs). Differential gene expression was performed using a threshold of 2-fold expression difference. Wilcoxon or Fisher's exact tests were used to compare the median of a continuous variable or the distribution of discrete variables across groups, respectively. Correlation coefficients were determined using Spearman's rank correlation.Results: Absence of the spleen signal on DWIBS was a frequent phenomenon in MM, seen in 71/295 (24%) patients. In all of these patients asplenia was excluded using alternative imaging techniques. Lack of a signal was highly positively associated with tumor-load parameters, such as the degree of BMPC infiltration (P=1x10-10) or International Staging System (ISS) 3 (P=0.0001). In contrast, it was not associated with age, gender, or the tumor progression markers gain(1q) and del(17p). Patients with absence of spleen signal experienced unfavorable outcome (hazard ratio of 1.8 for PFS and OS). In order to further investigate the biological underpinnings of this phenomenon we performed a differential gene expression analysis of purified CD138 MM cells. No differentially expressed genes were found between patients with and without spleen signal, suggesting that the absence of the spleen signal mainly reflected increased tumor burden rather than specific tumor features. As a proof of concept, we addressed the spleen signal in individuals with MGUS, and longitudinally in MM patients who presented with absence of the spleen signal at diagnoses. Indeed, in all 78 individuals with MGUS the signal was preserved, and the majority of MM patients showed re-appearance of the spleen on DWIBS during treatment as the tumor burden declined. Interestingly, re-appearance of the spleen was helpful to distinguish between malignant and non-malignant hyperintensities in the BM, making the spleen signal a promising parameter for MM follow-up investigations.Conclusions: Due to the low frequency of abnormalities affecting the spleen, this organ is often overlooked during abdominal examinations using cross-sectional imaging techniques. Here we show that the spleen signal on DWIBS provides clinically useful information on tumor burden in MM. Moreover, it opens the avenue to distinguish between malignant and reactive BM hypercellularity on imaging in patients receiving treatment. Our observation strongly supports the hypothesis, that the spleen signal is suppressed by a high BMPC involvement. Yet, the reasons for this phenomenon are still elusive. [Display omitted] DisclosuresRoy Choudhury:University of Arkansas for Medical Sciences: Employment, Research Funding. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:Dana Farber Cancer Institute: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Celgene: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; Multiple Myeloma Research Foundation: Other: travel stipend. Davies:Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Senescent CD8+ T Cells in Myeloma Patients: Implications for Cellular Therapies

▪Background: For autologous engineered T cell therapies such as CAR-T cells, the characteristics of the input donor-derived T cells impact the final T cell product. Early less-experienced T cells are thought to generate a T cell product with optimal functions whereas, terminally differentiated T cells, displaying features of cellular senescence, are unlikely to generate an ineffective product. An increase in senescence within the CD8+ T cell population has been described in multiple myeloma (MM) (Leukemia 2016;30:1716). To better understand the challenges that may be encountered in the production of engineered T cells in MM, we evaluated the overall T cell frequency and features of senescence in the CD8+ T cells in the peripheral blood of 14 MM patients. For those patients who underwent stem cell mobilization (n = 11) we assessed the impact of this procedure on the characteristics of the T cells collected.Methods: Peripheral blood samples were collected during treatment from patients in steady-state. Patients who subsequently underwent stem cell mobilization (G-CSF/plerixafor or Cyclophosphamide) as part of their clinical care and provided a second blood sample on the day of stem cell collection (mobilized sample).PBMCs were isolated and characterized by flow cytometry using the BD ACCURI C6 flow cytometer. Staining of surface antigens was performed by using fluorescently labeled CD3, CD8, CD28, CD57, and KLRG. Senescent T cells were defined as CD3+/CD8+CD28-/CD57+, and also as CD28-/KLRG+. Statistical analyses were performed with MedCalc.Summary of Results: Steady-state samples were collected from 14 MM patients (10M, 4F) with a median age of 66.5 years (range, 52-82) and a median time from diagnosis of 7.5 months (3-72). All patients had been treated with a proteasome inhibitor, an IMiD, and dexamethasone, and 4 were receiving daratumumab. At the time of sample collection, disease response was variable with 5 PR, 7 VPGR, 1 CR, and 1 refractory disease.In the steady state PBMC, CD3+ T cells were 49.1% and CD3+/CD8+ 5.1%. Median numbers of CD28-/CD57+ and CD28-/KLRG+ T cells within the CD8+ population were 24.1% (2.5-74.4) and 31% (2.9-73%) respectively. Two subgroups of MM patients were observed. Four patients had a significantly increased frequency of senescence within the CD8+ T cells (53-74% CD28-) and 10 had a much lower frequency of senescence (2.5%-25.9%). The group with elevated senescence within the CD8+ T cells included the oldest patient in the cohort (82 years), the patient with the longest duration of disease (6 years), a heavily pre-treated patient on daratumumab with severe lymphopenia, and a patient whose myeloma was a post-transplant lymphoproliferative disorder occurring a year after heart transplant on chronic immunosuppression with tacrolimus and prednisone.Following mobilization, the percentage of T cells within the PBMCs dropped significantly from 49.1% to 8.3%. Mobilization particularly impacted the CD8+ T cell population which dropped from a median 5.4% of PBMCs in the steady-state sample to 0.8%. In the paired samples, we did not observe a consistent impact on frequency of CD28- or CD57+ (senescent) cells within the CD8+ T cells (19.1% vs 21.1%).Conclusions: A subset of MM patients exhibited a significant increase in markers of senescence within the peripheral CD8+ T cells. Older patients, those on chronic immunosuppression, and those treated for an extended time appeared to be at increased risk. T cells from such patients may be suboptimal for manufacture of engineered T cell therapies. Further, stem cell mobilization resulted in a significant decrease in T cell frequency, particularly in CD8+ T cells although senescence markers were not consistently changed. Thus, mobilized blood collections may not be optimal starting material either. DisclosuresLee:Unum Therapeutics: Research Funding. Ma:Tufts Medical Center: Patents & Royalties: Patent: 9593332.