scholarly journals Significant Plasma Cell Loss (up to 90%) Despite Preserved Overall Cell Viability - Time Dependent Changes Observed in Multiparametric Flow Cytometry Analysis of Bone Marrow Samples in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4006-4006
Author(s):  
Tukten Rolfe ◽  
Quirine O'Loughlin ◽  
Heather Campbell ◽  
Jordan Barr ◽  
Fiona Shawyer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Yield ◽  
Relative Reduction ◽  
Sample Processing ◽  
Multiparametric Flow Cytometry

Abstract Multiparametric flow cytometry (MPFC) is a mainstream laboratory method used in the diagnosis of multiple myeloma. Minimal residual disease (MRD) assessment by EuroFlow next-generation flow cytometry allows assessment down to an assay sensitivity of 1x10 -5. Delayed sample processing remains a common challenge due to logistical limitations. Specialized tests performed in central pathology laboratories are frequently located a considerable distance from healthcare providers. Our study aims to evaluate the impact of delayed sample processing on plasma cell yield and bone marrow sample stability. There is little published data available. Plasma cell yield and bone marrow sample stability were investigated in patients with multiple myeloma who underwent bone marrow biopsy. Participants were included based on ³10% plasma cell burden by morphological quantification on the bone marrow aspirate smear. Bone marrow aspirates were collected in EDTA (with three samples also collected in lithium heparin) and stored at four degrees Celsius. Samples were analyzed by MPFC within four hours of collection, at 24 and at 48 hours after collection. CD138 and CD38 co-expression were used to identify plasma cells, and absence of 7-AAD to determine cell viability. Mean fluorescence intensity (MFI) of CD138 and CD38 was recorded. Statistical analyses were performed using two-tailed Wilcoxon signed-rank tests and repeated measures ANOVA with significance assigned at p<0.05. Bone marrow aspirate samples of nine participants were evaluated. Significant reduction in plasma cell yield was observed over time (p<0.001) while sample integrity remained unchanged (p>0.05). The most marked reduction in plasma cell detection was seen between initial processing and 24 hours (median absolute reduction 9%, range 0 to 23% and median relative reduction 37%, range -8 to 90%, p<0.01). Further significant reduction of plasma cells occurred after an additional 24 hours (p=0.025). At 48 hours, the median absolute reduction in plasma cell yield from initial testing was 12% (range 1 to 24%) and median relative reduction was 40% (range 18 to 90%). Sample integrity remained constant. The median viability at collection, 24 hours and 48 hours was 91%, 93% and 95% respectively. The most significant specimen deterioration observed was 13% viability reduction to 75% overall by 48 hours. Three of the participants had additional samples collected in lithium heparin anticoagulant media that were analyzed in parallel with their EDTA samples. Plasma cell yield remained similar across the two different anticoagulants with overall cell viability remaining high in lithium heparin (³90%). A trend of time-dependent reduction of CD138 MFI was observed with lithium heparin but not with EDTA. This study demonstrates the significance of time to processing as a pre-analytical variable in MPFC in multiple myeloma. The greatest loss of plasma cells occurs within the first 24 hours after collection but continues to fall significantly out to 48 hours. Reductions of up to 90% were observed in our small cohort and represent a potential 1 log reduction in yield. This decrease in plasma cell yield raises questions of reliability and validity of flow cytometry, whereby the sensitivity depth may be compromised if the sample cannot be processed on the same day of collection. It is a technical limitation of flow cytometry in comparison to polymerase chain reaction methods where sensitivity is unaffected by delays in processing. The overall viability of cells within the samples remained stable over time, despite the decline in plasma cells. A reduction in CD138 MFI is observed in lithium heparin storage medium that may impact on standardized gating techniques. Further validation studies are warranted to explore these phenomena. MRD monitoring in multiple myeloma is rapidly becoming an accepted standard of care in the evaluation of treatment response and represents an independent prognostic maker of progression free survival that can be used to guide further therapy. Our findings indicate the potential of false negative MRD results with delays in sample processing. This questions the current consensus guidelines that recommend samples can be processed up to 2 days after collection. These guidelines may need to be revised in the near future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Daratumumab Interferes with Flow Cytometric Evaluation of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5630-5630 ◽  
Author(s):  
Sudhir Perincheri ◽  
Richard Torres ◽  
Christopher A Tormey ◽  
Brian R Smith ◽  
Henry M Rinder ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Population ◽  
Plasma Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
Kappa Light Chain ◽  
Positive Events ◽  
History Of

Abstract The diagnosis of multiple myeloma (MM) requires the demonstration of clonal plasma cells at ≥10% marrow cellularity or a biopsy-proven bony or extra-medullary plasmacytoma, plus one or more myeloma-defining events. Clinical laboratories use multi-parameter flow cytometry (MFC) evaluation of cytoplasmic light chain expression in CD38-bright, CD45-dim or CD138-positive, CD45dim cells to establish plasma cell clonality with a high-degree of sensitivity and specificity. Daratumumab, a humanized IgG1 kappa monoclonal antibody targeting CD38, has been shown to significantly improve outcomes in refractory MM, and daratumumab was granted breakthrough status in 2013. Daratumumab is currently approved for treatment of MM patients who have failed first-line therapies. It has been noted that daratumumab can interfere in blood bank assays for antibody screening, as well as serum protein electrophoresis (SPEP). We describe for the first time daratumumab interference in the assessment of plasma cell neoplasms by MFC; daratumumab interfered with both CD38- and CD138-based gating strategies in three MM patients. Patient A is a 68 year old man with a 10 year history of MM who had failed multiple therapies. He had then been treated with daratumumab for two months, stopping therapy 25 days prior to bone marrow assessment. Patient B is a 53 year old man with a 3 year history MM who had failed numerous treatments. He had been receiving daratumumab monotherapy for two months at the time of his bone marrow studies. On multiple marrow aspirates at times of relapse prior to receiving daratumumab, both patients had demonstrated CD38-bright positive CD45dim/negative plasma cells expressing aberrant CD56, as well as kappa light chain restriction; mature B cells were polyclonal in both. Patient C is a 65 year old man with a four-year history of MM status post autologous stem cell transplantation, who had been receiving carfilzomib and pomalidomide following relapse and continues to have rising lambda light chains and rib pain. He now has abnormal plasma cells in blood worrisome for plasma cell leukemia. Bone marrow aspirates from patients A and B, and blood from patient C demonstrated near absence of CD38-bright events as detected by MFC (Figure 1). Hypothesizing that these results were due to blocking of the CD38 antigen by daratumumab, gating on CD138-positive events was assessed; surprisingly, virtually no CD138-positive events were detected by MFC. All 3 samples demonstrated a CD56-positive CD45dim population; when light chain studies were employed using specific gating on the CD56-positive population, light chain restriction was demonstrated in all patients (Figure 1). Aspirate morphology confirmed numerous abnormal, nucleolated plasma cells (Figure 2A), thus excluding a sampling error. CD138 and CD38 expression was also tested on the marrow biopsy cores from both patients. In contrast to MFC, immunohistochemistry (IHC) showed positive labeling of plasma cells with both CD138 (Figure 2B) and CD38 (Figure 2C). The reason for the labeling discrepancy between MFC and IHC is unknown. The different antibodies in the assays may target different epitopes; alternatively, tissue fixation/decalcification may dissociate the anti-CD38 therapeutic monoclonal from its target. Detection of clonal plasma cell populations is important for assessing response to therapy. Laboratories relying primarily on MFC to assess marrow aspirates without a concomitant biopsy may falsely diagnose remission or significant disease amelioration in daratumumab-treated patients. MFC is generally highly sensitive for monitoring minimal residual disease (MRD) in MM, but daratumumab-treated patients should have their biopsy evaluated to confirm the MRD assessment by MFC. We were able to detect large numbers of plasma cells and also demonstrate clonality in our patients based on an alternative MFC marker, aberrant CD56 expression, an approach that may not be possible in all cases. Figure 1 Flow cytometry showing near-absence of CD38-bright elements in the marrow of patient A (top panels). Gating on CD56-positive cells in the same sample reveals a kappa light chain-restricted plasma cell population (bottom panels). Figure 1. Flow cytometry showing near-absence of CD38-bright elements in the marrow of patient A (top panels). Gating on CD56-positive cells in the same sample reveals a kappa light chain-restricted plasma cell population (bottom panels). Figure 1 The marrow aspirate from Fig. 1 shows abnormal plasma cells (A). Immunohistochemistry on the concomitant biopsy shows the presence of numerous CD138-positive (B) and CD38-positive (C) plasma cells. Figure 1. The marrow aspirate from Fig. 1 shows abnormal plasma cells (A). Immunohistochemistry on the concomitant biopsy shows the presence of numerous CD138-positive (B) and CD38-positive (C) plasma cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Molecular Characterization of Bone Marrow and Peripheral Blood Compartments from Patients with Plasma Cell Leukemia in the Mmrf Commpass Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1782-1782
Author(s):  
Sheri Skerget ◽  
Austin Christofferson ◽  
Sara Nasser ◽  
Christophe Legendre ◽  
The MMRF CoMMpass Network ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Cycle Control ◽  
Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma (MM) where neoplastic plasma cells (PCs) escape the bone marrow (BM) and circulate in the peripheral blood (PB). Traditionally, PCL is defined by the presence of >20% circulating plasma cells (CPCs), however, recent studies have suggested that PCL be redefined as the presence of >5% CPCs. The Multiple Myeloma Research Foundation CoMMpass study (NCT01454297) is a longitudinal, observational clinical study with 1143 newly diagnosed MM patients. BM-derived MM samples were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. When >5% CPCs were detected by flow cytometry, PCs were enriched independently from both compartments, and T-cells were selected from the PB as a control for WGS and WES. This substudy within CoMMpass provides the largest, most comprehensively characterized dataset of matched MM and PCL samples to date, which can be leveraged to better understand the molecular drivers of PCL. At diagnosis, 813/1143 CoMMpass patients had flow cytometry data reporting the percent PCs in PB, of which 790 had <5%, 17 had 5-20%, and 6 had >20% CPCs. Survival analyses revealed that patients with 5-20% CPCs (median = 20 months) had poor overall survival (OS) outcomes compared to patients with <5% CPCs (median = 74 months, p < 0.001), and no significant difference in outcome was observed between patients with 5-20% and >20% (median = 38 months) CPCs. Patients with 1-5% CPCs (median = 50 months, HR = 2.45, 95% CI = 1.64 - 3.69, p < 0.001) also exhibited poor OS outcomes compared to patients with <1% CPCs (median = 74 months), suggesting that patients with >1% CPCs are a higher risk population, even if they do not meet the PCL threshold. Using a cutoff of >5% CPCs, 23/813 (2.8%) patients presented with primary PCL (pPCL) at diagnosis. Of these patients, 7 (30%) were hyperdiploid (HRD), of whom 1 had a CCND1 and 1 had a MYC translocation; while 16 (70%) were nonhyperdiploid (NHRD), all of whom had a canonical immunoglobulin translocation (6 CCND1, 5 WHSC1, 3 MAF, 1 MAFA, and 1 MAFB). Of 124 patients with serial sample collections, 5 (4%) patients without pPCL had >5% CPCs at progression, and thus relapsed with secondary PCL (sPCL). Of the 5 sPCL patients, 2 (40%) were NHRD with a CCND1 or MAF translocation; while 3 (60%) were HRD, 1 with a WHSC1 translocation. Median time to diagnosis of sPCL was 22 months (range = 2 - 31 months), and patients with sPCL (median = 22 months) and pPCL (median = 30 months) exhibited poor OS outcomes as compared to MM patients (74 months, p < 0.001). Sequencing data was available for 15 pPCL and 5 sPCL samples. For 12 patients with WES, WGS, and RNAseq performed on their PCL tumor sample, an integrated analysis identified recurrent, complete loss-of-function (LOF) events in only CDKN2C/FAF1, SETD2, and TRAF3. Five pPCL patients had complete LOF of a gene involved in G1/S cell cycle control, including CDKN2C, CDKN2A, CDKN1C, and ATM. These LOF events were not observed in NHRD t(11;14) PCL patients, suggesting that CCND1 overexpression and LOF of genes involved in G1/S cell cycle control may represent independent drivers of PCL. Comparing WES and WGS data between matched MM and PCL tumor samples revealed a high degree of similarity in mutation and copy number profile. However, differential expression analysis performed for 13 patients with RNAseq data comparing their MM and PCL tumors revealed 27 up- and 39 downregulated genes (padj < 0.01, FDR = 0.1) in PCL versus MM. Pathway analysis revealed an enrichment (p < 0.001) for genes involved in adhesion and diapedesis, including upregulation of ITGB2, PF4, and PPBP, and downregulation of CCL8, CXCL12, MMP19, and VCAM1. The most significantly downregulated gene in PCL (log2FC = -6.98) was VCAM1, which plays a role in cell adhesion, and where loss of expression (TPM < 0.01) was observed across all PCL samples. Upregulation of four S100 genes including S100A8, S100A9, S100A12, and S100P, which have been implicated in tumor growth, metastasis, and immune evasion, was also observed in PCL. Interestingly, a S100A9 inhibitor has been developed and may represent a novel treatment option for PCL patients. In summary, PCL was found to be associated with molecular events dysregulating G1/S cell cycle control coupled with subtle changes in transcription that likely occur in a subclonal population of the MM tumor. Disclosures Lonial: Genentech: Consultancy; GSK: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy.

High Frequency of Aberrant Phenotype in Multiple Myeloma Patients Detected by Multiparametric Flow Cytometry.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4862-4862
Author(s):  
Luiz Arthur Calheiros ◽  
Eliza Y.S. Kimura ◽  
Manuella S.S. Almeida ◽  
Maria de Lourdes L.F. Chauffaille ◽  
Jandey G. Bigonha ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
High Frequency ◽  
Plasma Cells ◽  
Residual Disease ◽  
Lymphoproliferative Disease ◽  
Multiparametric Flow Cytometry ◽  
Normal Plasma ◽  
Aberrant Phenotype

Abstract Multiple Myeloma (MM) is a B cell lymphoproliferative disease with clonal plasma cell accumulation in bone marrow. Multiparametric flow cytometry (MFC) is an usefull tool to distinguish MM cells from normal plasma cells. Normal plasma cells are characterized by the expression of CD19+, CD45++, CD38++, CD138++, cytoplasmic immunoglobulin light chains (κ and λ) and CD56- while most MM plasma cells lose CD19, CD45 and gain CD56. In addition, many other antigens may be expressed by myeloma cells such as myeloid or lymphoid lineage associated antigens and these abnormal antigen expression is known as aberrant phenotype (AP). We studied 29 MM patients at diagnosis, in attempt to evaluate AP, it’s frequency and relation to prognostic parameters. The following monoclonal antibodies were used: CD45, CD38, CD138, CD56, CD19, CD20, CD22, CD10, CD13, CD14, CD33, CD117, CD28 and CD40, conjugated to FITC, PE, PerCP and APC) and acquisition / analysis were done through flow cytometer (FACS calibur, BD, San Jose) using CELL QUEST software (BD). Plasma cells were identified by the expression of CD38, CD138 and CD45 and the monoclonality confirmed by immunoglobulin light chain restriction. Our results showed presence of at least 2 AP in all cases : 2 AP (7 patients), 3 AP (12 patients), 4 AP( 5 cases), 5 AP (4 cases) and 8 AP in one case. The most frequent APs were CD45−, CD56+, CD117+, CD13+, CD33+, and were observed in 88% of patients. The most frequent AP association was CD56+/CD45− (40%), followed by myeloid antigen associated phenotypes (CD117, CD33, CD13). The lymphoid antigens expression was more observed in patients with large number of AP (>4 AP). CD56- patients presented serum β2-microglobulin and ionic calcium labeling levels higher than CD56+ patients (p=0,02) showing the usefulness of this antigen as prognostic marker. Morphological analysis showed that the majority (55%) of plasmablastic cases expressed >2 myeloid antigens against 18% of mature plasma cell morphology cases. These results allow us to conclude that MM express high frequency of AP, highlighting the importance of CD56 as a prognostic factor. MFC may be useful to the immunological detection of minimal residual disease in a great majority of MM patients and we suggest the panel CD45, CD56, CD117, CD33 and CD13 for this purpose, in addition to CD38 and CD138.

CD66 Expression on Malignant and Normal Plasma Cells: A Potential Target for Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5073-5073 ◽  
Author(s):  
Deborah Richardson ◽  
Elizabeth Hodges ◽  
Adnan Mani ◽  
Kim Orchard
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Normal Individual ◽  
Conditioning Regimen ◽  
Becton Dickinson ◽  
Multiparametric Flow Cytometry ◽  
Plasma Cell Disorders ◽  
Dual Expression

Abstract CD66, a member of the carcinoembryonic antigen family, is known to be expressed on cells of myeloid and monocytic origin and has also been demonstrated on blasts from patients with B-lineage acute lymphoblastic leukaemia. An analysis of CD66 expression has been undertaken in bone marrow samples from patients with plasma cell disorders. Diagnostic bone marrow aspirate samples from 53 patients with multiple myeloma or monoclonal gammopathy were examined by multiparametric flow cytometry in order to demonstrate and quantitate plasma cells. Samples were analysed initially using a primary screening panel of antibodies including a combination of antiCD19 fluoroscein isothiocyanate (FITC)(Pharminogen), antiCD5 phycoerythrin (PE)(in-house), antiCD45 peridin chlorophyll protein (PerCP)(Becton Dickinson) and antiCD38 allophycocyanin (APC)(Phar). Samples shown to contain a CD45 negative, CD19 negative, CD38 positive population, consistent with the presence of plasma cells were then examined with a myeloma panel, including antiCD66. Samples from 41 patients were analysed using antiCD66 naked antibody (TheraPharm, GmBH) double-layered with sheep anti-mouse IgG F(ab’)2 fragment conjugated to FITC, antiCD138 PE, antiCD38 APC and antiCD45 PerCP. The correlation between CD38/CD138 and CD38/CD66 dual expression was 0.997. A further 13 samples were analysed using a commercial antiCD66 antibody conjugated to FITC (Dako). Again the correlation between CD38/CD138 and CD38/CD66 dual expression was 0.990. All patients apart from one, with morphologically detectable plasma cells, co-expressed CD66 with CD38. One patient with highly plasmablastic morphology did not express either CD38/138 or CD38/66 on plasma cells.The bone marrow of a normal individual was also examined and found to contain plasma cells which co-expressed CD19, CD138, CD38 and CD66. Plasma cells express CD66 in almost all patients with plasma cell disorders and expression has also been demonstrated on plasma cells in a normal individual. CD66 is therefore an attractive target for immunotherapy. A clinical trial using anti-CD66 targeted radiotherapy as part of the conditioning regimen for patients undergoing autologous or allogeneic stem cell transplantation, as therapy for multiple myeloma, is currently being conducted at our institution.

The Location of Multiple Myeloma Adhesion Variants in Diverse Niches within the Bone Marrow Affects Plasma Cells Enumeration.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5063-5063
Author(s):  
Liat Nadav ◽  
Ben-Zion Katz ◽  
Shoshana Baron ◽  
Lydia Lydia ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Gene Expression Profile ◽  
Plasma Cell ◽  
Expression Profile ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Morphological Evaluation

Abstract Background - The diagnosis of multiple myeloma (MM) is based on clinical and laboratory criteria combined with bone marrow (BM) plasmocytosis, estimated by inspection of bone marrow aspirates. Recent advances in flow-cytometry (FCM) have provided an additional tool for the diagnosis of MM and for monitoring response to therapy. However, significant discrepancy has been reported regarding the enumeration of plasma cells in marrow samples of MM patients using these two methods. Aims - In this study we compared the bone marrow plasmocytosis by microscopic examination of BM aspirates, to the flow cytometry results in samples obtained form MM patients. We tested whether the noted discrepancy between these two methods applies only to MM, or represents a trend in other hematopoietic malignancies as well. We defined this discrepancy and explained it. Methods - The number of plasma cells or blasts from BM aspirates of 41 MM or seven acute myeloid leukemia (AML) patients respectively were analyzed simultaneously by morphological evaluation and by FCM. Each sample was assessed independently by two qualified laboratory specialists and/or hemato-pathologist. In MM we found plasma cell fractions that were characterized by FCM and gene expression profile. Results - In MM it was evident that FCM under-estimated the number of BM plasma cells samples by an average of 60%, compared with conventional morphological evaluation. On the other hand in AML there was a good correlation between the morphological and FCM assessments of the blast cell population, indicating that the discrepancy observed in the MM BM samples may be related to unique characteristics of the malignant plasma cells. Since flow cytometry is performed on the bone marrow fluid which is depleted of fat tissue-adhesive plasma cells, we disrupted spicules from MM BM samples (by repeated passages through 21g needle) and found a 40% increase in plasma cell compared with the fluid of the same BM samples. In order to determine the FCM profile of the cells in these two fractions, we isolated BM derived spicules from aspirates of MM patients and treated them with extracellular matrix (ECM) degrading enzymes followed by mechanical shearing. This combination released the highly adhesive plasma cells from the spicules. The released myeloma cells displayed a different FCM profile and in particular had a higher level of CD138 expression. Gene expression profile, which was performed on similar adhesion variants of cultured MM cells, demonstrated distinct oncogenic and transcriptional programs. Summary - We have shown a major discrepancy between the percentage of MM cells obtained by routine BM morphology and flow cytometry counts. It is possible that this discrepancy is partially attributable to the two distinct microenvironmental components occupied by MM cells in the BM sample - the lipid spicules, and the fluid phase. MM cells located in different niches of the BM also differ in their FCM and gene expression profile. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells. These sub-populations may play distinct roles in the biological and clinical manifestations of the disease and differ in their response to anti-myeloma therapy.

Multiparameter Flow Cytometry For Detection Of Minimal Residual Disease In Multiple Myeloma After T-Cell Depleted Allogeneic Stem Cell Transplant

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4647-4647
Author(s):  
Satyajit Kosuri ◽  
Katherine M Smith ◽  
Deborah Kuk ◽  
Sean M. Devlin ◽  
Peter G. Maslak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry

Introduction Multiparameter flow cytometry (MFC) has been shown to be a sensitive, reproducible and broadly applicable method for the early detection of minimal residual disease (MRD) in the bone marrow (BM) of pts with multiple myeloma (MM) following induction chemotherapy and/or autologous stem cell transplantation. In this study, we were interested in assessing the potential of MFC as a reliable and potentially predictive marker in pts with multiple myeloma who have undergone T-cell depleted allogeneic hematopoietic stem cell transplantation (TCD HSCT). Methods We analyzed the results of MFC obtained in 35pts with multiply relapsed MM, who also have high-risk cytogenetics undergoing allo TCD-HSCT from HLA compatible related (n= 15) and unrelated (matched (n=8), mismatched (n=12) donors. We compared these results to standard myeloma markers obtained from the blood and marrow of these pts at days 30, 60-90, 120-180, 12 and 24 months routinely and as clinically indicated thereafter post TCD HSCT. Disease evaluation included serologic immunoglobulin levels, serum protein electrophoresis/immunofixation, and serum analysis of free light chains, bone marrow biopsy and aspirate. Bone marrow specimens from each time point were also analyzed by MFC with a panel including CD38, CD56, CD45, CD19, CD138, cyKAPPA, and cyLAMBDA by gating on distinct populations of bright CD38+/CD45- plasma cells at 200,000 acquired events total or at least 100 gated plasma cell events. Malignant plasma cells (MPC) were defined as CD38+/CD138+/CD56+/CD45- and/or positive for light chain clonal excess. MPC were detected in the BM sample at the MFC sensitivity of 10-4(>1 MPC in 104normal cells). Results Thirty-five pts with multiply relapsed MM undergoing allo TCD HSCT were analyzed over median follow up of 27 months (range 6.2 – 53.3). Eighteen/35 pts did not relapse during the follow up period and none of these pts had a detectable CD38+/CD138+/CD56+/CD45- cell population by MFC. Seventeen/35 pts developed relapsed disease at a median of 12.5 months (range 3.2 – 52.5) post allo TCD-HSCT by standard serologic markers and all pts were found to be positive by MFC. The percentages of bright CD38+/CD45- cells in these pts ranged from 0.01% to 16.05% at time of first detection. In 14/17 pts, MFC became positive concurrently with standard serologic myeloma markers at relapse. In 3/17 pts, MFC detected a malignant plasma cell population with aberrant phenotype of 0.068%, 0.043% and 0.012% at 48.2, 24 and 25.4 months, respectively, post TCD HSCT in the absence of other positive markers in blood and bone marrow. These pts were also immunofixation (IF) negative at conversion to MFC positivity. Subsequent follow up of studies of these 3 pts lead to detection of recurrence by IF and/or M-spike/ aspirate at 3.8, 1.8 and 8.7 months with median follow up of 150 days after first MFC detection. The populations of MPC initially detected by MFC had increased upon relapse to higher levels. Interestingly, in 2 pts we detected 6 and 8% plasma cells by bone marrow aspirate at 90 days and 180 days, respectively, post TCD HSCT, while flow cytometry detected only CD138+/CD56-/CD45+ cells. These 2 pts never relapsed and continued to remain in CR without further intervention. Conclusions These analyses demonstrate that MFC performed on marrow specimen of pts with relapsed MM who underwent a TCD HSCT provides additional important results to assess the overall disease status. A negative MFC indicated non relapse 100% of the time attesting to its negative predictive value. In all of our patients diagnosed with relapsed MM by traditional parameters, MFC was concurrently positive. Importantly, in 3/17 pts (18%) MRD detected MPC prior to overt relapse. Interestingly, MFC was able to detect false positive marrow relapses as well. Therefore, MFC permits the detection of MRD preceding frank relapse and can distinguish a malignant plasma cell population from proliferating recovering marrow post transplant. In the post allo TCD-HSCT setting MFC may serve as an early marker which can help formulate the timing of therapeutic interventions, such as adoptive immunotherapeutic approaches, as MFC detection provides a window of several weeks to initiate treatment before disease recurrence by serology. Disclosures: No relevant conflicts of interest to declare.

Minimal Residual Disease Analysis in Multiple Myeloma Patients By 6-Color Flow Cytometry: An Experience from Tertiary Care Center of North India

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5343-5343
Author(s):  
Praveen Sharma ◽  
Man Updesh Singh Sachdeva ◽  
Neelam Varma ◽  
Parveen Bose ◽  
Pankaj Malhotra
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Light Chain ◽  
Bone Marrow Aspirate ◽  
Plasma Cells ◽  
Residual Disease ◽  
Color Flow ◽  
Multiparametric Flow Cytometry ◽  
Minimal Residual

Abstract Therapeutic advances in multiple myeloma (MM) incorporating the use of high-dose melphalan, novel therapeutic immunomodulatory agents, proteasome inhibitors and supporting autologous stem-cell transplantation (ASCT) have improved response rates and overall survival. The detection of minimal residual disease (MRD) is recognized as a sensitive and rapid approach to evaluate treatment efficacy as a tool for predicting patient outcomes and guiding therapeutic decisions. MRD analysis is reflected by many different techniques, however, multiparametric flow cytometry is a sensitive, feasible and adequate method for monitoring residual disease. Studies from India related to this context are lacking. In the present study, we compare MRD levels in patients of multiple myeloma after chemotherapy/ASCT assessed by multiparametric flow cytometry, with M band status, immunofixation (IFE) and percentage of plasma cells on bone marrow aspirate. Seventeen patients of multiple myeloma were included in the study over a duration of one year, (Male=13, Female=4) with mean age of 56.8 years (range 44-80 years). MRD was analyzed using a dual laser 6 color-flow cytometer in 9 patients of ASCT (day 100) and 8 patients on chemotherapy alone (post-induction). Pre-titrated cocktail of CD38, CD138, CD19, CD45, cytoplasmic Kappa light chain, cytoplasmic lambda light chain, CD81, CD27, CD28 CD200 and CD10 were used in 6-color combination of three tubes for MRD analysis. MRD was detectable in 5 patients, mean of 0.61% (range of 0.07 - 6.44%). M band and IFE were positive in 2 patients, each. Bone marrow plasma cells ranged from 0 to 22%. MRD levels did not show significant correlation with percentage of plasma cells in bone marrow aspirate, however it had an statistical agreement with presence or absence of serum M-band and IFE. Patients are on regular follow up for their clinical and hematological response. Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiple myeloma: circulating lymphocytes that express plasma cell antigens

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 352-356
Author(s):  
GJ Ruiz-Arguelles ◽  
JA Katzmann ◽  
PR Greipp ◽  
NJ Gonchoroff ◽  
JP Garton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Peripheral Blood Lymphocytes ◽  
Tumor Burden ◽  
B Cell Differentiation ◽  
Blood Lymphocytes ◽  
Bone Marrow Plasma

The bone marrow and peripheral blood of 14 patients with multiple myeloma were studied with murine monoclonal antibodies that identify antigens on plasma cells (R1–3 and OKT10). Peripheral blood lymphocytes expressing plasma cell antigens were found in six cases. Five of these cases expressed the same antigens that were present on the plasma cells in the bone marrow. Patients that showed such peripheral blood involvement were found to have a larger tumor burden and higher bone marrow plasma cell proliferative activity. In some patients, antigens normally found at earlier stages of B cell differentiation (B1, B2, and J5) were expressed by peripheral blood lymphocytes and/or bone marrow plasma cells.

scholarly journals Myeloma Cell Associated Therapeutic Protein Discovery Using Single Cell RNA-Seq Data

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Lijun Yao ◽  
Reyka G Jayasinghe ◽  
Tianjiao Wang ◽  
Julie O'Neal ◽  
Ruiyang Liu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Plasma Cell ◽  
Tissue Specificity ◽  
Plasma Cells ◽  
Expression Data ◽  
Intracellular Proteins

Multiple myeloma (MM) is a hematological cancer of the antibody-secreting plasma cells. Despite therapeutic advancements, MM remains incurable due to high incidence of drug-resistant relapse. In recent years, targeted immunotherapies, which take advantage of the immune system's cytotoxic defenses to specifically eliminate tumor cells expressing certain cell surface and intracellular proteins have shown promise in combating this and other B cell hematologic malignancies. A major limitation in the development of these therapies lies in the discovery of optimal candidate targets, which require both high expression in tumor cells as well as stringent tissue specificity. In an effort to identify potential myeloma-specific target antigens, we performed an unbiased search for genes with specific expression in plasma and/or B cells using single-cell RNA-sequencing (scRNAseq) of 53 bone marrow samples taken from 42 patients. By comparing &gt;40K plasma cells to &gt;97K immune cells across our cohort, we were able to identify a total of 181 plasma cell-associated genes, including 65 that encode cell-surface proteins and 116 encoding intracellular proteins. Of particular interest is that the plasma cells from each patient were shown to be transcriptionally distinct with unique sets of genes expressed defining each patient's malignant plasma cells. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to B-Cell receptor (BCR) signaling, protein transport, and endoplasmic reticulum (ER) stress, involving genes such as DERL3, HERPUD1, PDIA4, PDIA6, RRBP1, SSR3, SSR4, TXNDC5, and UBE2J1. To note, our strategy successfully captured several of the most promising MM therapeutic targets currently under pre-clinical and clinical trials, including TNFRSF17(BCMA), SLAMF7, and SDC1 (CD138). Among these, TNFRSF17 showed very high plasma cell expression, with concomitant sharp exclusion of other immune cell types. To ascertain tissue specificity of candidate genes outside of the bone marrow, we analyzed gene and protein expression data from the Genotype-Tissue Expression (GTEx) portal and Human Protein Atlas (HPA). We found further support for several candidates (incl. TNFRSF17,SLAMF7, TNFRSF13B (TACI), and TNFRSF13C) as being both exclusively and highly expressed in lymphoid tissues. While several surface candidates were not found to be lymphocyte-restricted at the protein level, they remain relevant considerations as secondary targets for bi-specific immunotherapy approaches currently under development. To further investigate potential combinatorial targeting, we examine sample-level patterns of candidate co-expression and mutually-exclusive expression using correlation analysis. As the majority of our detected plasma cell-specific genes encode intracellular proteins, we investigated the potential utility of these epitopes as therapeutic targets via MHC presentation. Highly expressed candidates include MZB1, SEC11C, HLA-DOB, POU2AF1, and EAF2. We analyzed protein sequences using NetMHC and NETMHCII to predict high-affinity peptides for common class-I and class-II HLA alleles. To correlate MHC allelic preference with candidate expression in our cohort, we performed HLA-typing for 29 samples using Optitype. To support our scRNAseq-driven findings, we cross-referenced gene expression data with 907 bulk RNA-sequencing samples, including 15 from internal studies and 892 from the Multiple Myeloma Research Foundation (MMRF), as well as bulk global proteomics data from 4 MM cell lines (TIB.U266, RPMI8226, OPM2, MM1ST) and 4 patients. We see consistent trends across both cohorts, with high positive correlation (Pearson R ranging between 0.60 and 0.99) for a majority of genes when comparing scRNA and bulk RNA expression in the same samples. Our experimental design and analysis strategies enabled the efficient discovery of myeloma-associated therapeutic target candidates. In conclusion, this study identified a set of promising myeloma CAR-T targets, providing novel treatment options for myeloma patients. Disclosures Goldsmith: Wugen Inc.: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals THE BONE MARROW ON STERNAL ASPIRATION IN MULTIPLE MYELOMA

Blood ◽  
1948 ◽  
Vol 3 (9) ◽  
pp. 987-1018 ◽  
Author(s):  
EDWIN D. BAYRD
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Multinucleated Cells ◽  
Cytoplasmic Bodies ◽  
Plasma Cell Type ◽  
Well Differentiated ◽  
Diagnostic Pitfalls

Abstract Generalizing, it can be said that the pathologic cells seen in smears of the bone marrow in multiple myeloma resemble the plasma cell and vary from the very anaplastic and immature cell to the well-differentiated and almost characteristic plasma cell. The feature which the "myeloma" cell shares with the plasma cell is the abundant, granular, basophilic cytoplasm which tends to be fragile and undergo the same degenerative changes in each; namely, the formation of Russell bodies and vacuolization. Fairly frequently a perinuclear clear area or Hof is present and the nucleus tends to be eccentrically placed. Cytoplasmic extensions or pseudopodia may also be seen in either case, but they occur more often and more dramatically in instances of multiple myeloma. Multinucleated cells are commonly seen. In addition, myeloma-plasma cells will often have a large clear nucleolus and a leptochromatic nucleus and will exhibit a tendency to the formation of isolated areas of condensed chromatin. Cytoplasmic extrusions, free cytoplasmic bodies, occasionally complete with Russell bodies and vacuoles are almost universally present. All cases were of the plasma cell type; there was no exception. In these cases, the myeloma-plasma cell constituted from 2.5 to 96 per cent of the leukocytic elements present. The opinion was expressed that all so-called types of multiple myeloma are merely variations in differentiation of this same cell. It was noted that anaplasia, hypernucleation and lack of plasma cell predominance in certain cases were diagnostic pitfalls. Additional evidence was adduced to confirm the reticulo-endothelial origin of the myeloma-plasma cell. It was further observed that certain prognostically valuable information could be gleaned from a careful review of the cytologic characteristics in these cases.

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