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.

Increased Circulating Plasma Cells On Multiparametric Flow Cytometry As An Independent Prognostic Biomarker In Newly Diagnosed Multiple Myeloma: Implications For Redefining High-Risk Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1842-1842
Author(s):  
Wilson I Gonsalves ◽  
Vinay Gupta ◽  
S. Vincent Rajkumar ◽  
William G Morice ◽  
Michael M Timm ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Overall Survival ◽  
High Risk ◽  
Peripheral Blood ◽  
Research Funding ◽  
Light Chains ◽  
Newly Diagnosed ◽  
Risk Status ◽  
Multiparametric Flow Cytometry

Abstract Background Prior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. Methods We evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. 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. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs 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 assessed using the log rank test. Results There were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). Conclusion Quantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

The Prognostic Significance of Polyclonal Bone Marrow Plasma Cells in Patients with Actively Relapsing Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1194-1194
Author(s):  
Toshi Ghosh ◽  
Wilson I Gonsalves ◽  
Dragan Jevremovic ◽  
S. Vincent Rajkumar ◽  
Michael M. Timm ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Research Funding ◽  
Plasma Cells ◽  
Labeling Index ◽  
Light Chains ◽  
Fish Cytogenetics ◽  
Kaplan Meier

Abstract Background: Prior studies suggest that the presence of >5% polyclonal plasma cells (pPCs) among total plasma cells (PCs) within the bone marrow (BM) is associated with a longer progression-free survival, higher response rates, and lower frequency of high-risk cytogenetic abnormalities in patients with newly diagnosed multiple myeloma (MM). However, the incidence and prognostic utility of this factor in patients with relapsed and/or refractory MM has not been previously evaluated. Thus, we evaluated the prognostic value of quantifying the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM. Methods: We evaluated all MM patients with actively relapsing disease (biochemical and/or symptomatic) seen at the Mayo Clinic, Rochester, from 2012 to 2013, who had BM samples evaluated by seven-color multiparametric flow cytometry. All patients had at least 24 months of follow-up from the date of flow evaluation. Cell surface antigens were assessed by direct immunofluorescence antibodies for CD45, CD19, CD38, CD138, cytoplasmic Kappa and Lambda Ig light chains, and DAPI nuclear stain. The flow cytometry data was collected using the Becton Dickinson FACSCanto II instruments that analyzed 150,000 events (cells); this data was then analyzed by multi-parameter analysis using the BD FACS DIVA Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Clonal PCs were separated from pPCs based on the differential expression of CD45, CD19, DAPI (in non-diploid cases), and immunoglobulin light chains. The percentage of pPCs was calculated in total PCs detected. Survival analysis was performed by the Kaplan-Meier method and differences were assessed using the log rank test. Results: There were 180 consecutive patients with actively relapsing MM who had BM biopsies analyzed via flow cytometry as part of their routine clinical evaluation. The median age of this group was 65 years (range: 40 - 87); 52% were male. At the time of this analysis, 104 patients had died, and the 2-year overall survival (OS) rate for the cohort was 58%. The median number of therapies received was 4 (range: 1 - 15). Of these patients, 61% received a prior ASCT, and almost all (99%) received prior regimens containing either immunomodulators or proteasome inhibitors. There were 55 (30%) patients with >5% pPCs among the total PCs in their BM. The median percentage of pPCs among total PCs in these 55 patients was 33% (range: 5 - 99). The median OS for those with >5% pPCs was not reached compared with 22 months for those with <5% pPCs (P = 0.028; Figure 1). Patients with <5% pPCs PCs had a higher likelihood of high-risk FISH cytogenetics compared with the rest of the patients. In a univariate analysis, increasing number of pPCs was associated with an improved OS, while higher labeling index, number of prior therapies, and the presence of high-risk FISH cytogenetics were associated with a worse OS. In a multivariate analysis, only the increasing number of pPCs (P = 0.006), higher labeling index (P = 0.0002) and number of prior therapies (P = 0.003) retained statistical significance. Conclusion: Quantitative estimation of the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM was determined to be a predictor of worse OS. As such, this parameter is able to identify a group of patients with MM with actively relapsing disease who have a particularly poor outcome. Further studies evaluating its biological significance are warranted. Figure 1 Kaplan-Meier curve comparing OS between patients with ≥5% pPCs and <5% pPCs among the total PCs in their BM. Figure 1. Kaplan-Meier curve comparing OS between patients with ≥5% pPCs and <5% pPCs among the total PCs in their BM. Disclosures Kapoor: Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Gertz:Prothena Therapeutics: Research Funding; Novartis: Research Funding; Alnylam Pharmaceuticals: Research Funding; Research to Practice: Honoraria, Speakers Bureau; Med Learning Group: Honoraria, Speakers Bureau; Celgene: Honoraria; NCI Frederick: Honoraria; Sandoz Inc: Honoraria; GSK: Honoraria; Ionis: Research Funding; Annexon Biosciences: Research Funding. Kumar:AbbVie: Research Funding; Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy.

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.

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.

scholarly journals Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 258-258 ◽  
Author(s):  
Hiroyuki Takamatsu ◽  
Naoki Takezako ◽  
Rachel K Wee ◽  
Takeshi Yoroidaka ◽  
Takeshi Yamashita ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Research Funding ◽  
Prognostic Value ◽  
Plasma Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Next Generation ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM. Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts. Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used. Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively. Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. Figure 1. Figure 1. Disclosures Takamatsu: Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

scholarly journals Next Generation Flow Cytometry Provides a Standardized, Highly Sensitive and Informative Method for the Analysis of Circulating Plasma Cells in Newly Diagnosed Multiple Myeloma: A Single Center Study in 182 Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4338-4338 ◽  
Author(s):  
Evangelos Terpos ◽  
Ioannis V. Kostopoulos ◽  
Aristea-Maria Papanota ◽  
Konstantinos Papadimitriou ◽  
Panagiotis Malandrakis ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Research Funding ◽  
Prognostic Value ◽  
Plasma Cells ◽  
Next Generation ◽  
Single Center ◽  
Disease Characteristics ◽  
Highly Sensitive ◽  
Matched Samples

Introduction: The apparent heterogeneity of multiple myeloma (MM) constitutes a key challenge in the clinical management and the design of effective therapeutic interventions, while it entails the identification of biomarkers with a strong prognostic value. In this context and taking into account patients' inconvenience to invasive bone marrow (BM) aspiration, the assessment of circulating plasma cells (CPCs) in liquid biopsies, at the time of diagnosis, has been proposed as a useful assay with prognostic value. Different methodologies have been applied for the detection of CPCs; the most common is the use of multicolor flow cytometry (MFC), mainly of 2-, 4-, or 6-color combination panels, which however yielded heterogeneous results due to variations in the detection efficacy of each approach. In the present study, we applied the standardized and highly sensitive Next Generation Flow Cytometry (NGF) approach, to detect CPCs in diagnostic MM peripheral blood (PB) samples, we compared their phenotypic characteristics with the aberrant clonal cells of BM matched samples and we correlated their presence with disease characteristics. Patients and Methods: PB and BM matched samples from 182 consecutive MM patients, at diagnosis, were evaluated for the presence of aberrant plasma cells (APCs) following the standard operating procedures (SOP) of NGF, according to EuroFlow guidelines. All these patients were diagnosed and treated in a single center (Department of Clinical Therapeutics, N.K. University of Athens, Greece). Samples were collected in EDTA-anticoagulated tubes and treated with the bulk-lysis procedure. Recovered cells were stained with antibodies against surface CD19-PEC7, CD27-BV510, CD38-FITC, CD45-PERCP, CD56-PE and CD138-BV421 and the intracellular CyIgκ-APC and CyIgλ-APCC750 to verify clonality. Six to ten million cells were acquired per sample, thus reaching a median Limit of Detection (LOD) of 3.5x10-6. Optimal PMT voltages were set according to the EuroFlow SOP for instrument set-up and daily performance status of FACSCANTOII was monitored with both CS&T (BD) and Rainbow beads (Spherotech Inc, Lake Forest, IL). Results: CPCs were detected in 158/182 (86.8%) MM diagnostic samples within a range of 0.0002% to 63.8% of total PB nucleated cells (PBNCs). The CPCs showed the same aberrant phenotype as the one detected in the BM for all cases, although with a significantly reduced intensity for the markers CD27, CD38, CD138 and CD56. When more than one phenotypically distinct subgroups were detectable in the BM, the same phenotypic subsets were present in the PB with the same relative frequency for >90% of bi/multi-phenotypic cases. The higher number of CPCs (>0.1% of all PBNCs) strongly correlated with an increased BM infiltration rate by myeloma cells (p<0.0001), with ISS-3 disease stage (p<0.0001) and with the presence of high-risk cytogenetics [t(4;14), t(14;16) and/or del(17p53); p<0.0001]. There was also weaker correlation between high number of CPCs and high serum creatinine levels (p=0.015). Inversely, the absence of CPCs or the presence of CPCs at numbers ≤0.001% correlated with lower serum β2-microglobulin (p<0.0001), with higher hemoglobin levels (p<0.0001) and with the presence of an elevated normal plasma cell compartment within the BM (i.e. ≥5% of all PCs; r2=0.84, p<0.0001). There was no association between the CPC number and the therapeutic response to induction treatment (IMWG criteria). Despite the short follow-up period (median of 16 months), there is a trend for inferior PFS in patients with high CPCs (p=0.16). Conclusions: The NGF approach using the EuroFlow protocol enables the detection of even rare CPCs in diagnostic MM PB samples, due to the high number of cells acquired and the elegantly elaborated 8-color marker combinations which allows for the detection of CPCs with even a non-typical phenotype. Our matched PB and BM analysis revealed that BM APCs and CPCs share very similar characteristics suggesting that liquid biopsy offers a representative alternative for the phenotypic characterization of BM APCs. The correlation of high CPCs with adverse disease characteristics suggests that the quantification of CPCs by standardized NGF may emerge as a valuable surrogate prognostic biomarker which could replace other invasive methods or other less informative assays. Disclosures Terpos: Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria. Gavriatopoulou:Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses. Kastritis:Genesis: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

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