Measurement of the proliferation of clonal plasma cells by multiparametric flow cytometry is a clinically useful tool in relapsed multiple myeloma

2015 ◽  
Vol 15 ◽  
pp. S238-S239
Author(s):  
Samith Kochuparambil ◽  
William Morice ◽  
S. Vincent Rajkumar ◽  
Angela Dispenzieri ◽  
Michael Timm ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Plasma Cells ◽  
Multiparametric Flow Cytometry

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.

Prognostic Value Of Quantifying Circulating Plasma Cells By Multiparametric Flow Cytometry In Patients With Relapsed Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 754-754
Author(s):  
Wilson I Gonsalves ◽  
Vinay Gupta ◽  
S. Vincent Rajkumar ◽  
William G Morice ◽  
Michael M Timm ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Research Funding ◽  
Prognostic Value ◽  
Plasma Cells ◽  
Light Chains ◽  
Plateau Phase ◽  
Multiparametric Flow Cytometry ◽  
Relapsed Disease

Abstract Background The presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. Methods We evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. Results There were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs. We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. Conclusion The utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Evolution of Multiparametric Flow Cytometry Testing for Minimal Residual Disease Assessment in Multiple Myeloma and Its Impact on Clinical Outcomes: A Single Institution Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2274-2274 ◽  
Author(s):  
Nischala Ammannagari ◽  
Paul K. Wallace ◽  
Theresa Hahn ◽  
Yali Zhang ◽  
Christine M. Ho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Clinical Outcomes ◽  
Research Funding ◽  
Plasma Cells ◽  
Residual Disease ◽  
Advisory Committees ◽  
Multiparametric Flow Cytometry ◽  
Minimal Residual

Abstract Minimal residual disease (MRD) after autologous hematopoietic cell transplant (AHCT) in multiple myeloma (MM) has been shown to be an important predictor of clinical outcomes, suggesting that MRD negativity may be a new goal of therapy. Multiparametric flow cytometry (MFC) is a commonly used method for MRD assessment, however this technique is still evolving and efforts are underway to standardize this testing. The key factors which enable detection of residual malignant plasma cells by MFC remain an area of active investigation. We performed a retrospective review of 172 consecutive MM patients who received AHCT between 10/1/2007 and 5/31/2015 at our institution and had undergone MRD assessment by MFC at day +100 post-AHCT. Day +100 post-AHCT response was determined using the International Myeloma Working Group (IMWG) Uniform Response Criteria (URC) and was correlated with MRD assessment as well as progression free survival (PFS) and overall survival (OS). Data were collected on the specific MFC panel utilized, including the epitopes analyzed and the total plasma cell number (PCN) counted (normal and malignant PC). These variables were correlated with clinical outcomes including day +100 MM response, PFS and OS. Of 172 patients, 30 were MRD-positive, 133 MRD-negative, and 9 were equivocal at day +100 post-AHCT, the latter of which were excluded from further analyses. Day+100 MRD-negative status by MM response was: 31/37(84%) for VGPR, 35/41 (85%) for CR, and 42/42 (100%) for sCR. Patients who achieved a CR or sCR had improved PFS and OS rates compared with patients who achieved ≤VGPR: 3-year PFS: 61% (95% CI 49-74%) vs 46% (95% CI 32-59%), P=0.03; 3-year OS: 96% (95% CI 91-100%) vs 69% (95% CI 56-81%), P=0.005)). Patients with MRD-negative disease at day +100 post-AHCT had significantly superior PFS and OS compared to those with MRD-positive disease: 3-yr PFS 62% (95% CI 52-72%) vs 33% (95% CI 12-53%), P <0.0001) (Figure 1); 3-year OS 85% (95% CI 78-93%) vs 64% (95% CI 44-85%), P=0.004). There was no association between MRD status and age (<60 vs ≥60 years), sex, race (white vs other), performance status (KPS ≤80 vs ≥90), or subsequent transplant (P>0.1). The details of the four different MRD MFC panels are shown in Table 1. Panels C and D were compared, at a similar PCN level, but different epitopes tested, and found no significant difference in PFS or OS. Further analysis of PCN within the MRD-negative cohort revealed a trend towards improved 3-yr PFS rates with increasing numbers of PCN analyzed: 42% (95% CI 20-63%) for PCN<250,000, 68% (95% CI 52-83%) for PCN=250,000-500,000, 59% (95% CI 42-76%) for PCN >500,000-1,000,000 and 89% (78-100%) for PCN>1,000,000 (P=0.099) (Figure 2). The 3-yr OS rates for MRD-negative patients were higher for increasing PCNs analyzed, but the PCN categories were not statistically significantly different: 74% (95% CI 54-94%) for PCN<250,000, 88% (95% CI 77-99%) for PCN=250,000-500,000, 85% (95% CI 73-98%) for PCN >500,000-1,000,000 and 100% for PCN>1,000,000 (P=0.2). Sensitivity analysis revealed similar trends when a cut-off of above or below 500,000 or 1,000,000 was used. Our results confirm that achievement of MFC MRD negativity at day +100 post-AHCT is associated with improved PFS and OS. Factors such as the long-half lives of immunoglobulins, the quality of the bone marrow aspirate obtained, and the presence of occult extramedullary disease may account for the patients who were MRD negative but did not achieve a CR at day +100 post AHCT by IMWG URC. MRD assessment by MFC at our institution has evolved over time to include higher numbers of acquired and analyzed events. Notably, there was a trend towards improved outcomes with greater numbers of plasma cells analyzed, suggesting that continued development of MRD assessment by MFC should focus on increasing PCN analyzed in order to improve detection of residual MM clones. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holstein:Millennium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Multiparametric flow cytometry profiling of neoplastic plasma cells in multiple myeloma

2010 ◽  
Vol 78B (5) ◽  
pp. 338-347 ◽  
Author(s):  
Hans E. Johnsen ◽  
Martin Bøgsted ◽  
Tobias W. Klausen ◽  
Peter Gimsing ◽  
Alexander Schmitz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Plasma Cells ◽  
Multiparametric Flow Cytometry

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 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&lt;0.05. Bone marrow aspirate samples of nine participants were evaluated. Significant reduction in plasma cell yield was observed over time (p&lt;0.001) while sample integrity remained unchanged (p&gt;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&lt;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 ABCG2 expression, function, and promoter methylation in human multiple myeloma

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3881-3889 ◽  
Author(s):  
Joel G. Turner ◽  
Jana L. Gump ◽  
Chunchun Zhang ◽  
James M. Cook ◽  
Douglas Marchion ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Protein Expression ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Plasma Cells ◽  
Cancer Resistance ◽  
Response To Chemotherapy ◽  
Mrna And Protein Expression

AbstractWe investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.

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