Plasmacytoma Research Articles

A Case of Extramedullary Plasmacytoma in Both Maxillary Sinuses

Plasmacytoma is a rare hematolymphoid malignancy. The three types of plasmacytoma, according to clinical features and morphological characteristics, include solitary bone plasmacytoma, extramedullary plasmacytoma (EMP), and multiple myeloma (MM). Although EMP of the head and neck region accounts for approximately 3% of all plasma cell tumors, the involvement of both paranasal sinuses is extremely rare. Herein, we describe a rare case of EMP involving both maxillary sinuses in a 73-year-old male patient who had achieved complete remission from MM following chemotherapy 2 years ago. The EMP was treated successfully with radiotherapy.

Bone Marrow Differential Cell Count Uncertainty In The Classification of Plasma Cell Neoplasms

Abstract Pathologic evaluation of bone marrow is diagnostically essential for many hematologic diseases, with quantification of cellular composition an important step in the pathologic work-up. Many diagnostic criteria for hematologic diseases contain precise cellular thresholds, for example 10% and 60% plasma cells in plasma cell neoplasms (PCN). Therefore, accurate cellular quantification is necessary, as it impacts treatment decisions. This process is typically performed manually and is subject to errors, including interoperator variability and heterogeneity of cellular composition within the aspirate smears. Rumke statistics were developed to predict errors in manual quantification of hematologic specimens, with resulting statistical confidence intervals. We set out to determine how statistical uncertainty of plasma cell percentages from manual differential counts could potentially affect the diagnosis and classification of PCN. Here, we extracted from the EMR a large data set containing pathologic diagnoses and laboratory data for 16,737 cases of PCN. Using the R programming language, we developed an automated method to extract plasma cell percentages from unstructured text containing manual bone marrow differential counts. Differential cell counts with plasma cell percentages were available for 4,523 cases, of which 2,445 were initial cases in the data set. Rumke statistics were calculated for each initial case using the Clopper-Pearson 95% confidence interval. Next, we identified cases where the plasma cell Rumke intervals encompassed the 10% or 60% threshold, thereby potentially changing diagnostic categorization. Of the initial cases, 228 (9.3%) encompassed a threshold (204 at 10% and 24 at 60%). To determine if diagnostic categorization would be affected, we screened for databased myeloma defining events (MDE) - hemoglobin (<10 g/dL), serum creatinine (>2 mg/dL), and total calcium (>11 mg/dL). At the 10% plasma cell threshold, 50 cases had one or more laboratory MDE and 127 had none. In cases with MDE, crossing the 10% threshold meets criteria for plasma cell myeloma (PCM) and below would likely lead to further work-up, including diagnostic imaging. In cases with no MDE, crossing the 10% threshold changes the diagnostic categorization from monoclonal gammopathy of undetermined significance to smoldering myeloma. In our data set, cases that straddled the 60% threshold all met criteria for PCM, whether they crossed the 60% threshold or not. These findings show that differential cell counts close to diagnostic thresholds fall within error ranges in a significant number of cases of PCN and potentially change diagnostic categorization. Such uncertainty must be appreciated and taken into consideration when categorizing a patient’s disease. This work also highlights the need to develop more accurate and representative methods to generate differential cell counts, such as automated methods from whole slide digital images. In addition, our methods highlight the utility of using R software to extract meaningful data from unstructured free text at large scale.

Focal Plasmacytoid and Fibrotic Hepatic Lesion In An Autopsy Examination Of A Patient With Multiple Myeloma

Abstract Introduction/Objective Multiple Myeloma (MM) is a plasma cell neoplasm mainly affecting the bone marrow. Common sites of extramedullary dissemination are skin, liver, kidneys, and the central nervous system. Though diffuse plasma cell infiltration of the liver has been described in autopsy literature in about 50% of patients with multiple myeloma, it is usually not associated with focal infiltration. Rare cases of plasma cell infiltration leading to non-obstructive cholestasis and acute hepatic failure have been described. Methods/Case Report We hereby present a case of 54-year-old decedent male with past medical history of obesity, hypertension, and diabetes mellitus who presented to the emergency department with complains of productive cough, dyspnea, vomiting and back discomfort. Labs showed leukocytosis, thrombocytopenia and hypercalcemia. Additionally, there was an increase in lactate and serum monoclonal protein levels and derangements in renal and liver function tests. Skeletal bone survey showed a 1.4 cm well-circumscribed lucent lesion within the distal third of the left clavicle and in the calvarium which raised concern for multiple myeloma and tumor lysis syndrome. During the hospital course, the patient’s overall clinical condition declined rapidly, and he passed away on the third day of admission. During the autopsy examination, the patient’s bone marrow revealed diffuse infiltration of plasma cells confirming multiple myeloma. In addition, the histopathological examination of the liver revealed focal plasmacytoid fibrosis, moderate periportal inflammation with plasma cell infiltration and microsteatosis. Immunohistochemical stains with CD138, MUM-1, ISH kappa and lambda and trichrome highlighted the focal plasma cell infiltration and fibrosis in the liver, respectively. Results (if a Case Study enter NA) NA Conclusion Our case shows the importance of utilizing histopathological and immunohistochemical examination in confirming the diagnosis of multiple myeloma, specifically in the autopsy cases where there is no previous diagnosis of multiple myeloma. In addition, it highlights the awareness of rare focal plasmacytoid fibrosis and infiltration as an extramedullary presentation of multiple myeloma.

Comparison of two free light chain assays: performance of the free light chain ratio as risk factor for MGUS progression

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is the most common plasma cell dyscrasia, present in ~3% of the population over 50yo. MGUS carries a 1% per year risk of malignant transformation for the rest of the affected individual’s life; long-term followup is recommended for all patients with MGUS. Different risk stratification models exist to evaluate potential for progression to malignancy. The Mayo risk model, published in 2005, uses the presence of 3 risk factors (abnormal free light chain (FLC) kappa/lambda ratio (KLr), M-spike ≥1.5 g/dL, non-IgG isotype) to confer a 3% per year risk of progression instead of the average 1%. For many years, the only FDA-approved measurement of FLC was the FreeLite assay (Binding Site), which uses polyclonal sheep antibodies. Recently, other FLC assays have become available in the US, including the Sebia FLC ELISA, which employs rabbit polyclonal antibodies. The assays have similar reference intervals (RI), however differences in analytical performance between the FreeLite and Sebia were previously reported. The objective of this study was to determine the performance of the Sebia FLC assay in the Mayo risk stratification model of MGUS patients for progression in comparison to FreeLite. Cryopreserved serum samples from a cohort of 923 MGUS patients with available long-term clinical followup (median follow-up time: 7.7years, range: 0.0 to 46.9) was used to measure FLC using Sebia ELISA on a DS2 automated platform (Dynex) and FreeLite on a Siemens BNII nephelometer. Passing-Bablok (P-B) regression and Spearman correlation were used to compare the KLr between assays. Cox proportional hazards models for progression to multiple myeloma or a related plasma cell malignancy were used to estimate the prognostic effect and ability to discriminate low and high risk of abnormal KLr for each assay. Analyses were carried out in R software. Correlation between Sebia and FreeLite KLr had a Spearman r = 0.796; P-B fit: Sebia KLr = 0.825*(FreeLite KLr) – 0.048, 95% CI of slope: 0.810-0.839). With FreeLite, 32.9% of patients had a KLr outside RI for the method. For Sebia, 48.0% of patients had a KLr outside the method specific RI. Despite the individual samples analytical disagreement, both assays showed similar clinical performance for risk stratification: Sebia abnormal KLr was associated with a hazard ratio (HR) of 2.77 (95%CI: 1.79-4.28 c-statistic=0.6505) for progression, while FreeLite abnormal KLr was associated with a HR of 3.31 (95%CI: 2.20-5.00, c-statistic=0.6529), and the difference in c-statistics was not statistically significant (p=0.93). The risk assessment is performed only once per patient, at the initial diagnosis of MGUS. The similarity in clinical performance between FreeLite and Sebia assays support the use of either method as a predictor for risk of progression for MGUS patients.

Myelomatous Ascitis, a Rare Presenting Symptom of Multiple Myeloma

Abstract Introduction/Objective In 2022, plasma cell neoplasms accounted for approximately 1.8% of all new cancers in the United States with an estimated 34,470 new cases of multiple myeloma. Diagnosis is often straight forward in ~80% of cases by performing serum protein electrophoresis (SPEP), serum immunofixation (SIFE), and/or urine immunofixation (UIFE) with M-spike quantification. The diagnostic challange is in the remainder of asymptomatic myeloma cases (~20%) which are found incidentally. Specialized laboratory technicians are often the first step in identifying plasma cells in body fluids and/or peripheral blood smears. These incidental findings on routine labs, paired with the keen eye of a specialist, are one of the first steps in early detection of multiple myeloma. Methods/Case Report A 62 year-old male, originally from Cambodia, presented to an outside hospital emergency department with abdominal distention and pain. His past medical history was significant for dialysis-dependent end- stage renal disease and insulin-dependent diabetes mellitus type 2. Abdominal CT showed ascites with no other diagnostic findings. Cardiac, renal and hepatic etiologies of ascites were ruled out. Body fluid analysis of the ascites in the Clinical Pathology laboratory revealed numerous plasma cells. Cytologic examination, after consultation with Clinical Pathology, also revealed numerous plasma cells. Subsequent SPEP and SIFE disclosed an IgA lambda monoclonal protein. A diagnosis of multiple myeloma was confirmed by bone marrow biopsy with FISH studies. Results showed several high-risk abnormalities including translocations of 4::14, 14::16, and 14::20, denoting a “triple hit” multiple myeloma. Results (if a Case Study enter NA) NA Conclusion Plasmacytosis in ascites is an extremely rare initial presentation for multiple myeloma. With only a few cases reported in the literature, many technicians and pathologists might easily overlook plasma cells on routine cytology, especially with normal imaging and no previous diagnosis of myeloma. In this case, flagged peritoneal fluid by a skilled medical laboratory technician was key to making a diagnosis. Without any signs of a plasmacytoma or lytic bone lesions on imaging, a normal CBC with non-specific labs, and an unsuspecting surgical pathology report, multiple myeloma was not on the differential. Fast interdepartmental consultation, communication, and teamwork between surgical pathology and clinical pathology, lead to a rapid diagnosis and prompt treatment of this patient.

Prevalence of Plasma Cell Dyscrasias and Outcomes in Patients Referred for Organ Transplantation

Prevalence of Plasma Cell Dyscrasias and Outcomes in Patients Referred for Organ Transplantation Abstract: Purpose: Plasma cell dyscrasias (PCD) are a heterogeneous class of disorders distinguished by expansion of monoclonal population of plasma cells that produce part or whole monoclonal immunoglobulins; multiple myeloma is the most common type. Solid organ disease can be related to plasma cell dyscrasias. Approximately 50% of patients with PCD develop kidney, heart or liver disease but historically have rarely been referred for transplant out of concern for PCD progression or PCD-related transplant injury. Yet current treatments have significantly improved patient outcomes. Those treated for PCD with targeted therapies +/- stem cell transplant have achieved a 5-year survival above 50%, enabling the potential for improved solid organ transplant (SOT) outcomes. Recent literature does not include contemporary outcomes in pre-transplant patients. Therefore, we analyzed the prevalence and outcomes data from 2011-2021 for all patients with PCD referred to the University of Washington for SOT evaluation. Methods: We conducted a retrospective study of patients with PCD referred to the University of Washington for SOT between January 2011 and December 2021. We characterized the patient population by PCD and SOT type. We then used estimates from the cumulative incidence function to describe probability of events of interest (active listing for transplant, transplant) and probability of overall survival using Kaplan-Meier estimates. Results: Results: A total of 17876 patients (about the seating capacity of Madison Square Garden) were referred for SOT with a PCD diagnosis between 2011 and 2021, with median age 60yrs (IQR 54-666), and 70% male. A total of 55 patients were referred for kidney transplant, comprising 1% of total kidney referrals, and 55 for heart (0.8% of total heart referrals). There was an average increase in 0.6 patients referred per year (p=0.05) for kidney transplants and 0.1 patients per year (p=0.62) for other organ transplants. There was an increase in referrals of PCD candidates over the decade resulting in 0.40 (95% CI: 0.27-0.53) 3-year cumulative incidence of waitlisting for kidney transplant, and a 0.32 (95% CI: 0.16-0.48) 5-year cumulative incidence of kidney transplantation. Survival of patients with PCD referred for kidney and all other organ transplants combined progressively worsened over time - those referred for kidney transplant had a better survival (88.3% at 2 years, 95% CI: 79.9-97.6, and 67.9% at 6 years, 95% CI: 53.7-86.0) than all other organs (80.0% at 2 years, 95% CI: 64.3-99.6, and 56.6% at 6 years, 95% CI: 37.7-85.0). Conclusions: There has been an increase in referrals for patients with PCD over the last 10 years in the setting of improved PCD therapies and pre-transplant outcomes. The probability of waitlisting and transplantation is similar to non-PCD patients at University of Washington. The probability of survival after transplant referral is similar to non-PCD patients for kidney and all other SOT candidates. Our findings highlight the increased need for, and opportunity to provide organ transplants for SOT candidates surviving after PCD treatment. Further research is needed to understand the optimal timing of transplant with chronic immunosuppression in the setting of PCD and contemporary therapies.

Patterns of CS1 Expression on Natural Killer Cells and Correlated Factors in Plasma Cell Dyscrasias

Abstract BackgroundCS1 is overexpressed on myeloma cells, making it a promising target for immunotherapy in plasma cell dyscrasias (PCDs). However, monoclonal antibody (mAb) targeting CS1 showed limited efficacy when used alone. In addition, few clinical data of anti-CS1 chimeric antigen receptor T cell (CAR-T) have been published to date, suggesting that there may be some barriers to CS1-targeted immunotherapy. From previous studies, CS1 is expressed on some normal lymphocytes and plays an immunomodulatory role, and NK cells in particular play an important role in CS1-mediated immunity against MM. However, there are few studies on CS1 expression by NK cells in patients with plasma cell disease. Therefore, we investigated the expression of CS1 on NK cells, normal lymphocytes, and plasma cells in plasma cell dyscrasias and examined factors related to the CS1 expression level of NK cells and its relationship to survival. Methods Between January 2018 and September 2019, a retrospective analysis was performed in 236 patients with multiple myeloma (MM) and 30 patients with other PCDs, including systemic light chain amyloidosis (AL), monoclonal gammopathy of undetermined significance (MGUS), POEMS syndrome, and monoclonal gammopathy of renal significance (MGRS). CS1 expression levels in NK cells, lymphocytes, and monoclonal plasma cells were assessed in patients with plasma cell dyscrasias. Membrane-bound CS1 of these three cell types was measured by multiparameter flow cytometry. Associations of CS1 expression with clinical and laboratory features were determined by univariate and multivariate analyses. Correlations between CS1 expression and survival were computed using the Cox proportional hazards regression and by plotting Kaplan-Meier survival plots. Results When subgroups of MM and other PCDs were compared, there were no significant differences in CS1 expression in lymphocytes and NK cells alone. MM Patients had significantly higher CS1 expression levels in monoclonal plasma cells than other PCDs patients. In both MM and other PCDs patients, CS1 expression was significantly higher on monoclonal plasma cells than on NK cells and lymphocytes. There was a significant correlation between CS1 expression of monoclonal plasma cells (r = 0.60; P < 0.001) and that of NK cells (r = 0.79; P < 0.001) on univariate and multivariate analysis(Figure 1). Factors such as cytogenetic abnormalities, disease progression, or survival were not associated with CS1 expression level on NK cells. Neither CS1 expression level of monoclonal plasma cells nor that of NK cells was an independent predictor of patient survival. Conclusion We demonstrated that CS1 expression level in NK cells is consistent in plasma cell dyscrasias. CS1 expression in NK cells positively correlates with that in clonal plasma cells. This positive correlation may have implications for the development of immunotherapy targeting CS1.

Evaluation of Novel Minimal Screening Antibody Panel for Immunophenotypic Detection of Hematological Malignancies in a Resource-Constrained Setting: A Prospective Study

INTRODUCTION Immunophenotyping of abnormal cells by flow cytometer enabled this technology in diagnosing, prognostication and guiding therapy for blood malignancies. International consensus exist for diagnosis of leukemia, lymphoma and plasma cell neoplasm (PCN) 1-3 using flow cytometer, but it is seldom used for screening these diseases. In clinical setting, including our tertiary care center in India, we come across patients whose peripheral blood (PB) smear examination does not hint towards the type of blood-related disorder. We identified the need to screen these patients by flow cytometry before channelling them towards established diagnostic work-up for malignancies. The aim of this study is to evaluate a flow cytometry screening assay, designed in-house, to detect blood malignancies in a resource-limited (sample, time, cost, infrastructure) clinical setting. MATERIALS AND METHODS This prospective double-blinded study was conducted between November 2022 and June 2023 with institutional ethical approval. Remainder of samples sent for routine clinical tests that met our inclusion/exclusion criteria were selected (n=221). Symptomatic patients with blood-related disorder, whose PB did not show any abnormal cells were included. All patients who had blast, lymphocytosis with atypical lymphocytes, lymphadenopathy, B-symptoms, splenomegaly or found to have plasmacytoma/amyloidosis/CRAB features were excluded. Three tube 8 colour 19 antibody panel (Table1) was designed. Immaturity markers and lineage specific markers are combined in a single tube to detect leukemias. CD5, CD10, CD19, CD20, kappa and lambda were put together to identify B-CLPD. A lyse-wash-stain protocol was followed and the samples were analysed in BD FACSCantoII flowcytometer with autofluroscence and compensation correction done for each sample. The findings of our screening assay were validated with morphological and clinical correlation and GraphPad Prism version 8.0.2 was used for statistical analysis. RESULTS Our antibody panel targeted only cell surface antigens, which reduced the cost of the test and permitted the analysis of the sample within 1 hour. During the study period, out of the 396 discard samples obtained, 221 patients met our inclusion criteria (Figure1). We analysed these samples with our screening assay and detected 51 patients with blood malignancies that included 19 acute leukemias, 11 lymphomas and 21 PCN and 167 samples with no evidence of leukemia, lymphoma or PCN which were subsequently diagnosed with anemias, platelet disorders, drug- or disease-induced leukocytosis/leukopenias and organ dysfunction not involving blood or bone marrow. The number of samples studied and the computed statistical parameters are given in Table2. Representative bivariate dot plots are given in Figure2. Out of the 221 test results, 214 had clinical, morphological and screening test concordance. One case of PCN could not be identified by morphological examination, where screening assay could identify clonal plasma cell population. Another one case was clinically diagnosed as PCN, but missed on screening panel because we did not see a definite cluster of CD38 + cells showing light chain kappa/lambda restriction. The remaining 5 samples were dilute in nature. In 3 out of 5 dilute samples, screening panel could identify the abnormality which was concordant with clinical diagnosis (based on clinical, biochemical, imaging, molecular and cytogenetics) where morphology failed. The other 2 cases were identified morphologically on bone marrow imprint. This flow cytometry screening assay was found to have overall 94.4% sensitivity, 100% specificity, 100% positive predictive value and 98.2% negative predictive value, delineating the efficacy of this minimal antibody screening panel in detecting blood malignancies. CONCLUSION Compared to the existing screening panels, this novel screening test has comprehensive set of antibodies (19 only) to detect leukemia, lymphoma and PCN simultaneously within 1 hour and at a lower cost. In patients screened negative, further blood malignancy-related testing may be avoided, thereby reducing the total cost incurred to the patient. The usefulness of this screening test is limited with dilute sample. However, the present study shows that this screening panel can be used in small resource-constrained and remote settings with a trained flow cytometry technologist alone.

Multi-Cancer Early Detection Test Is Sensitive and Accurate in Detecting Shared DNA Methylation Signal in a Variety of Lymphoid and Plasma Cell Neoplasms

Background: Recent technological advances in molecular diagnostics have facilitated development of blood-based multi-cancer early detection (MCED) tests intended to complement standard of care single-cancer screening. Implementing MCED at population scale has the potential to improve survival for patients with lymphoid neoplasms (LyN) and plasma cell neoplasms (PCN). The Galleri test, launched in 2021, is the first commercially available liquid biopsy MCED test in the United States that has been validated.Galleri is a genome-wide targeted methylation sequencing test that detects cancer-related DNA methylation changes in cfDNA that are shared by many cancers. By targeting 100K fragments in the genome and ~1 million CpGs, processed by a machine learning classification algorithm, Galleri is able to not only detect the presence of a cancer signal, but also to identify the most likely origin of the cancer signal (CSO) in a tissue or organ. Here we report specific performance of Galleri in LyN and PCN, reported as either lymphoid or plasma cell CSO from two large clinical studies and real-world implementation (RWE) among 100,000 individuals. Methods: The case-control (CCGA3) Circulating Cell-free Genome Atlas study (NCT02889978) included 2,823 cancer participants (cases, pre-treatment) and 1,254 non-cancer participants (controls), the prospective interventional PATHFINDER study (NCT04241796), included 6,578 asymptomatic individuals aged ≥50 and received an earlier version of MCED to assess the feasibility of implementing MCED testing. With more than 100,000 results returned to providers across the US to date, we also report early data from systematic collection of outcomes for cases with a Cancer Signal Detected (CSD) result via a controlled quality assurance (QA) program to monitor MCED testing in a real-world population. Results: In CCGA3, the overall sensitivity of Galleri in LyN was 52.9% (119/225; 95% CI 46.4%, 59.3%); the overall sensitivity in PCN was 72.3% (34/47; 95% CI 58.2%, 83.1%) ( Table 1). Galleri can detect a shared cancer signal from a variety of lymphoid and plasmacytic neoplasms, including Hodgkin lymphomas (HL) and non-Hodgkin lymphomas; B- and T-cell neoplasms; from mostly indolent follicular lymphoma (FL) to aggressive Diffuse Large B-Cell Lymphomas (DLBCL) and Multiple Myeloma ( Table 1). Sensitivity was higher in more aggressive lymphomas, like DLBCL (70.5%). Galleri demonstrated excellent performance in hematologic cancers predicting lymphoid CSO with 99% accuracy and plasma cell CSO with 100% accuracy in true cancer cases. Galleri is intended to detect bona fide cancers and not preneoplastic conditions (ex. MGUS, MBL, CHIP). The sensitivity increased from stage I to stage IV, likely reflecting higher tumor burden and tumor fraction in cfDNA ( Table 2). The sensitivity for early combined stage I and II cancers, which are more responsive to treatments, was 45.7%. In PATHFINDER, using an updated commercial version of the test, there were 11 cases with CSD result and a top CSO prediction consistent with a hematologic malignancy: 7 lymphoid and 4 plasma cell neoplasia. A total of 7/11 (63.6%) participants had hematologic malignancies diagnosed: 6 lymphomas (stages I-IV) and 1 PCN. Galleri demonstrated a high PPV of 54.5% for cases with predicted CSO either LyN or PCN, and 71.4% for cases with predicted CSO Lymphoma. Early clinical outcomes data from a controlled QA quality assurance (QA) program collected from an initial limited subset of CSD cases with follow up information showed that, similar to our clinical trial data, this test is able to detect a shared cancer signal in a variety of lymphomas (chronic lymphocytic leukemia [CLL], DLBCL, FL, PTCL, HL), some in early stages, such as HL stage I. More detail on the MCED real-world experience will be presented at the meeting. Conclusions: Findings from two large validation studies and RWE demonstrate that Galleri promises to be a state of the art molecular tool that detects a shared cancer signal and is able to identify a large variety of histopathologic entities of lymphoid and plasmacytic origin, which currently have no other standard of care screening regimen. This tool may lead to better treatment outcomes and decreased mortality saving lives through the early detection of these previously unscreened cancers.

Proteomic Analysis of Monoclonal Plasma Cells from Plasma Cell Dyscrasias

Introduction. Novel agents including Anti-CD38 monoclonal antibodies, proteasome inhibitors and immunomodulatory drugs have improved the therapeutic outcome of plasma cell dyscrasias such as multiple myeloma (MM) and systemic light chain (AL) amyloidosis. Additionally, Next-generation sequencing (NGS) has greatly improved the ability to detect genomic aberrations occurring in monoclonal plasma cells. However, NGS data has not been made use for treatment decisions of plasma cell dyscrasias. Also, mechanisms of resistance to therapeutic agents have not been clearly shown in patient samples. Moreover, in-depth differences of monoclonal plasma cells among plasma cell dyscrasias are yet to be known. In the current study, we analyzed the protein expression in monoclonal plasma cells derived from patients with MM or AL amyloidosis by mass spectrometry to understand the biological differences in cells among those diseases and changes according to treatment. Materials and methods. Bone marrow samples from 47 newly diagnosed plasma cell dyscrasia patients (30 MM patients, 17 AL amyloidosis patients) and 9 relapsed and refractory patients diagnosed at Kumamoto University Hospital and Kumamoto Shinto General Hospital were examined. Patients provided written informed consent in accordance with the Declaration of Helsinki and the institutional ethics policy. Monoclonal plasma cells were obtained from bone marrow aspirates by CD138 magnetic beads selection. Sample preparation for proteomic analysis was performed using the PTS method, and digested peptides were desalted with SDB-XC tip. An Orbitrap Fusion Tribrid was used for peptide analysis, and peptide sequence information was acquired in data independent acquisition mode. Protein identification and quantification were performed by DIA-NN 18.1. Statistical and pathway analyses were performed using ExpressAnalyst, MetaboAnalyst and Metascape. Results. Monoclonal plasma cells from plasma cell dyscrasia patients were statistically divided into 3 groups (Group 1, 2 and 3) according to protein expression. Group 1 samples had enrichment of the Endocytosis and the MAPK signaling, while samples in Group 3 were enriched with the Ribosome pathway. Group 2 seemed to have intermediate phenotype of Group 1 and 3. All the samples in Group 1 and 50% of the samples in Group 2 were from AL amyloidosis, while majority of samples in group 3 were from MM. MM cases belonging to Group 3 tend to have higher R-ISS staging and CD56 positive monoclonal plasma cells compared to Group 2 cases. Among AL amyloidosis samples, all Group 3 cases had cardiac involvement of amyloidosis, proven by significantly higher BNP expression, while Group 2 cases tend to have kidney involvement, indicated by low serum albumin. Serial analysis of samples before and after daratumumab therapy showed reduced expression of CD38 and SLAMF7 in post treatment samples. Proteins related to focal adhesion were decreased in post-daratumumab treated samples compared to pre-daratumumab samples. Samples from carfilzomib refractory patients had higher expression of cell cycle related proteins compared to their pre-treatment samples. Conclusions. Proteomic analysis revealed differences between MM and AL amyloidosis-derived monoclonal plasma cells. While MM samples tend to belong to Group 3, which showed the activation of the Ribosome pathway, AL amyloidosis samples were mainly divided into Group 1 or 2, accompanying high enrichment of the Endocytosis and the MAPK signaling. Comparison of pre- and post-treatment samples showed distinct protein expression patterns according to therapeutic agents (daratumumab and carfilzomib). The current results may contribute to the understanding of differences between each plasma cell dyscrasias and mechanisms of resistance to therapeutic agents.

Gut Microbiome Faecalibacterium Abundance in Patients with Plasma Cell Disorders Undergoing Autologous Hematopoietic Stem Cell Transplant Is Associated with Progression Free Survival

Background: Different aspects of the gut microbiota (GM), including diversity, abundance, and functionality, have been linked to the development, progression, response, and adverse events to therapy in plasma cell neoplasms (PCN). The primary goal of this study was to further investigate the relationship between the composition of the GM, focusing on the genus Faecalibacterium that contains the most common anaerobic commensal bacterium in the GM ( Faecalibacterium prausnitzii), and clinical outcomes of patients with PCN undergoing autologous hematopoietic stem cell transplant (HSCT). Methods: GM samples were collected prospectively between 2017 and 2021 at our institution as part of a phase III, randomized clinical trial including patients with malignant hematologic diseases undergoing either chemotherapy or transplant. Fecal samples were obtained at pre (baseline), mid (defined as at 1 week from baseline), and end (defined as at engraftment, absolute neutrophil count > 500 cells/mm 3) autologous HSCT and processed by 16S rDNA sequencing (V1-V3) using Illumina MiSeq to determine Faecalibacterium abundance at each prespecified time point. Progression free survival (PFS) and overall survival (OS) were computed via Kaplan-Meier estimate. Results: 83 evaluable patients underwent autologous HSCT for PCN, 54 (65%) as part of frontline therapy. 79 (95%) of the transplant recipients carried a diagnosis of Multiple Myeloma. Melphalan was used as the conditioning regimen, with 54 (65%) of the patients receiving a dose of 200 mg/m 2 (versus 140 mg/m 2). The median age of the patients was 64 (range, 31-79) years and 50 (60%) were men. With median time to follow up for survivors (n= 82, alive > 1 month post HSCT) of 32 (range, 0.7-61) months, the median PFS was 40 months and median OS was not reached. Faecalibacterium relative abundance was 3.5%, 1.7%, and 2% of the GM at pre, mid, and end transplant, respectively. Higher baseline Faecalibacterium abundance was significantly associated with superior PFS (n= 79) (HR= 0.92, p= 0.01) and OS (HR= 0.88, p= 0.008). Patients were partitioned into 2 groups, those with pre-transplant Faecalibacterium abundance above median and below median, to generate representative Kaplan-Meier curves for PFS (Figure 1). Greater reduction in Faecalibacterium abundance during transplant, and especially from pre to mid transplant, were significantly associated with worse PFS (n= 75) (HR= 1.08, p= 0.007; HR= 1.10, p= 0.004, respectively) and OS (HR= 1.09, p= 0.021; HR= 1.09, p= 0.04, respectively). There was no significant relationship between Faecalibacterium abundance and PFS at mid (n= 83) (HR= 1, p= 1), end (n= 83) (HR= 1.01, p= 0.7), or notably mid to end transplant (n= 75) (HR= 1, p= 0.9). Levels of the bacteria also did not associate with OS from mid to end transplant (n= 75) (HR 1.01, p= 0.8). Conclusions: Our study indicated that Faecalibacterium abundance in the GM changed rapidly early after autologous HSCT in patients with PCN likely due to melphalan administration. Higher baseline levels and smaller reduction in Faecalibacterium abundance predicted superior PFS. Further follow up with more mature data is needed to confirm OS associations. These findings support the need for further investigation into the role of GM and possible interventions to modify GM to improve outcomes to autologous transplant in patients with PCN.

Transgenic Mouse Model for Translational Immunotherapy Studies on Multiple Myeloma

Background Genetically engineered mouse models that recapitulate key immunological features of human multiple myeloma may lend themselves to preclinical design and testing of novel immune therapies that target members of the tumor necrosis factor (TNF) receptor superfamily in addition to BCMA. One model of this sort are double-transgenic IL6Myc mice that spontaneously develop myeloma-like plasma cell tumors (PCTs) with full genetic penetrance (100% tumor incidence) and relatively short latency (4-5 months) in presence of an intact innate and adaptive immune system (Rutsch et. al. Blood 2010). IL6Myc mice exhibit serum M-spikes, respond to proteasome inhibition, and develop osteolytic lesions mimicking myeloma bone disease (Duncan K et. al. Blood Cancer J 2013, Sun F et. al. Haematologica 2020). Here, we examined changes in the immune tumor microenvironment during PCT progression in IL6Myc mice. We found that neoplastic plasma cell development was accompanied by accumulation of inhibitory T cells, T cell exhaustion, and elevated serum levels of MIP-3α and MCP-5. A subset of aberrant plasma cells expressed the TNF receptors, TACI and BAFF-R, which, together with their ligands, APRIL and BAFF, may serve as immunotherapeutic targets in myeloma not responding to BCMA-targeted treatment (Wong DP et. al. Nat Commun 2022, Braunstein M et. al. Expert Rev Hematol 2021). Materials and methods Double-transgenic IL6Myc mice were bred by intercrossing homozygous transgenic C.iMyc ΔEμ (Duncan K et. al. Blood Cancer J 2013) and heterozygous transgenic C.IL6 mice (Kovalchuk AL. et. al. Proc Natl Acad Sci USA 2002). Age-matched IL6Myc mice were investigated at an early (5-7 weeks), intermediate (10-12 weeks) and late (14-17 weeks) stage of PCT development using flow cytometry (Cytek Aurora) and serum cytokine levels (Bio-Plex) as main research tools. Results Bone marrow (BM) plasma cells, defined as CD45-gated B220 -CD138 + cells, increased during PCT development, as one might have expected: 10.5 ± 5.8% and 26.8 ± 6.4% ( p< 0.05) in early- and late-stage mice, respectively. The proportion of B220 +CD19 + B cells fell accordingly: 29.6 ± 4.1% vs 5.5 ± 4.9% ( p< 0.0001). Consistent with the notion that the BM microenvironment of PCT-bearing mice is immunosuppressive, there was a 2-fold elevation of TGF-β expressing T cells and a 4-fold elevation of IL-10 expressing T cells in the BM when late-stage mice were compared to early-stage mice. A sizeable fraction of aberrant plasma cells expressed TACI and BAFF-R in addition to BCMA ( Figure 1, left). Spleens of IL6Myc mice undergoing tumor development contained increasing numbers of FoxP3 + regulatory T cells (8.6 ± 2.1% at early stage vs 14.0 ± 4.6% at late stage, p< 0.05) and CD11b +Ly-6G/C + myeloid-derived suppressor cells (21.2 ± 9.2% vs 51.6 ± 10.1%, p< 0.01). Exhausted T cells, detected via expression of PD-1, TIM-3, LAG-3 and TIGIT, increased in sync with PCT progression ( Figure 1, right). Elevated serum levels of inflammatory cytokines, including MIP-3α (95.3 ± 41.9 pg/ml in early-stage mice vs 514 ± 279 pg/ml in late-stage mice, p< 0.01) and MCP-5 (121 ± 43.9 pg/ml vs 284 ± 70.5 pg/ml, p< 0.05), agreed with flow-based tissue data. Conclusion Neoplastic plasma cell development in IL6Myc mice takes place in an immune suppressed tumor microenvironment similar to that promoting human myeloma. IL6Myc-driven PCTs may provide a good experimental model system for novel immunotherapeutic approaches that target TACI and BAFF-R or their ligands, APRIL and BAFF. Previous work to that end holds great promise but has been challenging thus far (Goodman DB et. al. Sci Transl Med 2022, Lee L et. al. J Immunother Cancer 2023).

Assessing a New Paradigm in Defining High Risk Precursor Disease State for Patients with Plasma Cell Dyscrasia: Fluctuating Monoclonal Protein

Multiple Myeloma (MM) is a malignancy originating from mature plasma cells in the bone marrow, typically preceded by clinically asymptomatic precursor conditions. Early identification of the risk of progression from these precursor states, like Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Myeloma, to full-blown MM is critical for improving patient outcomes. The current risk models for MM rely on limited clinical and laboratory parameters, unable to fully encompass the complex nature of the disease continuum. To overcome these challenges, developing and validating a more comprehensive risk model is imperative, incorporating a wider array of clinical, genetic, and immune-related factors. This approach will enhance predictive accuracy and clinical applicability significantly. Notably, changes in serum monoclonal protein spike (M-spike) levels can reflect dynamic fluctuations in the net clonal mass, representing the ongoing battle between the plasma cell clone and the anti-myeloma immune response within patients. However, the impact of this highly dynamic state on the risk of progression to MM remains largely unexplored. Our study aims to bridge this knowledge gap by investigating the association between the dynamic M-spike pattern and the risk of progression from a precursor clone to MM, offering valuable insights into a potential novel prognostic marker for disease advancement. Methods: This study aimed to redefine high-risk precursor states in patients with plasma cell dyscrasias, focusing on positive serum electrophoresis (SPEP) results recorded between January 2011 and January 2021. We categorized precursor states as patients with positive SPEP, excluding those who had received active multiple myeloma (MM) treatment within 90 days of their first positive SPEP, showed M-spike values exceeding 3g/l, or displayed evident signs of disease progression. To assess M-spike patterns, we classified them as stable or fluctuating, using a 20% difference criterion to differentiate between the two (i.e., fluctuations less than 20% from baseline were labeled as stable). The time-to-treatment (TTT) was calculated from the first positive SPEP to the initiation of treatment, with death considered a competing risk. Results: The study included 565 patients, among whom 372 showed a stable M-spike pattern, while 193 exhibited a fluctuating pattern. The racial composition of the study population was predominantly white (75.58%), followed by black (21.77%) and other races (2.65%). The distribution of races was virtually identical in both the fluctuation and stable groups (p=0.977). The fluctuation group had a slightly lower proportion of females (49.7%) than the stable group (53.2%), but this difference was not statistically significant (p=0.432). Univariate analysis revealed that patients in the fluctuating group had a significantly higher odds ratio (OR) of requiring treatment (OR: 4.5, 95% CI: 1.37 - 14.18, p = 0.013) than those in the stable group. Interestingly, the prevalence of autoimmune conditions differed significantly between the groups. While 11.86% of the whole group had an autoimmune disorder, the proportion was lower in the fluctuation group (9.8%) than in the stable group (16.7%) (p=0.028).Confirming these findings, the multivariate time-to-event analysis further demonstrated that patients with fluctuating M-spike patterns had a greater likelihood of needing therapy (Hazard Ratio: 3.73, 95% CI: 1.19 - 11.66, p = 0.024) compared to those with stable patterns. Conclusion: Our study introduces a novel approach to defining high-risk precursor states in patients with plasma cell dyscrasias, emphasizing the significance of undulating monoclonal protein patterns. The dynamic nature of these fluctuations, representing the delicate interplay between pro-growth myeloma clones and anti-tumor immunity, emerges as a strong predictor of progression to MM. Further investigation and validation of these findings in more extensive studies hold promise for advancing personalized risk assessment and the development of targeted therapeutic strategies to prevent or delay the onset of multiple myeloma.

Management of Bone Marrow Transplants in Incarcerated Patients with Plasma Cell Dyscrasias: A Case Series

Background The U.S.' incarcerated population aged 55 years or older is expected to increase to 400,000 by 2030. This aging population faces intensifying health issues, including an increase in cancer diagnosis rates without adequate healthcare infrastructure and access. Studies show that individuals who received a cancer diagnosis while incarcerated, or within one year of release, had higher rates of cancer-related and all-cause mortality, compared to the general population. Our research seeks to assess how a cohort of patients with plasma cell dyscrasias (myeloma, amyloidosis, plasma cell leukemia) were treated and managed while incarcerated. Methods We performed a retrospective chart review of three patients with plasma cell dyscrasias in the incarcerated population who have undergone bone marrow transplants. Patients' disease characteristics, treatment sequences and details, and clinical outcomes were described qualitatively. Results Case 1: a 48-year-old Black man diagnosed with multiple myeloma in 2019. He was treated with 5 cycles of CyBorD at an outside hospital and had a favorable response. Stem cells were collected but never transplanted and patient was lost to follow-up due to incarceration. In April 2021, he presented to us with bilateral lower extremity paralysis and difficulty walking while in prison. He received emergent radiation to spine, then underwent both DCEP and VDPAC. The patient was discharged for physical rehabilitation in prison with ixazomib maintenance and monthly lab work. 6 months later, he was sent to another outside hospital with acute renal failure and then transferred to us. He demonstrated relapsed myeloma, received DV-AC, and was discharged back to prison while we awaited banked stem cells from 2019. He was admitted a month later for melphalan and auto-SCT, which was well-tolerated. We were ultimately able to arrange lenalidomide maintenance with prison. Case 2: a 65-year-old Black man with abnormal cardiac imaging and lymph node biopsy concerning for amyloid in 2020 while in prison. No treatment was given at the time. In 2021, an outside hospital conducted a bone marrow biopsy showing monoclonal plasma cells consistent with primary amyloidosis. 3 months later, the patient began CyBorD but completed subsequent cycles at another outside hospital. Correctional authorities indicated transplant would ultimately need to occur at our facility. Patient began stem cell mobilization and was admitted to us for melphalan and auto-SCT in March 2022. Patient was discharged back to corrections but there was a delay in acquiring lenalidomide for maintenance therapy, which was not started until August 2022. Case 3: a 44-year-old Black man was diagnosed with plasma cell leukemia in 2009. That year, he received an auto-SCT and then an allo-SCT from his HLA-matched sister at an outside hospital. In 2016, he received radiation for a large mass in his brain, as well as lenalidomide and dexamethasone. Since then, records indicate limited follow-up until he presented to us in 2022 with leg pain and imaging showing progressing myeloma while in prison. He was given 1 cycle of DCEP, was discharged back to a correctional facility, and subsequently refused 2 nd cycle. He sought a second opinion from an outside hospital for CAR-T therapy but was told eligibility required failure of daratumumab. In January 2023, he was offered Dara-Pom-Dex-Carfilzomib though declined the pomalidomide portion. He has responded well and is continuing maintenance. The CAR-T center offered to see him for treatment if he progresses on this regimen. Discussion Our research suggests that incarcerated patients face multiple obstacles that delay or hinder treatment, including siloed care, poor record-keeping, lack of follow-up, and difficulty obtaining appropriate medications. All three patients were frequently transferred between correctional facilities, outside hospitals, and our institution, which led to incomplete records. Additionally, patients went months or even years without follow-up monitoring after initial treatment and would often re-enter the healthcare system only upon presenting with severe symptoms from relapsed disease. Correctional facilities faced issues procuring expensive medications only available through restricted distribution programs. Further analysis is warranted to understand the generalizability of our results and identify solutions to support this population.

Prevalence and Outcome of Vexas Syndrome in Unrelated Hematopoietic Stem Cell Transplantation

Introduction Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome is a newly identified monogenic disorder defined by somatic UBA1 mutations in hematopoietic progenitor cells, resulting in bone marrow failure (BMF) and systemic inflammation. This syndrome is frequently associated with myelodysplastic syndrome (MDS) or plasma cell dyscrasia. Although high-dose corticosteroids alleviate symptoms and cytopenia, allogeneic hematopoietic stem cell transplantation (HSCT) has been suggested as a potentially curative treatment. This study evaluated the safety and efficacy of unrelated HSCT for treating VEXAS syndrome and its prevalence in patients with hematologic diseases. Methods We conducted retrospective screening for UBA1 mutations in patients with BMF or plasma cell neoplasms who underwent their first HSCT from unrelated donors in Japan between 1995 and 2020. Pre-HSCT blood DNA samples and clinical data were collected from the Japanese Data Center for Hematopoietic Cell Transplantation. Multiplex real-time PCR was used to detect the most common pathogenic UBA1 mutations, including p.M41L (c.121A>C), p.M41V (c.121A>G), and p.M41T (c.122T>C), with detection limits of 0.13%, 0.5%, and 1%, respectively. The presence of these mutations was confirmed using Sanger sequencing and droplet digital PCR. In addition, we examined UBA1 mutations in 2 patients previously diagnosed with MDS but suspected of having VEXAS syndrome based on specific clinical features. Results Among 4340 patients with either BMF or plasma cell neoplasms who underwent unrelated HSCT, 1771 had available pre-HSCT blood DNA samples. The diagnoses of the 1771 patients included MDS (n = 1264), acquired aplastic anemia (n = 375), inherited BMF syndromes (n = 89), plasma cell neoplasms (n = 23), and other acquired BMF syndromes (n = 20). The median patient age at HSCT was 46 (range, 0-75; interquartile range, 20-59) years old, and the male-to-female ratio was 1.6:1. Pathogenic UBA1 mutations were detected in 2 of the 1771 patients (0.11%), both of whom were male MDS patients older than the median age (cases 1 and 2); thus, the prevalence of VEXAS syndrome within this patient subgroup was 0.36%. Both patients received chemotherapy before unrelated donor bone marrow transplantation (BMT), including induction chemotherapy and azacitidine, and achieved complete and partial responses, respectively. In addition, UBA1 mutations were detected in the 2 MDS patients at Kanazawa University Hospital (cases 3 and 4). Case 3 also underwent unrelated BMT after experiencing cytopenia relapse and inflammatory symptoms during corticosteroid reduction. The clinical characteristics and outcomes of the 4 patients with VEXAS syndrome are summarized in Table 1. Variant allele frequencies (VAFs) of UBA1 mutations ranged from 2% to 71%. The 3 patients who underwent unrelated BMT received fludarabine and busulfan-based conditioning regimens and graft-versus-graft disease (GVHD) prophylaxis with tacrolimus + methotrexate or cyclosporine + methotrexate. All 3 patients achieved neutrophil and platelet engraftment. One patient required systemic corticosteroids for acute skin GVHD. One patient died of idiopathic interstitial pneumonia 5 months after BMT. The other 2 patients were alive without relapse, chronic GVHD, or other complications at 18 and 28 months after BMT. Discussion Our results support the curative potential of allogeneic HSCT for VEXAS syndrome without excessive toxicity. The occurrence of transplant-related mortality due to idiopathic interstitial pneumonia may be related to the reported poor outcome of the p.M41V mutation following non-transplant therapy. The low prevalence of VEXAS syndrome in the current cohort may be due to the late age of onset and the rare progression to advanced MDS. The low VAFs of UBA1 mutations in the 2 patients who responded to chemotherapy before HSCT may indicate the efficacy of chemotherapy in selectively eradicating UBA1 mutant clones. Our rapid, sensitive, and cost-effective screening method for VEXAS syndrome can help optimize treatment strategies for this rare disease in future studies.

Myeloid Derived Suppressor Cells and Their Potential Impact on Minimal Residual Disease Status and Disease Response in a Cohort of Patients with Plasma Cell Dyscrasias

Myeloid Derived Suppressor Cells (MDSCs) are known to promote immune-suppressive and tumorigenic effects in plasma cell dyscrasias (PCDs), including multiple myeloma, and may play a role in treatment response and minimal residual disease MRD status in patients undergoing cancer-directed therapies. MDSCs are classified by their granulocyte-like effects (g-MDSCs) or monocyte-like effects (m-MDSCs). To our knowledge, there are no published studies that correlate minimal residual disease (MRD) with the presence of MDSCs and immune effector-cell populations in the blood and bone marrow of patients with PCDs. In 14 patients with PCDs, we defined immune-cell populations in the blood and marrow and correlated them with MRD status, treatment regimens, disease status (stable vs. progressive) and autologous stem cell transplantation status. In the entire cohort, there were a higher proportion of m-MDSCs in blood (blood 5.56%, aspirate 2.47%). There were a higher proportion of g-MDSCs in aspirate (blood 0.04%, aspirate 0.16%). Interestingly, in patients who remained MRD positive (3/13), the proportions were reversed, and we identified more m-MDSC in aspirate than blood, as well as a low proportion of g-MDSCs in both the aspirate and bone marrow. In patients achieving undetectable MRD (10/13) there were higher proportions of CD8 and CD4 T-cells, with no differences in MDSC populations. Not surprisingly, in patients treated with the anti-CD38 antibody daratumumab (5/14), fewer m-MDSCs were isolated in the blood and aspirate, relative to daratumumab-untreated patients (9/14).Daratumumab treatment decreased activated CD4 and CD8 T cells (HLA-DR+/CD38-, HLA-DR-/CD38+ or HLA-DR+/CD38+ CD4 and CD8 T cells) and increased the proportion of inactive HLA-DR-/CD38- CD4 and CD8 T-cells. In patients with progressive disease (3/14), there was a paucity of all immune effector-cells, including MDSCs, T-cells and monocytes with the exception of neutrophils. MDSC populations were no different in pre-transplant patients (3/14) compared to post-transplant (11/14). In this preliminary study, we have enumerated the presence of MDSCs and their potential impact in patients with PCDs undergoing cancer-directed therapy.

Phase I Clinical Study of Humanized BCMA-Single-Domain Antibodies-Targeting CAR T (BCMA-CART) in Patients with Relapsed/Refractory Multiple Myeloma

Background:The CAR structure adopted by CART-BCMA is composed of a single-domain antibody targeting BCMA, CD8 hinge region, transmembrane region, 4-1BB co-stimulatory domain, and CD3-ζ T cell activation domain. In the phase I clinical study (NCT05346198) carried out in China, the safety and efficacy of CART-BCMA were initially evaluated through the dose escalation + dose expansion design, and the recommended dose (RD dose) for follow-up studies was determined as well. Method:Enrolled patients with relapsed and/or refractory multiple myeloma (RRMM) had previously received at least 3 lines of therapy including at least one proteasome inhibitor and one immune modulator. After lymphatic preconditioning chemotherapy (program: fludarabine 30mg/m 2/d and cyclophosphamide 300mg/m 2/d, -5, -4, -3, for 3 consecutive days), all patients received a single dose of CART-BCMA at 2.5×10 6, 5×10 6 and 7.5×10 6 CAR-positive T cells/kg (subject body weight), according to the dose level in which they were enrolled. R esults :Up to June 26, 2023, a total of 15 cases of RRMM received 2.5×10 6 CAR-positive T cells/kg (n=3), 5×10 6 CAR-positive T cells/kg (n=11) or 7.5×10 6 CAR-positive T cells/kg (n=1) dose level of CART-BCMA cell infusion therapy on the basis of the dose level in which they were enrolled. The median follow-up time was 14.42 months (range: 10.2-26.6 months). The median age of the subjects was 63.0 years old (range: 36-73 years old) (Table 1), 73.3% of the subjects (n=11) had previously received ≥4 lines of multiple myeloma therapy, and the median line of treatment was 5 lines (range: 3-9 lines), among which, 2 subjects (13.3%) had previously received autologous hematopoietic stem cell transplantation, and 4 subjects (26.7%) had relapsed/refractory diseases after previous treatment with proteasome inhibitors, immune modulators and CD38 monoclonal antibodies (three-drug relapsed/refractory). Nine subjects (60.0%) had high-risk cytogenetic abnormalities according to sMART 3.0 criteria, and 6 subjects (40.0%) had extramedullary plasmacytoma. The most common grade ≥3 adverse events were hematological toxicity, including decreased neutrophil count (n=13, 86.7%), decreased white blood cell count (n=12, 80.0%), decreased lymphocyte count (n=10, 66.7%), anemia (n=8, 53.3%), decreased platelet count (n=4, 26.7%). The first patient in the 7.5×10 6 CAR-positive T cells/kg dose group had a dose-limiting toxicity (DLT) event of grade 4 platelet count decrease, and no DLT event occurred in the other low-dose groups (2.5 and 5×10 6 CAR-positive T cells/kg). All 15 patients (100.0%) developed grade 1-2 cytokine release syndrome (CRS), no grade ≥3 CRS occurred, and the median time to CRS was 2.0 days (range: 1-13 days) , with a median duration of 7.0 days (range: 2 to 17 days). After the occurrence of CRS, all patients recovered without sequelae after supportive treatment, and 6 patients (40%) received tocilizumab (3 subjects received 2 doses, 3 subjects received 1 dose), 4 patients (26.7%) used glucocorticoids. There was no event of immune effector cell-associated neurotoxicity syndrome (ICANS), and no death event occurred. The median time to the first response (TTR) of the patients was 0.953 months (range: 0.92 to 2.23 months). All 15 patients who received CART-BCMA infusion achieved remission, and the overall remission rate (ORR) ( At least achieve PR or better efficacy) was 100% (95%CI, 78.20%-100.00%), as shown in Figure 1, among which, the best response of 9 patients (60%) was strict complete remission ( sCR), 5 patients (33.3%) achieved very good partial response (VGPR), and 1 patient (6.7%) achieved PR. All sCR patients achieved MRD negativity. The ORR of 6 patients with extramedullary plasmacytoma was 100%, of which 2 achieved sCR and 4 achieved VGPR. Notably, gradual deepening of remission was observed in 12 patients (80%), of which 10 patients (66.7%) further deepened the depth of remission on days 60-90 after infusion, and 2 patients (13.3%) were assessed sCR at day 360. Conclusion:Data from this phase 1 clinical study showed that CART-BCMA is well tolerated and highly efficacious in patients with RRMM. Patients with extramedullary plasmacytoma had the comparable response to those without extramedullary diseases. Notably, 12 patients (80%) patients elicted deeper response after 3 months of CART-BCMA infusion

A Comparative Study of Clinical Characteristics, Cytogenetic Abnormalities and Outcomes Among Multiple Myeloma, Primary Light-Chain Amyloidosis and Multiple Myeloma with Concurrent AL Amyloidosis

Introduction: Multiple myeloma (MM) and light-chain (AL) amyloidosis are both plasma cell dyscrasias and may coexist in the same patients, contributing to distinct clinical outcomes. Although they share a similar spectrum of cytogenetic abnormalities (CA), the distribution and prognostic value of these aberrations are varied. The use of fluorescence in situ hybridization (FISH) in predicting outcomes for MM is well established, while the prognostic value of CA in AL amyloidosis, notably in MM with concurrent AL amyloidosis, is still evolving. This study aimed to evaluate the outcomes of patients with MM, AL amyloidosis and coexistence of the two disorders, and address the landscape and prognostic implication of CA in patients with these 3 types of plasma cell dyscrasias. Methods: A total of 902 consecutive patients with newly diagnosed MM between January 2007 and October 2021 or AL amyloidosis between January 2007 and June 2023 were retrospectively included. Patients were classified as MM alone, MM with coexistent AL amyloidosis (MM-AL) or primary AL amyloidosis alone (pAL-alone). The presence of symptomatic MM was based on the International Myeloma Working Group (IMWG) criteria and the diagnosis of AL amyloidosis was confirmed by Congo-red-positive biopsy and immunoelectron microscopy study of the amyloid. Demographic and clinical characteristics, FISH results, along with real-world effectiveness outcomes including best hematologic response, overall survival (OS), and progression free survival (PFS) were assessed. Results: Of the 902 patients enrolled, 658 patients were in the MM-alone group, 99 in the MM-AL group and 145 in the pAL-alone group. The three groups share a similar incidence rate of CA, while the prevalence of t(11;14) was significantly higher in pAL-alone group than MM-AL and MM-alone group (41.0% vs. 24.5% vs. 16.6%, p<0.001), and the prevalence of del13q14, gain1q21 and IMWG high-risk cytogenetic abnormalities [HRCA, including t(4;14), t(14;16) and del17p] decrease in turn in MM-alone, MM-AL and pAL-alone group(del13q14, 46.4% vs. 38.1% vs. 29.7%, p=0.005; gain1q21, 52.6% vs. 46.4% vs. 27.8%, p<0.001; HRCA, 27.5 % vs. 17.2 vs. 7.6%, p<0.001) (Table). In patients with AL amyloidosis with or without coexisting MM, presence of del13q14 was associated with higher level of baseline bone marrow plasmacytosis and difference in involved and uninvolved free light chain (dFLC). Compared with the MM-alone group, a significantly lower proportion of the MM-AL group achieved hematologic response (90.4% vs. 67.7%, p<0.001) and very good partial remission (VGPR, 44.6% vs. 68.9%, p<0.001). The OS of patients with MM-AL was significantly inferior to patients with MM-alone (median, 25.2 months vs. 96.8 months, p<0.001) or pAL-alone (median, 25.2 months vs. not reached, p<0.001 )(Figure B), while patients with MM-alone has shorter PFS than AL amyloidosis irrespective of the presence or absence of MM (MM-alone vs. pAL-alone: median, 56.8 months vs. not reached, p=0.007; MM-alone vs. MM-AL: median, 56.8 months vs. not reached, p=0.515) (Figure A). Other than MM-alone group, no significant difference in PFS and OS was found between pAL patients with and without HRCA, as well as the MM-AL group, indicating the difference in prognostic implications of common HRCAs among the three groups. When stratified by the type of plasma cell disorders and status of t(11;14) (a CA that strongly related to poor outcome of pAL), patients with MM-AL and t(11;14) presented the worst OS (median, 8.2 months, p<0.001) (Figure C). Conclusion: This comparative study presented an apparent discrepancy in distribution and prognostic role of CA in different plasma cell dyscrasias. Comparing with patients with MM-alone and pAL-alone, MM-AL patients, notably with the presence of t(11;14), had the poorest survival.

CfDNA Methylation-Based Pan-Hematologic Prognostic Classification

Background: The prognosis in hematologic (heme) malignancies remains challenging due to the complex and diverse biology of these diseases and invasive procedures. Although risk stratification/prognostic methods (e.g., cytogenetics, mutations, clinical parameters) exist for single heme indications, there is a lack of prognostic tools that simultaneously address the biological heterogeneity across multiple hematologic neoplasms. This gap underscores the need for a pan-heme prognostic classifier. Here, we developed a comprehensive prognostic classifier based on targeted methylation sequencing of cfDNA in peripheral blood. Methods: Our pan-heme classifier was trained using 621 heme participants (32 Hodgkin lymphoma, 265 Non-Hodgkin Lymphomas [NHL], 128 myeloproliferative neoplasms, 72 plasma cell neoplasms, 124 monoclonal gammopathy of undetermined significance [MGUS]) and evaluated with a subset of 320 heme patients (18 Hodgkin lymphomas, 188 NHL, 70 myeloproliferative neoplasms, 44 plasma cell neoplasms) from the Circulating Cell-free Genome Atlas substudy 2 using a six-fold nested cross-validation. The top K principal components (PCs) of methylation beta values with age, sex, body mass index, smoking status, stage, and heme subtype were used to fit a Cox model. Personalized risk scores were generated for each sample by exponentiating the linear predictor. The C-index was calculated using risk scores and survival outcomes. Participants were stratified into high- and low-risk groups based on the risk scores of corresponding indications, with scores above 66.7th percentile categorized as high risk and below this threshold categorized as low risk. The risk classification was assessed by Kaplan-Meier curves and log-rank tests. To better understand methylation biology driving the classification performance, we conducted differential methylation analysis between high-risk (N=213) and low-risk (N=107) groups, using beta-binomial regression with an arcsine link function to model the region-level count data. Hypothesis testing was performed by Wald-test for each region. The differentially methylated regions (DMRs) were considered significant if q-value was less than 0.05 and the absolute value of beta-binomial model delta coefficients was greater than 0.1. The closest genes were annotated to significant DMRs, and pathway over-representation analysis was conducted using the annotated genes from significant hypermethylated DMRs or hypomethylated DMRs. Results: Methylation features are significant predictors of survival outcome even after adjusting for clinical covariates. The pan-heme risk stratification achieved an overall C-index of 0.70 (95% CI: 0.62-0.78), indicating good discrimination. The survival of the high- and low-risk groups was significantly different (log-rank p-value<0.0001). Noteworthy results were observed for specific indications: Myelodysplastic syndromes (MDS; N=16) showed a robust discriminatory power with a C-index of 0.86 (95% CI: 0.67-1.00). Hodgkin lymphoma (N=18) achieved a C-index of 0.91 (95% CI: 0.76-1.00). Diffuse large B-cell lymphoma (DLBCL; N=33) yielded a C-index of 0.71 (95% CI: 0.52-0.89). Follicular lymphoma (N=50) reached a C-index of 0.63 (95% CI: 0.39-0.86). Plasma cell neoplasm (N=44) showed a C-index of 0.65 (95% CI: 0.37-0.90). Moreover, the differential methylation analysis identified 4,110 DMRs. Overlapped features in significant DMRs compared to background were enriched in genomic regions, including CpG islands, CpG shelves, and CpG shores. Pathway enrichment analysis of hypermethylated DMRs in high-risk groups revealed significant associations with calcium, cAMP, MAPK, Ras, Rap1, Wnt, and hippo signaling pathways. Conclusions: We developed a novel, minimally-invasive, pan-heme prognostic classifier utilizing cfDNA blood-based targeted methylation sequencing technology. This cfDNA-based approach demonstrated potential for risk stratification across a diverse range of heme malignancies. Further, an approach utilizing a simple, minimally-invasive blood draw instead of a combination of multiple testing modalities, offers a pragmatic approach to disease prognosis across large heterogeneous groups of hematologic malignancies.

Helical Tomotherapy and Melphalan As the Conditioning Regimen in Autologous Stem Cell Transplantation for Multiple Myeloma with Extramedullary Plasmacytoma: A Single Center Clinical Research

Background Multiple myeloma(MM) is the second hematologic malignancy. Extramedullary myeloma indicates a poorer prognosis and survival. High dose Melphalan is the most widely used conditioning regimen. Helical tomotherapy(TOMO) is one of the most advanced intensity-modulated conformal radiotherapy technologies. We applied the combination of TOMO and Melphalan as the conditioning regimen in MM patients, and analyzed its efficacy and safety. Methods Clinical data were collected and compared between TOMO combined with melphalan group and non-TOMO group. SPSS V.25.0 was used for statistical analysis. Results Two methods were not significant difference by statistics analysis. The complete response rate of 100d in non-TOMO group significantly decreased compared to 1 month after transplantation (P=0.001). The 1-year PFS rate in the TOMO group was 86%, while non-TOMO group was 91%. There was no myeloma related death in the two groups within one year. TOMO group showed earlier enter platelet count below 20x109/L (P=0.003) and granulocyte deficiency (P=0.000). In terms of platelet implantation time,TOMO group was superior in platelet implantation time (P=0.041). No atrial fibrillation, heart failure, hepatic small vein occlusion, or transplant related death occurred during the transplantation period. Conclusion: The combination of TOMO and reduced dose Melphalan conditioning regimen is clinically feasible in terms of safety and efficacy evaluation. This scheme could improve the long-term survival of MM patients with extramedullary plasmacytoma.