The hypereosinophilic syndrome (HES) is a group of rare disorders characterized by persistently high peripheral blood eosinophiles (≥ 1.5x109/L), and related signs or symptoms of organ involvement without secondary causes. Eosinophilia with recurrent genetic abnormalities (PDGFRA/B, FGFR1) comprises a minority of these patients. In this report, we aimed to point out a case with 4q12 deletion whose diagnosis and treatment were delayed for quite a while. The patient was followed for bronchial asthma for a long time and the recognition of hypereosinophilia yielded a suspicion for HES / Chronic eosinophilic leukemia (CEL). During the initial part of his diagnostic evaluation, there was an unawareness of the cryptic deletion which was a target for tyrosine kinases. The symptoms resolved and complete cytogenetic response was achieved with 100 mg imatinib continuing for 57 months.

Demagnetization of permanent magnet machines is a fault that evolves over time leading to an eventual catastrophic failure. This condition is of particular interest in renewables, with emphasis on remote applications such as offshore wind, tidal and wave energy harvesting. Moreover, the demagnetization fault can be either uniform or non-uniform. The strong majority of research papers are focusing on non-uniform demagnetization and typically study the case of a single faulty magnet. This is because, the uniform demagnetization does not create harmonics that can be detected with spectral methods. Despite the above, the study of single magnet demagnetization studies have been criticized due to being unrealistic to describe most practical cases where permanent magnet machines are subjected to demagnetization due to overloading and/or overheating conditions and leading to uniform demagnetization. This paper sheds some light into such theories, while studying a, realistically induced in the lab, demagnetization case of a direct-drive C-GEN permanent magnet generator.

Purpose/Objective(s)The COVID19 pandemic required radiation oncologists (ROs) to consider shorter treatment courses to minimize patient and staff exposure and conserve healthcare resources. Hematologic ROs adopted hypofractionated radiation therapy (hRT) regimens according to guidelines published by the International Lymphoma Radiation Oncology Group (ILROG). We report for the first time the preliminary efficacy and toxicity of these novel hypofractionated regimens in the treatment of hematologic malignancies.Materials/MethodsWe conducted a multicenter, multinational retrospective study under the direction of the ILROG. All patients receiving hRT according to ILROG guidelines from 1/1/2020 to 8/31/2020 were included. Patient and treatment details were abstracted from separate institutional databases. Toxicity was graded using CTCAE v5.0.ResultsNinety-three patients from 4 institutions treated with 114 RT courses were included. Patient and treatment details are displayed in Table 1. Median follow up for the cohort was 179 days, and 77 patients (82%) were alive at last follow up. Maximal toxicity experienced by patients included Grade 1 (n = 16), Grade 2 (n = 1) and Grade 3 (n = 1) toxicities. Of 80 sites with response assessment within the RT field, 69% of patients achieved a complete response (n = 55), 20% partial response (n = 16), 9% stable disease (n = 7), and 2% progressive disease (n = 2). No COVID19 infections during or after RT have been documented in this patient cohort.ConclusionHRT according to ILROG guidelines resulted in low rates of acute toxicity and reasonable short-term treatment efficacy. Longer follow up and comparison with control groups is needed to draw more definitive conclusions and will be presented at the Annual Meeting.

Relapsed or refractory central nervous system lymphoma (CNSL) after high-dose methotrexate-based therapy is a challenging clinical entity with limited treatment options. Salvage systemic therapy and whole brain radiation therapy (WBRT) are commonly employed but are associated with significant toxicity and poor outcomes. Prior studies have shown that primary CNSLs frequently exhibit increased expression of PD-L1, and immune checkpoint inhibitor (ICI) therapy may be active in this setting. We assessed the safety and efficacy of concurrent ICI therapy and hypo-fractionated stereotactic radiation therapy (SRT) in patients with relapsed or refractory CNSL.We retrospectively analyzed the records of patients with relapsed or refractory CNSL treated with SRT + ICI at our institution from 2017 to 2020. ICI therapy included pembrolizumab 200 mg IV q3weeks or nivolumab 3 mg/mg IV q2weeks. Linac-based SRT was delivered in 5 daily fractions of 5 Gy. CTVs included all grossly enhancing disease on T1 weighted post-contrast MR scans, with 3 mm uniform PTV expansions. Patients were followed for symptomatic and radiographic response. Progression-free survival (PFS) and overall survival (OS) were calculated via Kaplan-Meier analysis.Seven consecutive patients with relapsed or recurrent CNSL were analyzed: 6 with primary CNSL, 1 with secondary CNSL. Patients presented at a median age of 72.2 years (range: 40.9 - 89.9) after a median of 2 prior courses of systemic therapy. Five patients had a solitary intracranial lesion and 2 had multiple lesions. None had active extracranial disease. Median pre-SRT Karnofsky Performance Status (KPS) was 60 (range 50 - 90). All 7 patients completed SRT and received concurrent and adjuvant ICI therapy. Median follow-up after completion of SRT was 9.1 months. Acute toxicities of SRT and ICI therapy were minimal, including fatigue (3 patients), diarrhea (2), and adrenal insufficiency requiring oral steroids (1). Five patients (71.4%) achieved symptomatic improvement after SRT including 2 patients (28.5%) with a clinical complete response (CR). Median post-SRT KPS was 70 (range: 60 - 100). Six patients had follow-up imaging, and all had an objective response including 3 (50%) with a radiographic CR. The median time to best radiographic response was 2.0 months (range 0.3 - 3.7 months), and all treated lesions remained controlled on all subsequent imaging. Three patients (42.9%) experienced out-of-field intracranial recurrence. Median PFS was 7.7 months (95% CI: 4.0 - 11.4 months) and median OS was 12.6 months (95% CI: 7.5 - 12.7 months) post-SRT.Among patients with relapsed or refractory CNSL, SRT + ICI appears to be well tolerated and offers excellent symptomatic and radiographic local control. Further studies are needed to identify appropriate candidates for salvage SRT + ICI, and to evaluate the clinical efficacy and neurocognitive impact of this approach.

<h3>Purpose/Objective(s)</h3> Painful osteolytic bone lesions are common in patients with multiple myeloma (MM). Radiotherapy (RT) is effective in providing pain relief from MM bone lesions in over 80% of patients. There is no consensus as to the most effective dose or fractionation for palliation. Shorter courses of RT are not only more convenient for patients and their families, but they also have less impact on timing of systemic therapies. There is precedent for using 2 Gy x 2 for palliation of lymphomas, which have similar radiosensitivity to myeloma. The primary objective is to determine whether treatment with 2 Gy x 2 to painful myeloma bone lesions achieves patient-reported pain reduction comparable to historical controls at 4 weeks. Secondary objectives will assess QOL endpoints, use of analgesia and time to pain relief, and duration of pain relief. <h3>Materials/Methods</h3> Patients who consent to participation will complete quality of life and pain questionnaires (Brief Pain Index, EORTC QLQ-BM22, and EORTC QLQ-C30) prior to treatment and at 2,4,8 weeks and 6 months following treatment. Pain response, as defined by the international consensus on palliative RT for bone metastases, will be measured based on BPI and daily oral morphine equivalent. Reirradiation at standard dose can be considered at ≥4 weeks following initial treatment for indeterminate pain response or pain progression. Cytogenetics and International Myeloma Working Group risk stratification and response criteria are recorded, when available, but are not required for patient participation. <h3>Results</h3> This trial, supported by ILROG, has opened at 7 institutions with one more in process of opening. Prior to COVID, accrual was 1.5 patients per month. Since COVID, enrollment has been at 0.7 patients per month. A total of 18 patients have been accrued. The median age of patients accrued is 65.5 years with 7/18 female patients. Fourteen patients are Caucasian. Twelve patients have an ECOG performance score of 1-2. Thirteen patients had pain response captured at 4 weeks following RT. Of the 5 patients that did not complete questionnaires at 4 weeks post-RT, 2 expired, 1 was lost to follow-up, 1 had a missed evaluation and 1 had pain progression). The most common site of treatment was the shoulder (4/18). <h3>Conclusion</h3> This ongoing prospective trial in palliation of multiple myeloma bone lesions is feasible and able to accrue at multiple institutions and will provide valuable information as to the role of low-dose RT in this population.

Multiple myeloma (MM) is characterized by a wide heterogeneity at both clinical and genomic level. Two major subgroups of MM have been identified: hyperdiploid MM (HMM) characterized by the trisomy of eight odd number chromosomes (3, 5, 7, 9, 11, 15, 19, and 21); and the non-hyperdiploid (NHMM) often associated with hemizygous deletion of chromosome 13, translocations involving the immunoglobulin heavy chain gene and copy number alterations (CNA) in other chromosome regions such as 1q, 6q, 8p, and 16q. Although, aneuploidy is a hallmark of MM, the sequence of occurrence of these complex genomic rearrangements during the MM development is unknown. Here we have studied, utilizing large datasets, when and in what sequence does each of these genomic events occurs, in both MM subgroup.We performed copy number profiling on 336 CD138+ MM cells from newly diagnosed patients and validated the results on another set of 103 samples. All patients were uniformly treated and have clinical data with median 6 years of follow up. We have used both cytoscan array as well as whole genome sequencing, as needed for the analysis. We have analyzed the data using dual quantile normalization, residual variability removal and segmentation using HG19. For each sample than we have extracted kernel smooth over a window of genomically contagious log2 ratio as smoothed signal. Clonality was then assessed by using deviation from two copies. We have also calculated clonality occurrence index which considers number of patients with clonal change for specific chromosome or cytoband.Our initial results showed that even though copy number gains were usually clonal and early events, while deletions, except 13q, were subclonal and also late events. Focused analysis on HMM samples showed that chromosome 15 is likely the first event with detected clonality in 82% of the cases, followed by occurrence of trisomies involving chromosomes 9 and 19 with clonality observed in 70 and 68% of the cases, respectively. For trisomies involving these three chromosomes, the clonality index was 42, 35 and 35 respectively, suggesting an early hyperdiploid events. Trisomies involving chromosomes 3, 5 and 11 occur subsequently and have clonality index of 25. Finally, chromosomes 7 and 21 trisomies are late events with clonality index of 15. This information is confirmed in an independent data set.In NHMM deletion 13 is a frequent and early clonal event while the other chromosomal aberrations such as del 1p, del 6q and del 17p are usually subclonal and hence late events. Del 13 is also observed in more than 25% of HMM patients where the clonality for del 13 was higher in the HMM chromosomes (chr 9, 15 and 19) compared to del13 (89% vs 65%, respectively) indicating that HMM occurs before del13 in these patients. Monosomy 14 is observed in 10% of patients and usually occurs at the same time with deletion 13, differently from other deletions such as 16q which is likely to happen after del13. Our analysis also showed that clonal or subclonal events on different regions across the genome actually carries much more information than just the global copy number data, e.g . clonality of chromosome 13 and chromosome 17 carry poor prognosis (p< 0.006, p <0.00026, respectively) while subclonality does not.Our decomposition analysis shows the potential order of occurrence of aneuploidy in MM and provides a new understand of the development of disease. This information now can be utilized to consider various genes resident within the early and late event chromosomes for their potential role in evolution of the disease. DisclosuresRichardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Moreau:Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Consultancy, Honoraria; Amgen: Honoraria; Millennium: Consultancy, Honoraria; Onyx Pharmaceutical: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Consultancy, Honoraria. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Attal:Sanofi: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; JANSSEN: Consultancy, Research Funding.

In these studies, we investigated the role of Pembrolizumab (anti-PD-1)to enhance the function of myeloma-specific CD8+ cytotoxic T lymphocytes (CTL) and maintain their antigens-specific CTL activities against multiple myeloma cells. Bone marrow or peripheral blood mononuclear cells (BMMC, PBMC) from healthy donors or MGUS, smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (MM), relapsed MM, relapsed/refractory MM patients (N=5, respectively) were evaluated for expression of immune checkpoints that regulate antigen-specific CTL development and function. Active MM patients had higher levels of CD138+ tumor cells as compared to MGUS and SMM patients. PD-L1 and PD-L2 expression levels on myeloma cells were also higher in active MM patients than healthy donors. CD4 Treg (CD3+CD4+CD25+FoxP3+), but not CD8 Treg (CD3+CD8+CD25+FoxP3+), frequencies were highest in both BMMC and PBMC from MM patients. PD-L1, but not PD-L2 nor PD-1, was expressed on the CD4 Treg in BMMC and PBMC from MM patients. G-type MDSC (CD11b+CD33+HLA-DRlowCD15+), but not M-type MDSC (CD11b+CD33+HLA-DRlowCD14+), was increased in MM as compared to MGUS, SMM or healthy donors. The MDSC from active MM patients also had high levels of PD-L1 and PD-L2 expression. BMMC and PBMC from newly diagnosed MM patients (N=3) treated in vitro with Pembrolizumab enhanced the CD3+ T cell proliferation as compared to the untreated group. Interestingly, Pembrolizumab treatment induced proliferation of T cells expressing immune agonists (OX40, GITR) beneficial for cell function and immune checkpoint (LAG3) involved in down-regulating T cell activities. XBP1/CD138/CS1 antigens-specific CTL (XBP1/CD138/CS1-CTL) generated in vitro with a cocktail of immunogenic peptides [heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV), and native CS1239-247 (SLFVLGLFL)] displayed increased proliferation of CD8+ CTL expressing the CD28 co-stimulatory and CD38 activation markers upon treatment with Pembrolizumab. Importantly, the XBP1/CD138/CS1-specific central memory and effector memory CTL subsets increased the cell proliferation, following treatment with the PD-1-specific antibody. In addition, XBP1/CD138/CS1-CTL treated with Pembrolizumab demonstrated increased cytotoxic activity and Th1-specific cytokines production against U266 myeloma cells. Thus, these studies suggest the potential benefit of Pembrolizumab (a-PD-1) therapy in MM patients to enhance XBP1/CD138/CS1 antigens-specific memory CTL proliferation and boost their anti-tumor activities through blockage of the PD-1 specific immune checkpoint specifically expressed on antigens-specific CD8+ CTL. These data provide the framework for an immunotherapeutic strategy encompassing a combination of multipeptide-based cancer vaccine with PD1-specific antibody to enhance the antigens-specific T cells efficacy and promote stronger and long-lasting CD8+ CTL immune responses in myeloma patients. DisclosuresBae:OncoPep: Other: Scientific Founder. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

IntroductionImmunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind to the CRL4CRBN ubiquitin ligase and trigger proteasomal degradation of IKZF1/3 and cytotoxicity of multiple myeloma (MM) cells. Previous studies show that downregulation of CRL4CRBN abrogates sensitivity to IMiDs in MM cell lines. Clinical studies, show that IMiDs achieve better clinical responses in MM patients when combined with proteasome inhibitor bortezomib, which would in principle inhibit proteasomal degradation of IMiDs-targeted proteins. Moreover, some MM cells are resistant to IMiDs treatment despite harboring high CRBN expression levels. Thus, the molecular mechanisms of intrinsic resistance to IMiDs are not yet fully understood. In this study, we found that dual roles of IMiDs in activating CRL4CRBN and a mechanism of action for combination therapy of IMiDs with bortezomib. We also show that non-canonical NF-κB pathway, independent of CRBN, is involved in the IMiDs resistance in MM.Methods and ResultsTo study the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells, using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. Twenty-eight genes and one miRNA were identified which were associated with resistance to IMiDs. All six sgRNAs targeting CRBN were also identified, consistent with CRBN as the direct target of IMiDs and required for their MM cytotoxicity. Among these validated genes, 7 of 9 components of the COP signalosome (CSN) subunits were found to be required for MM cell sensitivity to IMiDs. Deletion of any of these CSN subunits in MM cells led to a dramatic decrease of CRBN protein level thereby inhibiting IKZF1/3 degradation induced by IMiDs.To investigate whether the CSN regulates CRBN level through the ubiquitin-proteasome pathway, we carried out ubiquitination assays and found that CRBN can be degraded by proteasomes via cullin E3, rather than CRL4, ubiquitin ligase. Moreover, we identified the interaction of CRBN with SCFFbxo7 complex, and demonstrated that CRBN interacted with both CRL4 and SCFFbxo7 with distinct domains. To determine if CRBN is a SCFFbxo7 substrate, we examined the ubiquitination and degradation of CRBN after altering SCFFbxo7 complex. CRBN, when unbound to CRL4, is ubiquitinated by SCFFbxo7, followed by proteasomal degradation. Importantly, deletion of Cul1 or Fbxo7 completely restored the CRBN protein in CSN2 or CSN6 knockout MM cells to levels found in wild type cells, as well as re-sensitized these CSN mutant MM cells to pomalidomide-induced cytotoxicity.Since CRBN protein turnover is controlled by a SCF ubiquitin ligase, we next tested whether proteasome inhibitor, in addition to their inherent anti-MM activities, might similarly enhance IMiDs-induced MM cell cytotoxicity. As expected, sequential treatment with bortezomib and pomalidomide primed MM cells to pomalidomide-induced downregulation of IKZF3 and cell death, associated with synergistic cytotoxicity. We also found that lenalidomide potently inhibited the ubiquitination of CRBN, which was abrogated by loss of lenalidomide binding on CRBN, downregulation of Fbxo7, or Cul1 expression. These data suggest that IMiDs stabilize the CRBN protein both by shifting its assembly with the SCF and CRL4 complexes and by directly blocking its ubiquitination and degradation targeted by SCFFbxo7.From the genome-scale CRISPR-Cas9 knockout screening, we also found that knock-out of TRAF2, a member of the TNF receptor associated factor protein family, conferred resistance of MM cells to IMiDs treatment, without effecting CRBN level and degradation of IKZF1/3 induced by IMiDs. TRAF2 -KO MM cells displayed increased processing of p100 NF-κB2 protein to p52, resulting in hyperactivation of the non-canonical NF-κB pathway. This finding suggests that the non-canonical NF-kB pathway may be involved in the intrinsic resistance of MM to IMiDs.ConclusionsTaken together, our findings therefore identify novel mechanisms of IMiDs resistance, involving the regulation of CRBN and the non-canonical NF-κB pathway, and provide a mechanistic framework to inform combination therapy strategies to overcome IMiDs resistance in MM. DisclosuresZhou:WuXi AppTec Inc.: Employment. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Anderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees.

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.

Multiple myeloma (MM), a plasma cell malignancy, is associated with immune dysfunction influenced by both direct and an indirect interaction between MM cells and its bone-marrow (BM) microenvironment. The most prominent immune deficiency lies in the humoral immune response, which manifests as low serum uninvolved immunoglobulin (UIg) levels, lack of responses to vaccination, and consequent infectious complications. The cellular and molecular processes driving suppressed normal B cell immunity remains unclear. Therefore, we investigated the hypothesis that observed low UIg levels and immune dysfunction are due to alterations in the developmental stages of the B cell-compartment with associated T and B cell subset alterations in MM BM (N=46) and peripheral blood mononuclear cells (PBMC, N=50) along with normal BM (N=17) and PBMC (N=33). We observed that B2 cells were significantly lower (42.7±4.3, p<0.05) and B1a cells were significantly higher (20.1±3.1, p<0.05) in myeloma BM compared with normal BM B2 cells (57±3.7) and B1a cells (10.5±2.5). Utilizing 12-color flow cytometry we integrated B cell-developmental stages in BM samples from normal donor) smoldering (SMM) (n=4), newly-diagnosed (NDMM) (n=10) and relapsed MM patients (RRMM) (n=6). We observed an altered B cell-development in BM with significantly lower proportion of pro-B cells (CD38+, CD34+, CD10+, CD19+ and IgM-) with renewal capacity in SMM (12±3.1; p<0.05), NDMM (19.2±4.9;) and RRMM (3.1±1.6; p<0.05) compared to normal BM (25.1±3; N=3) suggesting that aberrations observed in B cell development may contribute to suppressed UIG in MM. Similarly, we observed that B2 cells were significantly lower (45.2±5.8, p<0.05) and B1a cells were significantly higher (30.3±5, p<0.05) in myeloma PBMC compared with normal PBMC B2 cells (69.6±2.9) and B1a cells (8.4±2). We next analyzed the impact of uniform treatment and response on B cell subsets in patients enrolled on IFM-DFCI 10-106 study. B cell subsets in PBMCs were evaluated at diagnosis and then following achieving complete response (CR) in 14 patients and those not achieving CR in 18 patients. We observed a significantly higher numbers of B2 cells at diagnosis (63.6±4.6l vs. 42.1±3.9; p<0.05), and significantly lower numbers of Bregs (19.8±2.6, vs. 30.4±2.6; p<0.05), IRA-B cells within B1a cells (8±2.2 vs. 32.8±6; p<0.05) and MZ cells within B2 cells (10.8±2.7 vs. 29.6±5; p<0.05) in patients achieving CR versus those not achieving CR respectively. The absolute B2 cell-numbers were also significantly higher (p<0.05) in patients achieving CR (123,872±29,226 cells/ml) versus those not achieving CR (33,174±17352 cells/ml). Importantly, the UIg levels were significantly higher (p<0.05) at diagnosis, in patients with higher B2 cells (682±108 mg/dL) compared with those with lower B2 cells (392±24 mg/d). Moreover, patients with higher B2 cells had longer EFS (115±28 weeks) compared to the patients with lower B2 cell numbers (82±15 weeks). In the T cell-compartment, amongst CD4 cells, Th2 cells were significantly higher (73±6.1) (p<0.05) in patients achieving CR compared with those not achieving CR (47.7±5.9); and amongst CD8 cells, PD-1 expressing CD8+ cells were significantly higher (63.2±8) (p<0.05) at diagnosis in patients achieving CR compared with those not achieving CR (39.5±6.1). We also observed that PD-1 expressing CD8+ cell numbers were significantly higher (p<0.05) in MRD-negative patients (68.4±11.3) compared with MRD-positive patients with (56.8 ± 8.5) or without achieving CR (35.5± 6.8). These results highlight possible B cell impact on immune T cell response in myeloma. Interestingly, our in vitro studies confirmed the above results when we activated normal PBMC via anti-CD3 antibody in the presence or absence of B cells, we observed that the absence of B cells significantly inhibited interferon-producing T cells compared to control (by 43%; p<0.05). In conclusion, we report a cellular explanation for observed suppression of B cell immunity in myeloma, specifically, suppression of uninvolved immunoglobulins. Here, for the first time we report significance of the B cell-subset in MM with possible impact also on T cell subsets as well as patients outcome. The observed relationship between B cell subset changes and ability to achieve complete response is being investigated in a large cohort of patients as potential bio marker of response and survival. DisclosuresRichardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Oncopep: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.