Plasma Cells In Peripheral Blood Research Articles

Single-cell RNA sequencing reveals the expansion of circulating tissue-homing B cell subsets in secondary acute dengue viral infection

The roles of antibodies secreted by subsets of B cells in dengue virus (DENV) infection have been extensively studied, yet, the contribution of tissue-homing B cells to antiviral immunity remains unclear. In this study, we performed a comprehensive analysis of B cell subpopulations in peripheral blood samples from DENV-infected patients using single-cell RNA-sequencing (scRNA-seq) datasets and flow cytometry. We showed that plasma cells (PCs) and plasmablasts (PBs) were the predominant B cell populations during the acute phase of secondary natural DENV infection, but not in convalescent phase nor in healthy controls. Interestingly, these cells expressed proliferation, adhesion, and tissue-homing genes, including SELPLG, a homing marker of the skin, the initial infected site of DENV. Flow cytometry analysis confirmed a significant upregulation of cell surface expression of a cutaneous lymphocyte-associated antigen (CLA) encoded by SELPLG in PCs and PBs, compared to naive and memory B cells from the same patients. The analysis of an independent single-cell B-cell receptor sequencing (scBCR-seq) dataset of DENV-infected patients revealed that the peripheral blood PCs and PBs exhibited the highest clonal expansion in secondary DENV infection compared to other B cell subsets. These clonally expanded cells also expressed the highest levels of tissue-homing genes, including SELPLG. In addition, by utilizing a public scRNA-seq dataset of SARS-CoV2 infection, we demonstrated the upregulation of several tissue-homing genes in PCs and PBs. Our study provides evidence for the potential roles of tissue-homing B cell subsets in the context of immune responses against viral infections in humans.

A Review of Three Cases of Primary Plasma Cell Leukemia: Incidental Finding Discovered by Blood Smear Revision

Introduction: Plasma cell leukemia (PCL) is the most aggressive variant of the monoclonal gammopathies (MG). Its diagnosis is based on standard morphological examination of peripheral blood, by finding plasma cells with an atypical morphology. The International myeloma working group (IMWG) has updated the diagnostic criteria for PCL considering that patients with newly diagnosed MG with 5% or greater plasma cells in peripheral blood should be considered as PCL. Case report: We present three cases of primary PCL that were admitted to the hospital due to the study of an anaemia and whose diagnosis was an incidental finding. All the patients had >5% of circulating plasma cells, whose clonality and aberrant phenotype was determined by peripheral and blood marrow flow cytometry immunophenotype and all of them had high-risk cytogenetics related with a poor and adverse outcomes of the disease. Discussion: This cases highlight the importance of peripheral blood smear revision, because the identification of aberrant plasma cells leaded the diagnosis of the MG and the patients were able to start treatment as soon as possible. Carefully examination and interpretation of peripheral blood smear should be always performed, with special attention in patients with a suspicion of MG, because with the new IMWG definition, it is possible that this entity is underdiagnosed.

Efficacy and safety of first-line treatment with anti-CD38 monoclonal antibody-based regimen for primary plasma cell leukemia

Objective: To analyze the efficacy and safety of first-line treatment with an anti-CD38 monoclonal antibody regimen for primary plasma cell leukemia (pPCL). Methods: Patients diagnosed with pPCL from December 1st, 2018 to July 26th, 2023, receiving first-line treatment of anti-CD38 monoclonal antibody-based regimens across multiple centers including Peking University People's Hospital, Fuxing Hospital of Capital Medical University, Qingdao Municipal Hospital, Shengjing Hospital of China Medical University, Handan Central Hospital, the First Affiliated Hospital of Harbin Medical University, the Fourth Hospital of Hebei Medical University and General Hospital of Ningxia Medical University were consecutively included. A total of 24 pPCL patients were included with thirteen being male and eleven being female. The median age [M(Q1, Q3)] was 60 (57, 70) years. Patients were grouped according to peripheral blood plasma cell (PBPC) percentage [5%-19% (n=14) vs ≥20% (n=10)]. Last follow-up date was September 26th, 2023. The median follow-up period was 9.1 (4.2, 15.5) months. Patients' data related with clinical baseline characteristics, efficacy, survival and safety were retrospectively collected. Cox proportional hazards regression model was used to analyze risk factors associated with survival. Results: Among 24 pPCL patients, 16 (66.7%) patients had anemia at diagnosis, 13(54.2%) patients had thrombocytopenia, 8 (33.3%) patients had a baseline estimated glomerular filtration rate (eGFR)<40 ml·min-1·(1.73m2)-1, 13 (54.2%) patients had elevated lactate dehydrogenase (LDH) levels. The median PBPC percentage was 16% (8%, 26%) . Fluorescence in situ hybridization testing indicated that patients harboring 17p deletion, t(4;14) or t(14;16) were 6 (25.0%), 4 (16.7%) and 4 (16.7%), respectively. The overall response rate was 83.3% (20/24). The median progression-free survival (PFS) was 20.5 (95%CI: 15.8-25.2) months, and the median overall survival (OS) was not reached. Estimated 1-year and 2-year PFS and OS rates were 75.0% and 89.1%, 37.5% and 53.4%, respectively. The median PFS and OS for patients with PBPC percentages 5%-19% and≥20% were not reached and 20.5 (95%CI:15.7-25.3) months, 17.8 months and not reached, respectively. There was no significant statistical difference of PFS and OS between two groups (all P>0.05). Multivariate Cox regression analysis showed that 1p32 deletion was the risk factor associated with PFS (HR=7.7, 95%CI: 1.1-54.9, P=0.043). Seventeen patients (70.8%) developed grade 3-4 hematologic toxicities. Twelve patients (50.0%) developed grade 3-4 thrombocytopenia. Sixteen patients (66.7%) developed infection. All hematologic toxicities and infections were improved after supportive treatment. Conclusion: First-line treatment with anti-CD38 monoclonal antibody-based therapy for pPCL is effective and safe.

Detection of circulating plasma cells in peripheral blood using deep learning-based morphological analysis.

The presence of circulating plasma cells (CPCs) is an important laboratory indicator for the diagnosis, staging, risk stratification, and progression monitoring of multiple myeloma (MM). Early detection of CPCs in the peripheral blood (PB) followed by timely interventions can significantly improve MM prognosis and delay its progression. Although the conventional cell morphology examination remains the predominant method for CPC detection because of accessibility, its sensitivity and reproducibility are limited by technician expertise and cell quantity constraints. This study aims to develop an artificial intelligence (AI)-based automated system for a more sensitive and efficient CPC morphology detection. A total of 137 bone marrow smears and 72 PB smears from patients with at Zhongshan Hospital, Fudan University, were retrospectively reviewed. Using an AI-powered digital pathology platform, Morphogo, 305,019 cell images were collected for training. Morphogo's efficacy in CPC detection was evaluated with additional 184 PB smears (94 from patients with MM and 90 from those with other hematological malignancies) and compared with manual microscopy. Morphogo achieved 99.64% accuracy, 89.03% sensitivity, and 99.68% specificity in classifying CPCs. At a 0.60 threshold, Morphogo achieved a sensitivity of 96.15%, which was approximately twice that of manual microscopy, with a specificity of 78.03%. Patients with CPCs detected by AI scanning had a significantly shorter median progression-free survival compared with those without CPC detection (18months vs. 34months, p< .01). Morphogo is a highly sensitive system for the automated detection of CPCs, with potential applications in initial screening, prognosis prediction, and posttreatment monitoring for MM patients. Diagnosing and monitoring multiple myeloma (MM), a type of blood cancer, requires identifying and quantifying specific cells called circulating plasma cells (CPCs) in the blood. The conventional method for detecting CPCs is manual microscopic examination, which is time-consuming and lacks sensitivity. This study introduces a highly sensitive CPC detection method using an artificial intelligence-based system, Morphogo. It demonstrated remarkable sensitivity and accuracy, surpassing conventional microscopy. This advanced approach suggests that early and accurate CPC detection is achievable by morphology examination, making efficient CPC screening more accessible for patients with MM. This innovative system has the potential to be used in the diagnosis and risk assessment of MM.

Distinct Genetic Features in Peripheral Blood Represent the Characteristics of Circulating Plasma Cells in Primary Plasma Cell Leukemia

Clonal plasma cells in peripheral blood (PB) are crucial for diagnosing plasma cell neoplasm. The diagnostic criteria for plasma cell leukemia (PCL) have recently been revised to 5% or more circulating plasma cells (CPCs) in PB. Considering that CPCs are an independent worse prognostic factor in multiple myeloma (MM), elucidating how many CPCs are sufficient for distinguishing patients between PCL and other plasma cell neoplasm is important yet uncertain. These CPCs have been recently discussed in MM, characterized by more immature phenotype, clonogenic potential, and high expression of cancer stem cell (CTC) markers. Though the exact mechanism of peripheral circulation still needs further studies, MM CTCs are considered subclones of bone marrow (BM) plasma cells. On the other hand, previous studies regarding PB plasma cells in primary PCL (pPCL) have been mainly focused on similar characteristics to BM plasma cells. Considering significant prognostic differences based on CPCs, PB from PCL patients should have distinct features. Previous studies on PCL revealed that high genetic heterogeneity and accumulation of diverse high-risk genetic abnormalities eventually led to the disease. Therefore, we aimed to investigate the genetic profile of CPCs in PCL to exhibit proper evidence for diagnostic criteria and improve the current practices. We retrospectively identified patients diagnosed with PCL from 2015 to 2017 at Seoul National University Hospital. Our study population included 4 pPCL and 2 secondary PCL (sPCL) patients. We retrospectively collected patients' clinical information and laboratory test results and performed targeted sequencing using the NGS panel of 647 genes related to hematologic malignancies. In all analyzed samples, 51 in PB and 40 variants in BM were detected in pPCL. The median number of variants in pPCL patients was 11 per patient (range 5-25) in PB and 10 per patient (range 3-17) in BM. When comparing variant allele frequency (VAF) between the paired samples, there was a moderate correlation (R=0.44, P&amp;lt;0.05). Similarly, a moderate correlation was observed in both pPCL (R=0.41, P&amp;lt;0.05) and sPCL (R=0.47, P&amp;lt;0.05) groups. When comparing paired variants between PB and BM, 38 were commonly detected in both samples in pPCL patients. Fifteen variants were identified in only one of the two samples, 12 in PB and 3 in BM. Most variants showed a heterogeneous distribution of genes. We checked the paired samples using an Integrative genomics viewer. As a result, twelve pPCL and thirteen sPCL variants were identified in the paired samples. These variants were initially excluded in the filtering process because of low VAF. Consequently, three unique variants were identified in each pPCL and sPCL group, which were considered truly unique. In pPCL patients, MED12, VPS13B variant from PB, and ZMYM3 variant from BM were identified in one of the paired samples. In sPCL patients, unique variants were identified in ARID1A, FANCE, and TP53. In addition, we determined that PB VAF is higher than BM VAF in shared variants of pPCL patients. Conversely, BM VAF tended to be higher than PB VAF in sPCL. However, plasma cell% was generally higher in BM than PB, regardless of pPCL or sPCL. Therefore, the differences in the percentage of plasma cells between PB and BM samples cannot explain the unique variants detected in only one of the paired samples. pPCL is an aggressive disease characterized by a markedly high diversity of heterogeneous mutation distribution and high mutational burden. The clinical outcomes of pPCL are still poor in the novel agent era. There is an urgent need to improve the current diagnosis and treatment strategy. As shown in this study, CPCs in pPCL are not simply part of the malignant plasma cells shed from the BM but have characteristics distinct from them. In this study, we exhibited that PB variants had higher VAF than BM variants, and some unique variants were identified only in the PB samples. Conversely, in sPCL, more diverse variants were detected mainly in BM samples, and VAF was also higher in BM than PB. Considering unique variants detected in PB and the high genetic heterogeneity of the disease, PB should be included in the diagnosis of PCL to determine the exact genetic profile.

A Prognostic Model Integrating Circulating Clonal Plasma Cells into R-ISS and PET-CT Based Tumor Burden in Transplant-Ineligible Multiple Myeloma

Introduction: Circulating clonal plasma cell (cCPC) is an easily obtained and non-invasive biomarker in multiple myeloma (MM), providing distinct prognostic information from the revised-international staging system (R-ISS). However, its prognostic relevance has been investigated primarily in transplant-eligible and clinical trial settings. Consequently, there is limited data on the impact of cCPC on outcomes and optimal cut-off values in transplant-ineligible (TIE) MM patients. Moreover, appropriate combinations to refine risk stratification, such as tumor burden measured by positron emission tomography-computed tomography (PET-CT), have yet to be fully explored. Methods: We performed a retrospective analysis on newly diagnosed TIE-MM patients with available peripheral blood (PB) flow cytometry (FCM) and PET-CT data from 2011 to 2021. Patients with primary plasma cell leukemia (n=8), defined as &amp;gt;5% monoclonal plasma cells in PB, were excluded from the study. Quantification of cCPC was performed using 6-color (2011-2016) or 8-color (2017-2021) FCM staining for CD19, CD56, CD38, CD138, cytoplasmic κ, and cytoplasmic λ to confirm monoclonality (Beckman Coulter, USA), which required at least 150000 mononuclear cell counts with the detectable limit at 0.01% (10 -4) based on previous reports (Gonsalves et al). Tumor burden was assessed by calculating total lesion glycolysis (TLG) using the following formula: the mean standardized uptake value (SUV) multiplied by the total tumor volume with an absolute SUV &amp;gt;2.5, using Metavol Software (Hokkaido University, Japan). Receiver operating curve analysis with the Youden index was employed to determine the optimal cutoff for predicting 2-year progression-free survival (PFS) and 5-year overall survival (OS). The predictive performance of R-ISS, cCPC, and TLG alone, along with their combinations, was compared using Harrell's concordance index (c-index). Results: 114 patients with TIE-MM were analyzed. The median age was 78 years (interquartile range [IQR] 73-83). The number of patients classified as R-ISS Ⅰ, Ⅱ, and Ⅲ was 12 (10.5%), 82 (71.9%), and 20 (17.5%), respectively. Most patients (85.1%) received proteasome inhibitor-based induction remission therapy. cCPCs were detectable in 90 patients (80.7%), with a median level of 6 ×10 -4 (IQR 1-22). Regarding tumor burden, 73 patients (64%) had at least one fluorodeoxyglucose avid lesion with a median TLG of 34.7 (IQR 0-268). cCPC levels were significantly correlated with CPC in bone marrow (R =0.24, P =0.01) but not with TLG (R =-0.01, P =0.88) and involved free light chain levels (R =0.07, P =0.41). The optimal cut-off values in predicting 2-year PFS and 5-year OS were 9 and 10 ×10 -4 for cCPC, and 325 and 343 for TLG, respectively. For simplicity, we classified &amp;gt;10 ×10 -4 for cCPC and &amp;gt;300 for TLG as high-risks. Notably, approximately two-thirds of the patients (62.3%) had at least one high-risk feature (Figure 1). Both high cCPC and TLG showed significant associations with worse OS and PFS (5-year OS rate: cCPC high vs. low, 40.2% vs. 69.3%; TLG high vs. low, 38% vs. 64.5%; 2-year PFS rate: cCPC high vs. low, 45.7% vs. 69.1%; TLG high vs. low, 46.4% vs. 64% all P &amp;lt; 0.05). These factors retained prognostic significance after adjustment for R-ISS on multivariate analysis. Finally, we constructed a novel predictive model integrating RISS, cCPC, and TLG. This model discriminated OS among patients with 0 (5-year OS rate, 80% [95% confidence interval 68.4%-93.6%]), 1 (50.5% [37.7%-67.7%]), and ≧2 factors (29% [13.3%-63.5%], P &amp;lt;0.001, Figure 2). The c-index was the highest (0.651) in this combined model compared to the single use of these risk factors (0.585, 0.594, and 0.569). Conclusions: We demonstrated the prognostic impact of cCPC, which is independently associated with R-ISS and TLG, on real-world TIE-MM patients. The new prediction model, integrated with these different clinical and biological features, may provide robust outcome discrimination.

Identification of signature genes and drug candidates for primary plasma cell leukemia: An integrated system biology approach

BackgroundPlasma cell leukemia (PCL) is one of the rare cancer which is characterized by the uncontrolled proliferation of plasma cells in peripheral blood and bone marrow. The aggressive behavior of the disease and high mortality rate among PCL patients makes it a thirst area to be explored. MethodsThe dataset for PCL was obtained from the GEO database and was analyzed using GEO2R for differentially expressed genes. Further, the functional enrichment analysis was carried out for DEGs using DAVID. The protein-protein interactions (PPI) for DEGs were obtained using STRING 11.5 and were analyzed in Cytoscape 3.7.2. to obtain the key hub genes. These key hub genes were investigated for their interaction with suitable drug candidates using DGIdb, DrugMAP, and Schrodinger's version 2022–1. ResultsOut of the total of 104 DEGs, 39 genes were up-regulated whereas 65 genes were down-regulated. A total of 11 biological processes, 2 cellular components, and 5 molecular functions were enriched along with the 7 KEGG pathways for the DEGs. Further, a total of 11 hub genes were obtained from the PPI of DEGs of which TP53, MAPK1, SOCS1, MBD3, and YES1 were the key hub genes. Oxaliplatin, mitoxantrone, and ponatinib were found to have the highest binding affinity towards the p53, MAPK1, and YES1 proteins respectively. ConclusionTP53, MAPK1, SOCS1, MBD3, and YES1 are the signature hub genes that might be responsible for the aggressive prognosis of PCL leading to poor survival rate. However, p53, MAPK1, and YES1 can be targeted with oxaliplatin, mitoxantrone, and ponatinib.

Secondary plasma cell leukemia: Case report

Plasma cell leukemia (PCL) is a rare lymphoproliferative disorder characterized by monoclonal proliferation of plasma cells in marrow and peripheral blood. It is defined by a blood plasma count greater than 2 G/l or a plasma cell count greater than 20% of white blood cells. There are two forms of PCL: primary PCL (primary plasma cell leukemia or PCL-P), which occurs de novo and is diagnosed by blood dissemination, and secondary PCL (secondary plasma cell leukemia or SPL), which is the result of the evolution of multiple myeloma (MM). We report an observation of secondary plasma cell leukemia in a 42-year-old female patient diagnosed at the hematology laboratory of the Mohamed VI University Hospital in Marrakech. The patient was being followed up for chronic end-stage renal failure in whom the etiological assessment of a hypercalcemia of fortuitous discovery revealed MM for which she was put on chemotherapy according to the Velcade-Thalidomide-Dexamethasone protocol with a good evolution after 6 courses. Two and a half years later, the patient presented with anemia and spinal pain. The biological work-up showed a normocytic normochromic anemia at 5.4 g/dl and hypercalcemia at 150 mg/l. The blood smear showed the presence of 22% of circulating plasma cells, the majority of which were dystrophic, in favor of secondary plasma cell leukemia. Patients diagnosed with multiple myeloma who have undergone treatment and have a stable disease should have regular surveillance, hence the interest of the blood smear examination in all multiple myeloma.

Circulating Tumor Plasma Cells in the Screened Istopmm Smoldering Multiple Myeloma Cohort

Background The presence and number of circulating tumor plasma cells (CTPCs) in peripheral blood (PB) as detected by flow cytometry has previously been associated with increased risk of progression from smoldering multiple myeloma (SMM) to multiple myeloma (MM). CTPCs may therefore be a marker for tumor burden and/or tumor aggressiveness in SMM that can, if the correlation to progression can be verified, potentially spare patients from bone marrow biopsies. However, the rate of detectable CTPCs in the overall SMM population is not known and the dynamics of CTPC numbers between diseases stages and risk categories in a screened cohort of individuals with SMM have not been studied. Aim To investigate the detection and quantification of CTPCs in individuals with SMM diagnosed in the screened population-based Iceland Screens, Treats, or Prevents Multiple Myeloma study (iStopMM), and to associate the findings to known clinical risk models of progression from SMM to MM. Methods Individuals diagnosed with SMM, monoclonal gammopathy of undetermined significance (MGUS) and MM in the iStopMM study were included. Next-generation flow (NGF) cytometry was used to assess CTPC numbers in PB and the limit of detection (LOD) was set at ≥20 tumor PC. An optimal cutoff value of the percentage of CTPC to discriminate between SMM and MM was found using receiver operating characteristic (ROC) curve analysis of individuals with CTPCs detected. In a subgroup of individuals with SMM, interphase fluorescent in situ hybridization (FISH) analysis was performed. The difference in frequency of CTPC was tested between risk groups according to the PETHEMA and Mayo 2/20/20 models, the groups with, and without high-risk FISH abnormalities and between clinical entities (MGUS, SMM and MM). The Mann-Whitney U test or the Kruskal-Wallis tests were used to assess differences observed between two or more than two groups, respectively. Results A total of 93 individuals with SMM, 29 with MM, and 103 with MGUS were included. CTPCs were detected in 64 (69%) of individuals with SMM, with a median frequency of 0.0009% (range 0.0002-0.078%), or 0.054 CTPC/μL (range 0.011-3.99 CTPC/μL). The median sensitivity reached for samples with undetectable CTPCs was 2.3x10-6 (range 4.8-1.7x10-6). The detection rate and concentration of CTPCs increased from MGUS to SMM and MM (p<0.001 for both, figure) and from low to higher risk categories according to both risk models (table and figure). The increase in frequency of CTPCs according to risk score was only significant for the PETHEMA model (p<0.001), but not the Mayo 2/20/20 model (p=0.19). However, when low-risk individuals were compared to the intermediate/high risk groups according to the Mayo 2/20/20 model, the difference reached statistical significance (p=0.05). An optimal cutoff of 0.0017% CTPCs for SMM vs. MM was found with a sensitivity of 0.70, specificity of 0.69, and area under the curve 0.76 (figure). In total, 20 (22%) individuals with SMM had CTPC over the cutoff. This proportion increased with higher Mayo 2/20/20 risk categories (20% in low-risk, 23% in intermediate-risk and 25% in high-risk SMM) and PETHEMA risk categories (18% in low-risk, 23% in intermediate-risk and 30% in high-risk SMM). In the 40 individuals with SMM who underwent FISH, 12 (30%) had high-risk FISH abnormalities (del(17p), t(4;14) or t(14;16)) present, and of those 75% had CTPCs detected vs. 71% in those without high-risk FISH abnormalities, no difference in CTPC frequency was found between the groups (p=0.11) Conclusion CTPCs are a potential marker of tumor burden or tumor aggressiveness in SMM and are present in 69% of the overall population with SMM. Both the detection and numbers of CTPCs increase progressively from MGUS, to SMM, and MM. A similar trend was observed with increasing risk score in SMM, according to two established risk stratification models. These results indicate that NGF of PB to detect CTPCs might have a role in the management of SMM. Future studies with serial monitoring of CTPCs will help to further define the clinical value of CTPCs in relation to the risk of progression, and to define how CTPC monitoring impacts the role of established markers, especially to identify individuals with low risk of progression where bone marrow sampling might be omitted. This would focus clinical care and treatments in SMM and MM to those who benefit the most from them while limiting interventions in those who do not need them. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment of Primary Plasma Cell Leukemia with Carfilzomib and Lenalidomide-Based Therapy: Results of the Final Analysis of the Prospective Phase 2 EMN12/HOVON-129 Study for Patients Aged 18-65 Years

Introduction. Primary plasma cell leukemia (pPCL) is a rare and aggressive plasma cell disorder with a very poor prognosis. The median PFS for transplant-eligible patients (pts) is approximately 9 months. The aim of the EMN12/ HOVON-129 study was to improve the outcome of pPCL by incorporating carfilzomib and lenalidomide in induction, consolidation, and maintenance. The trial enrolled 61 pts aged 18 years and older, with different treatment for those aged 18-65 (n=36) and ≥66 years (n=25). Here we report the results of the final analysis for pts aged 18-65 years. Methods. The EMN12/HOVON-129 study is a prospective, non-randomized, phase 2, multicenter study, for previously untreated pPCL pts. Inclusion criteria were newly diagnosed pPCL (defined as >2x109/L circulating monoclonal plasma cells or plasmacytosis >20% of the differential white cell count) and WHO performance status 0-3. Main exclusion criteria were severe cardiac or pulmonary dysfunction; and creatinine clearance of <15 ml/min. There were no restrictions based on blood counts. Pts aged 18-65 years were treated with induction therapy with four 28-day cycles of carfilzomib-lenalidomide-dexamethasone (KRd; carfilzomib 20/36 mg/m2 on days 1,2,8,9,15,16; lenalidomide 25 mg days 1-21; dexamethasone 20 mg on days 1,2,8,9,15,16,22,23). KRd induction was followed by tandem autologous stem cell transplantation (ASCT), 4 cycles of KRd consolidation, and then maintenance with carfilzomib (27 mg/m2 on days 1,2,15,16) and lenalidomide (10 mg on days 1-21/28 days) until progression. Pts who were eligible for allo-SCT, could also receive one ASCT, followed by 2 KRd consolidation cycles, reduced-intensity conditioning allo-SCT, and carfilzomib/lenalidomide maintenance. The primary endpoint was PFS, and secondary endpoints were response rate, OS, and toxicity. Results. From October 2015 till August 2021, we enrolled 36 pts with pPCL aged ≤65 years. Median age was 60 years; 67% had bone disease; and WHO performance status was ≥2 in 33% of pts. Pts had a high tumor burden with a median plasma cell percentage in BM of 80%. The median peripheral blood plasma cell count was 4.1x109/L (range 1.6-60.0); median platelet count was 111x109/L (range 20-517); and median eGFR was 54.0 ml/min (range 15.0-123). Pts had high-risk disease as evidenced by elevated LDH in 58%; and high frequency of high-risk genetic lesions (del(17p) in 47%, t(4;14) in 9%, t(14;16) in 19%, gain/ampl(1q) in 61%). Two or more high-risk cytogenetic abnormalities were present in 38%. The frequency of t(11;14) was 26%. In addition, 17% of pts had extramedullary plasmacytomas. ISS stage 3 was present in 64%, and revised ISS stage 3 in 50%. Response to induction was ≥PR in 72%, ≥VGPR in 64%, and ≥CR in 14%. Twenty-four pts (67%) received high-dose melphalan (HDM)/ASCT; 12 pts (33%) underwent a second course of HDM/ASCT; and 5 (14%) received allo-SCT after first HDM/ASCT. Eighteen pts (50%) received maintenance treatment. Best response on protocol was ≥PR in 83%, ≥VGPR in 81% and ≥CR in 50%; 10 (28%) pts achieved MRD-negative (10-5) CR. With a median follow-up of 40.8 months (range 8-2-74.5), the median PFS was 15.5 months (95% CI 9.4-38.4), which was sufficient to reject our null hypothesis (median PFS=9 months). With 19 pts having died (14 due to disease progression, 4 due to infections, and 1 due to cardiac disorder), median OS was 28.4 months (95% CI 15.1-not reached). In exploratory analyses, the presence of elevated LDH, t(14;16), and del(17p) were associated with both inferior PFS and OS. For patients who underwent first HDM/ASCT, median PFS and OS were 32.9 and 34.1 months from date of ASCT, resp. Adverse events mainly occurred during the first cycle of KRd, and decreased thereafter. Overall, grade ≥3 adverse events were observed in 67% of pts, including infections (25%) and cardiovascular disorders (14%). One patient developed a second primary malignancy (MDS). As of July 24 2022, 25 pts (69%) pts discontinued protocol treatment, mainly because of progression, but none because of treatment-related toxicity. Conclusions. KRd induction provides efficient and rapid disease control, which allows two-thirds of pts to undergo a first course of HDM/ASCT. Toxicity occurred mainly during the first cycle of induction, and was manageable with appropriate interventions. However, median PFS and OS remain low in pPCL, compared to MM. This trial was registered at https://trialsearch.who.int as NTR5350 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Carfilzomib and Lenalidomide for the Treatment of Primary Plasma Cell Leukemia: Final Results of the Prospective Phase 2 EMN12/HOVON-129 Study for Patients Aged ≥66 Years

Introduction. Primary plasma cell leukemia (pPCL) is a rare and aggressive plasma cell disorder with a very poor prognosis, requiring urgent action to prevent early mortality and to control the disease and complications. The EMN12/HOVON-129 study assessed the efficacy of carfilzomib and lenalidomide as part of first-line therapy in pPCL. The trial enrolled patients (pts) aged 18 years and older, with different treatment for those 18-65 and ≥66 years. Here we report the results for pts in the age-group ≥66 years. Methods. The EMN12/HOVON-129 study is a prospective, non-randomized, phase 2, multicenter study for previously untreated pPCL pts. Inclusion criteria were newly diagnosed pPCL (in this study defined as >2x109/L circulating monoclonal plasma cells and/or plasmacytosis >20% of the differential white cell count) and WHO performance status 0-3. Main exclusion criteria were severe cardiac or pulmonary dysfunction and creatinine clearance <15 ml/min. There were no restrictions based on blood counts. Pts ≥66 years received induction therapy with eight 28-day cycles of carfilzomib-lenalidomide-dexamethasone (KRd; carfilzomib 20/36 mg/m2 on days 1,2,8,9,15,16; lenalidomide 25 mg on days 1-21; dexamethasone 20 mg on days 1,2,8,9,15,16,22,23), followed by maintenance with carfilzomib (27 mg/m2 on days 1,2,15,16 for the first twelve cycles; then 56 mg/m2 on days 1,15) and lenalidomide (10 mg on days 1-21/28 days) until progression. The primary endpoint was progression-free survival (PFS); secondary endpoints were response rate, overall survival (OS), and toxicity. Results. From October 2015 to August 2021, we enrolled 61 pts with pPCL: 36 aged ≤65 years and 25 aged ≥66 years. Among the 25 pts aged ≥66 years, median age was 71 years (range 66-84); 68% had bone disease; and WHO performance status was 2 in 24% and 3 in 16% of pts. Median plasma cell percentage in bone marrow biopsy was 80% (10-100). Median peripheral blood plasma cell count was 3.8x109/L (range 2.0-52); median platelet count was 142x109/L (range 13-384); and median GFR was 56 ml/min (range 24-95). Most of pts had high-risk disease features, as proved by elevated LDH in 52% and high frequency of poor-risk genetic lesions (del(17p) in 20%, t(4;14) in 4%, t(14;16) in 4%, and gain/amp(1q) in 48%). In addition, 12% of pts had extramedullary plasmacytomas. ISS stage III was present in 68% and Revised ISS stage III in 40%. At the data cut-off (July 1, 2022), among the 25 pts, 17 (68%) received the planned 8 cycles of induction treatment, achieving ≥partial response (PR) in 80%, ≥very good (VG)PR in 68%, and ≥complete response (CR) in 32% of pts. Sixteen pts (64%) received maintenance treatment. Best response on protocol was ≥PR in 80%, ≥VGPR in 68% and ≥CR in 36%; 3 out of 4 pts in CR who could be evaluated for minimal residual disease (MRD) achieved MRD negativity (10-5) by flow cytometry. With a median follow-up of 24.6 months (range 7.9-59.6), the median PFS was 13.8 months (95% CI 9.2-35.5), which was sufficient to reject our null hypothesis (median PFS=6.5 months). With 16 pts having died (8 due to disease progression, 2 unknown, 1 pneumonia, 1 sepsis, 1 systemic aspergillus infection, 1 small intestinal SPM, 1 disseminated intravascular coagulation, 1 respiratory failure due to COPD exacerbation), median OS was 24.8 months (95% CI 14-not reached [NR]; Figure). Adverse events mainly occurred during the first two cycles of KRd and decreased thereafter. Overall, 36% of pts had grade (G)3 and another 36% G4 adverse events. G3 and G4 hematologic toxicity rates were 8% and 12%, respectively. Infections (20% and 16%) and respiratory events (16% and 4%) were the most common G3 and G4 non-hematologic toxicities. Twenty-two pts (88%) discontinued protocol treatment, mainly because of progression (13 pts, 59%). Conclusions. KRd is a potent treatment strategy to control disease in pPCL pts over 65 years of age, with not negligible, but generally manageable, treatment-related toxicities. Of note, along with a significant improvement of PFS, median OS substantially doubled compared to what has been reported in recent retrospective studies and, particularly, in the only other prospective trial (with the doublet lenalidomide-dexamethasone) so far conducted in transplant ineligible, elderly pts with pPCL (Musto P et al. Leukemia. 2014;28:222-5). This trial was registered at www.trialregister.nl (until June 2022) and https://trialsearch.who.int/ as NTR5350. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 641: Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation

Abstract Background: Multiple Myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM). MM is almost always preceded by the precursor stage smoldering multiple myeloma (SMM). BM biopsies are useful to monitor disease progression, but they are invasive and not routinely collected from patients for disease monitoring during precursor stages. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) could aid early detection, disease monitoring, and biomarker identification to predict patients at high risk of progression that may benefit from early therapeutic intervention. Methods: Paired PB and BM aspirates were collected from 40 SMM patients enrolled in the PCROWD study (IRB #14-174) at Dana-Farber Cancer Institute. Malignant PCs were enriched by magnetic bead-based methods and underwent 5’ single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics). Results: We analyzed 105,246 BM PCs and 33,234 PB PCs from 15 patients. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements, assessed by concurrent scBCR-seq. A total of 86,986 BM tumor cells and 8,718 CTCs were captured. A median of 5, 26, and 47 CTCs were present per mL of blood from low, intermediate, and high-risk SMM patients as defined by the International Myeloma Working Group (IMWG) “20/2/20” criteria, suggesting sequencing-based CTC enumeration corresponds to prognosis. High levels of driver genes commonly upregulated in patients with specific translocations, including CCND1 and MAF, were detected in both BM tumor and CTC clusters in 3 patients with t(11;14) and t(14;16) confirmed by fluorescence in situ hybridization (FISH) clinical testing, and 2 additional patients with inconclusive FISH results (Wilcoxon, q &amp;lt;10-3), supporting the idea of CTC-based prognostication. Differential expression (DE) analysis revealed 8 genes that were significantly upregulated and 3 genes that were significantly downregulated in CTCs compared to BM tumor cells robustly across 15 paired samples. Gene set enrichment analysis (GSEA) revealed genes DE in CTCs are associated with TNF-α and NF-κB signaling, which are commonly induced by extrinsic factors in the bone marrow milieu, providing insight into the biology of tumor cell circulation. Conclusions: This study highlights the utility of scRNA-seq for molecular profiling of CTCs, even in asymptomatic low tumor burden disease. Additional analyses are ongoing in the expanded cohort of 40 patients with paired samples to help gain further insight into CTC heterogeneity. Overall, this study will help enable the design of new molecular liquid biopsy-based approaches to diagnosis, disease monitoring, and biological insights to improve treatment strategies for precursor myeloma patients. Citation Format: Elizabeth D. Lightbody, Danielle T. Firer, Romanos Sklavenitis-Pistofidis, Michael Agius, Ankit K. Dutta, Michelle Aranha, Jean-Baptiste Alberge, Laura Hevenor, Nang Kham Su, Cody Boehner, Erica Horowitz, Jacqueline Perry, Anna Cowan, Hadley Barr, Anna Justis, Daniel Auclair, Catherine R. Marinac, Gad Getz, Irene Ghobrial. Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 641.

Clinical Significance of Circulating Clonal Plasma Cells Detected by a Novel Microfluidic Chip in Multiple Myeloma

Circulating Clonal Plasma cells (cCPCs) in peripheral blood is emerged as an important biomarker in evaluating the Minimal Residual Disease (MRD) status in Multiple myeloma (MM). The elevation of cCPCs is closely related to the recurrence of disease. Comparing to the conventional bone marrow aspirate, it can be accessed regularly in a noninvasive manner. Over the past decade, microfluidic techniques have been widely explored as a platform to segregate the rare cells in blood with the advantages of easy manipulation and low cost. Recently, we and several other groups have reported the successful development of cCPCs-specific microfluidic chip. However, there had been no comparative study of the performance of microfluidic chip to currently available clinical MRD prognostic tools, so we used our previously developed microfluidic platform to explore its clinical utility in comparison with existing methods of Multiparameter Flow Cytometry (MFC) and Serum Protein Electrophoresis analysis. The MRD test results from the 19 MM patients showed 89.47% of overall agreement between MFC and microfluidic chip. Through the monitoring of 2 MM patients before and after treatment, a similar fluctuation pattern was observed between the cCPCs level detected by microfluidic chip and the paraprotein level detected by Serum Protein Electrophoresis.

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

Genomics of Plasma Cell Leukemia.

Plasma cell leukemia (PCL) is a rare and highly aggressive plasma cell dyscrasia characterized by the presence of clonal circulating plasma cells in peripheral blood. PCL accounts for approximately 2-4% of all multiple myeloma (MM) cases. PCL can be classified in primary PCL (pPCL) when it appears de novo and in secondary PCL (sPCL) when it arises from a pre-existing relapsed/refractory MM. Despite the improvement in treatment modalities, the prognosis remains very poor. There is growing evidence that pPCL is a different clinicopathological entity as compared to MM, although the mechanisms underlying its pathogenesis are not fully elucidated. The development of new high-throughput technologies, such as microarrays and new generation sequencing (NGS), has contributed to a better understanding of the peculiar biological and clinical features of this disease. Relevant information is now available on cytogenetic alterations, genetic variants, transcriptome, methylation patterns, and non-coding RNA profiles. Additionally, attempts have been made to integrate genomic alterations with gene expression data. However, given the low frequency of PCL, most of the genetic information comes from retrospective studies with a small number of patients, sometimes leading to inconsistent results.

A broadly neutralizing human monoclonal antibody against the hemagglutinin of avian influenza virus H7N9.

Background:The new emerging avian influenza A H7N9 virus, causing severe human infection with a mortality rate of around 41%. This study aims to provide a novel treatment option for the prevention and control of H7N9.Methods:H7 hemagglutinin (HA)-specific B cells were isolated from peripheral blood plasma cells of the patients previously infected by H7N9 in Jiangsu Province, China. The human monoclonal antibodies (mAbs) were generated by amplification and cloning of these HA-specific B cells. First, all human mAbs were screened for binding activity by enzyme-linked immunosorbent assay. Then, those mAbs, exhibiting potent affinity to recognize H7 HAs were further evaluated by hemagglutination-inhibiting (HAI) and microneutralization in vitro assays. Finally, the lead mAb candidate was selected and tested against the lethal challenge of the H7N9 virus using murine models.Results:The mAb 6-137 was able to recognize a panel of H7 HAs with high affinity but not HA of other subtypes, including H1N1 and H3N2. The mAb 6-137 can efficiently inhibit the HA activity in the inactivated H7N9 virus and neutralize 100 tissue culture infectious dose 50 (TCID50) of H7N9 virus (influenza A/Nanjing/1/2013) in vitro, with neutralizing activity as low as 78 ng/mL. In addition, the mAb 6-137 protected the mice against the lethal challenge of H7N9 prophylactically and therapeutically.Conclusion:The mAb 6-137 could be an effective antibody as a prophylactic or therapeutic biological treatment for the H7N9 exposure or infection.

Patients with treated autoimmune hepatitis and persistent suppression of plasmacytoid dendritic cells: A different point of view.

Objectives: Plasmacytoid dendritic cells (pDCs) have been shown to have a role in autoimmune diseases, but their role in Autoimmune Hepatitis (AIH) is not completely clear. In the present study, we assessed the frequency of pDCs in peripheral blood of AIH patients under long-term standard immunosuppressive therapy. Methods: This cross-sectional analysis enrolled 27 AIH patients and 27 healthy controls. We analyzed and compared their proportion of pDCs, CD4+, CD8+, γδ T cells, CD25+ regulatory T (Treg) cells, FoxP3+, Foxp3+CD39+ Treg cells, total B (CD19+) cells, and plasma cells (CD38+) in peripheral blood using flow cytometry immunophenotyping. Results: AIH patients had a lower percentage of pDCs (median frequencies of 0.2% vs. 0.4%; p = .002) and higher expression of CD8 T cells (32.5% vs 28.6%; p = 0.008) in peripheral blood, when compared to healthy controls. We did not find statistically significant differences between the groups regarding the other cell subtypes.Conclusion: Our data suggest a persistent suppression of pDCs in AIH patients, along with increased CD8 T cell activity, years after AIH diagnosis and despite of good clinical response to treatment, thus pointing to a role of pDCs in the AIH pathogenesis.

Identification of HBEGF+ fibroblasts in the remission of rheumatoid arthritis by integrating single-cell RNA sequencing datasets and bulk RNA sequencing datasets

BackgroundFibroblasts are important structural cells in synovium and play key roles in maintaining the synovial homeostasis. By single-cell RNA sequencing (scRNA-seq), subpopulation of synovium-resident cells has been reported to protect intra-articular structures from chronic inflammation and promote tissue repair. However, a significant number of researchers have concentrated on the role of fibroblasts in the progress of rheumatoid arthritis (RA) while few reports had described the contribution of distinct fibroblast subsets in the RA remission. It is helpful to understand the role of fibroblast subpopulations in the RA process to provide predictive biomarkers and address RA remission mechanisms. Here, we found HBEGF+ fibroblasts that contributed to RA remission by integrating scRNA-seq datasets and bulk RNA sequencing (bulk RNA-seq) datasets.MethodThree single-cell RNA datasets of cells harvested from RA patients were processed and integrated by Seurat and Harmony R packages. After identifying cell types by classic marker genes, the integrated dataset was used to run CellChat for analysis of cell-cell communication. Specially, EGF signaling pathway was found and HBEGF+ fibroblasts were identified based on HBEGF expression. Differential expressed genes of HBEGF+ were shown in heatmap and volcano plot and used to run gene ontology (GO) enrichment analysis. Next, bulk RNA-seq datasets of synovium under different conditions (health, osteoarthritis (OA), rheumatoid arthritis, before and after classical treatment) were compared to show expression change of HBEGF and gene markers that are mainly expressed by HBEGF+ fibroblasts such as CLIC5, PDGFD, BDH2, and ENPP1. Finally, two single-cell RNA sequencing datasets of synovial cells from mice were integrated to identify Hbegf+ fibroblasts and calculate the population of Hbegf+ fibroblasts under different joint conditions (health, K/BxN serum transfer arthritis (STA), and remission of STA).ResultAfter integrating three single-cell RNA sequencing datasets, we identified 11 clusters of synovial cells, such as fibroblasts, mural cells, endothelial cells, CD4+ T cells, CD8+ T cells, natural killer cells, synovium macrophage, peripheral blood macrophages, plasma cells, B cells, and STMN1+ cells. We found fibroblasts had an extensive communication network with other clusters and interacted with synovial macrophages through EGF signaling pathway via analysis of cell-cell communication between synovial cells. HBEGF, ligand to EGF signaling pathway, was highly expressed by a subset of fibroblasts and macrophages, and EGFR, receptor to EGF signaling pathway, was highly expressed by fibroblasts and meniscus cells. Moreover, HBEGF was downregulated under RA state and it had an increase after classical treatment. We then defined fibroblasts with high expression of HBEGF as HBEGF+ fibroblasts. In addition, we also found that HBEGF+ fibroblasts highly expressed CRTAC1, ITGB8, SCARA5, THBS4, and ITGBL1, genes relative to encoding extracellular matrix proteins and engaged in positive regulation of cell migration and motility, cellular component movement, and cell growth by GO enrichment analysis. We eventually identified HBEGF+ fibroblasts specially expressed CLIC5, PDGFD, BDH2, and ENPP1, which positively correlated with the expression of HBEGF. Moreover, the expression of CLIC5, PDGFD, BDH2, and ENPP1 was downregulated under RA state and elevated by classical therapy. On the contrary, the HBEGF+ macrophages specially expressed SLAMF8, GK, L1RN, and JAK2, which negatively correlated with the expression of HBEGF. The expression was upregulated in SLAMF8, GK, L1RN, and JAK2 under the RA state, whereas it was decreased after classical treatment. In mice, the number of Hbegf+ fibroblasts was reduced in the RA synovium but increased in the RA remitting synovium.ConclusionsHBEGF+ fibroblasts play a role in the remission of rheumatoid arthritis, and HBEGF has potential to become a novel biomarker for prediction of RA progress.

Primary plasma cell leukemia: consensus definition by the International Myeloma Working Group according to peripheral blood plasma cell percentage

Primary plasma cell leukemia (PCL) has a consistently ominous prognosis, even after progress in the last decades. PCL deserves a prompt identification to start the most effective treatment for this ultra-high-risk disease. The aim of this position paper is to revisit the diagnosis of PCL according to the presence of circulating plasma cells in patients otherwise meeting diagnostic criteria of multiple myeloma. We could identify two retrospective series where the question about what number of circulating plasma cells in peripheral blood should be used for defining PCL. The presence of ≥5% circulating plasma cells in patients with MM had a similar adverse prognostic impact as the previously defined PCL. Therefore, PCL should be defined by the presence of 5% or more circulating plasma cells in peripheral blood smears in patients otherwise diagnosed with symptomatic multiple myeloma.

High-Risk Gene Expression Subtype Provides Molecular Basis for Different Clinical Presentation of Primary and Secondary Plasma Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma where neoplastic plasma cells (PCs) lose dependence on the bone marrow (BM) and circulate in the peripheral blood (PB). PCL is clinically defined by diagnosis of myeloma with ≥20% circulating plasma cells (CPCs), however, several groups have proposed a ≥5% CPC cutoff. PCL is classified as primary (pPCL) if it presents at myeloma diagnosis or secondary (sPCL) if it arises at a later progression event. These presentations of PCL are clinically distinct, with sPCL patients responding poorly to novel therapies and having inferior outcomes compared to pPCL patients. Despite recent advances in myeloma therapy, PCL prognosis remains poor, and the molecular drivers of PCL remain poorly understood.The MMRF CoMMpass study (NCT01454297) is a longitudinal, observational clinical study of 1171 newly-diagnosed myeloma patients. Tumors were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. PCs were isolated from BM-derived tumors and when >5% CPCs were detected, PCs were also isolated from the PB, creating a subcohort of patients with sequencing data from both the BM and PB compartments, with some patients assayed longitudinally.The percent CPCs determined using flow cytometry was reported for 982 patients at myeloma diagnosis and 194 patients at progression. Patients with 5-20% CPCs (median = 19 months) at diagnosis had poor overall survival (OS) outcomes compared to those with less than 5% CPCs (median = 95 months, p<0.001). No outcome difference was observed between patients with 5-20% and >20% CPCs (median = 41 months), confirming the findings of previous independent studies. A ≥5% CPC cutoff identified 947 myeloma, 29 pPCL, and 6 sPCL patients in the CoMMpass cohort. Compared to myeloma, pPCL and sPCL patients had poor OS (p<0.001), and after sPCL detection patients had a median OS of only 53 days (range = 0-169 days). For 10 pPCL patients, the percent CPCs was reported at diagnosis and at least one progression event, and patients with persistent CPCs (n = 5, median = 16 months, p<0.01) had poor OS compared to patients with no detectable CPCs at progression (n = 5, median not met, median follow up = 64 months). This underscores the benefit of early eradication of CPCs and repeated CPC measurements in pPCL.The proliferative (PR) gene expression subtype of myeloma has been previously described and defines a high-risk group of patients with diverse genetic backgrounds and inferior outcomes. For PCL patients, we determined the subtype of all BM and PB tumor samples characterized using RNAseq. There was high subtype concordance between paired BM and PB tumor samples (12/13, 92.3%). Overall, 6/23 (26.1%) pPCL patients were in the PR subtype, and PR pPCL patients had poor OS outcomes (median = 10 months, p<0.001) compared to non-PR pPCL patients (median = 55 months). PR emerged as a robust predictor of risk in pPCL, outperforming other molecular and clinical variables including high BM or PB PCs, plasmacytomas, renal failure, high LDH, high B2M, low platelets, t(11;14), del(1p), amp(1q), del(13q), and del(17p), suggesting that RNA subtyping CPCs may represent a non-invasive tool to predict risk in pPCL.At myeloma diagnosis, all sPCL patients with RNAseq data were classified in non-PR subtypes. However, at sPCL, 5/6 (83.3%) patients were in the PR subtype, indicating that sPCL is associated with transition to PR. Two sPCL patients that transitioned to PR acquired biallelic deletion of CDKN2C, and a third acquired biallelic deletion of RB1. Overall, a subset of pPCL (26.1%) but the majority of sPCL (83.3%) patients were in the PR subtype at PCL diagnosis, providing a molecular basis for the different clinical presentations observed between these two groups, including the highly-aggressive nature of sPCL. In summary, this study supports using a lower percent CPC cutoff to clinically define PCL and highlights the importance of repeated CPC measurements in prognosticating pPCL patients. Further, PR RNA subtype emerged as a predictor of risk in pPCL and, given that the majority of sPCL patients were in the PR subtype, provides a molecular basis for the different clinical features observed between pPCL and sPCL patients. DisclosuresMikhael: Amgen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Sanofi: Consultancy; GSK: Consultancy; Oncopeptides: Consultancy.