scholarly journals Eight-Color Flow Cytometry Phenotypic Markers and Disease Progression in Monoclonal Gammopathy of Unknown Significance

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2713-2713
Author(s):  
Naveen K Yarlagadda ◽  
Meera Mohan ◽  
Shebli Atrash ◽  
Sravani Gundarlapalli ◽  
Shadiqul Hoque ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Disease Progression ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Clone ◽  
Clone Size ◽  
Color Flow ◽  
Unknown Significance ◽  
M Spike

Abstract Introduction: Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification, and monitoring of plasma cell disorders. Herein, we seek to study the clinical significance of expression of phenotype markers in monoclonal gammopathy of unknown significance (MGUS). Methods: We identified a cohort of patients with a diagnosis of MGUS from the institutional myeloma database. Bone marrow (BM) aspirate assessment was performed using 8-color immunophenotypic next-generation flow cytometric (NGF) analysis with a minimum sensitivity of 10 -5 cells at the time of diagnosis or first visit to our institution. BM aspirate samples were immunophenotyped on a FACSCanto II flow cytometer using antibodies (BD) to delineate normal and abnormal plasma cells [CD138 (V-500), CD38 (FITC), CD19 (PE-Cy7), CD45 (V-450), CD27 (PercpCy5.5), CD81 (APC-H-7), CD56 (APC) and CD20 (PE)]. The sensitivity or the Limit of Detection (LOD) for this assay was validated to 20 cells in 2 ×10 6 events (0.001%), and the reproducibility or Lower Limit of Quantitation (LLOQ) is 50 cells in 2 ×10 6 events. Clinical and laboratory variables were also collected. Based on previously published data, expression (CD19, CD45, CD81), and lack of expression (CD56, CD27, CD20) of the above-mentioned surface antigens were analyzed. Additional variables such as IgA isotype, size of M-protein (≥15 g/L), and abnormal free light chain ratio(abnFLR) (defined as <0.1 or >10) were included in regression fitting models. Results: A total of 157 patients with MGUS were included in this analysis. The median age at diagnosis was 60 years (range 24- 84), 84 (53 %) patients were female and 25 (16%) were African American. Overall, IgG Kappa (75/148, 50%) was the most common isotype. Fluorescent-in-situ hybridization (FISH) data were available in 35 patients with t (4:14) and t (14;16) seen in 3 patients each. At a median follow-up of 18.15 years (quantiles 11.35, 33.62), 28 patients experienced disease progression (25 to MM, 2 to Waldenstrom macroglobulinemia, and 1 Smoldering myeloma). The median progression-free survival of this cohort was 17.3 years. Among these, occurrences of the bone lesion (8/28; 28.6%) were the most common pattern of disease progression to MM. This analysis showed lower odds of progression with the expression of CD27 (OR-0.39, 95% CI 0.15-0.99) (figure 1A). Disease progression was more common in patients with an abnormal plasma cell clone size ≥ of 3.1% at diagnosis (60% vs. 12.5%, p=0.0005). An abnormal plasma cell clone of ≥3.1% at diagnosis, was associated with increased odds of progression (OR-10.79, 95% CI 4.02-28.98) and a shorter PFS (12.5 years versus NR, p=0.01) (figure 1B). Serum M-spike ≥1.5 g/dL (OR-3.54;95% 1.30-9.62) and abnFLR (OR-2.30, 95% CI 1.00-5.32) were also associated with a higher odds of progression. However, in this population, the presence of IgA isotype did not increase the odds of MGUS progression. In a stepwise regression model, serum M-spike≥1.5 g/dL, abnFLR, and the lack of expression of CD27 were associated with the risk of disease progression. Conclusion: In addition to previously published risk factors, our cohort shows that the expression of CD27 antigen by eight-color flow cytometry confers a lower risk of disease progression of MGUS. This is consistent with our previous report that CD27 is progressively down-regulated in the transition from normal plasma cells (NPC) to MGUS to MM (Zhan et al, Blood 2006). Furthermore, we show that size of the myeloma clone (≥ 3.1% ) is a possible surrogate marker for disease progression in MGUS. Figure 1: 1A shows forest plot of odds ratios for flow cytometry markers, IgA isotype, size of M protein, abnFLR and plasma cell clone size. 1B shows the Kaplan Meier estimates of PFS for patients stratifies by plasma cell clone size. Figure 1 Figure 1. Disclosures Mohan: Medical College of Wisconsin: Current Employment. Atrash: GSK: Research Funding; AMGEN: Research Funding; Jansen: Research Funding, Speakers Bureau.

Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

Multi-Parameter Flow Cytometry(FC) Can Detect Proliferating Myeloma Cells After Treatment: Implications for Targeting the Myeloma Proliferative Component

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4995-4995
Author(s):  
John Lust ◽  
Shaji Kumar ◽  
Michael Timm ◽  
Kathleen Donovan ◽  
Philip R. Greipp ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Autologous Transplantation ◽  
Cell Labeling ◽  
Growth Fraction ◽  
Proliferating Cells ◽  
Myeloma Cells

Abstract Abstract 4995 Background: Multiple myeloma results from an accumulation of monoclonal nonproliferating plasma cells arising from a small subpopulation of proliferating myeloma cells. In an effort to optimize detection of the human myeloma cell growth fraction and obviate the need for slide review, a novel FC strategy was developed combining elements of light chain restriction, surface antigen expression, and ploidy analysis. Methods: Bone marrow cells from 41 patients with plasma cell proliferative disorders were lysed with ACK and resuspended in 3% BSA. Cells were stained using a 6-color assay with anti-CD45, anti-CD38, anti-CD138, and anti-CD19. Cells were washed and 100 ul Caltag solution A was added for 15 min. Cells were washed and 100 ul of Caltag solution B, anti-kappa and anti-lambda were added for 10 min. Cells were washed and 100 ul PBS and 3 ul RNAse are added for 15 min. Cells were washed and 400 ul of a 1:1000 solution of 3uM DAPI in Tris 0.1% NP-40 was added. Cells were incubated at 4°C for 45 minutes before running on a FACSCanto instrument for ploidy determination. All patients were analyzed both by flow cytometry and the slide based plasma cell labeling index. Results: Forty-one patients were studied; 34 demonstrated a proliferative fraction and 7 had too few plasma cells for analysis after therapy. Of the 34 patients, 6 had MGUS/SMM, 5 newly diagnosed MM, 7 amyloid, and 16 were treated MM. The mean percent proliferating cells were 1.1% (range 0 – 8.6%) with PCLI and 1.4% (range 0.1 – 12.7%) by flow. The correlation between PCLI and flow gave a RSquare value of 0.54. Twelve patients with a PCLI of 0% had a flow proliferation between 0.1 – 1.4% (mean 0.49%). Treated patients received lenalidomide, dexamethasone, bortezomib, and/or autologous transplantation. All 16 treated myeloma patients with adequate plasma cells had a flow proliferation between 0.2 – 12.7% (mean 2.2%). Conclusion: Flow cytometry offers a useful way to detect the proliferative myeloma component at diagnosis and after treatment. The continued presence of proliferating myeloma cells after treatment may explain why most patients relapse and offers another important marker to monitor and cell population to target in patients with active disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

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.

scholarly journals Detection and Characterization of Plasma Cell and B Cell Clones in Patients with Systemic Light Chain Amyloidosis Using Flow Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2068-2068
Author(s):  
Stefan Schönland ◽  
Ute Hegenbart ◽  
Christoph Kimmich ◽  
Katarina Lisenko ◽  
Dirk Hose ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Cell Count ◽  
Plasma Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
Cell Clone ◽  
Flow Analysis ◽  
Al Amyloidosis ◽  
Cell Clones

Abstract Introduction: AL amyloidosis is a rare and life-threatening protein-deposition disorder caused by a small B cell (mostly plasma cell) clone which produces amyloidogenic light chains. The goal of therapy is to target this clone and halt the uncontrolled release of free light chain, which might subsequently lead to improvement of organ function. In routine diagnostic some of these B cell clones are missed as they might be extremely small. However, specific treatment can only be applied if the clone is well characterized. Hardly any data on the characteristics of these cells using flow cytometry have been reported. (e.g. Paiva et al., Blood 2011). Study design: We performed a retrospective analysis of consecutive patients who were referred to our amyloidosis center (March to July 2014) and have been thoroughly studied (immunhistology of amyloid, free light chain assay, immunofixation, bone marrow diagnostic: cytology, flow cytometry and interphase-FISH cytogenetics (iFISH)). Patients and Methods: Twenty-two patients were included (all untreated, 21 AL patients, one pt with monoclonal gammopathy of renal significance (MGRS)). Plasma cells were detected by their co-expression of CD38 and CD138 antigens. Differentiation between malignant and normal plasma cells was achieved by analysis of aberrant CD45 and CD19 expressions and proof of intracellular light chain restriction (see Figure 1). To evaluate potential targets for an antibody-based immunotherapy, we stained CD20, CD22, CD30, CD52 and CS-1 on these plasma cells. Overall, positivity was defined as >20% expression of the antigen. iFISH was done after CD138 selection as previously described (Bochtler et al., Blood 2011). Results: Main characteristics and results are shown in Table 1. Median dFLC was 304 mg/l, three patients had a dFLC of less than 50 mg/l. Median plasma cell count in cytology was 10%, 3 patients had less than 5%. Median plasma cell count by flow was 3.8%, three patients had less than 1%. Correlation between dFLC, plasma cell count in cytology and flow was low (FLC vs. flow: spearman=0.25, p=0.26; FLC vs. cytology: spearman=0.49, p=0.02; flow vs. cytology, spearman=0.36, p=0.1). Detection of the amyloidogenic clone by flow was possible in all but one patient (95%). In this patient we were not able to show a light chain restriction although we detected a relevant aberrant plasma cell clone (CD45low, CD19low). In one patient we found a B cell lymphoma as underlying disease for MGRS type IgG lambda (CD19+, CD20+, lambda+, CD5-, CD22+, FMC7-, CD23-, CD25+, CD103-, CD38+ typical for marginal zone lymphoma). In all 21 patients the light chain restriction demonstrated by flow was confirmed by immunofixation, FLC, and immunohistology of the amyloid. All patients analyzed for the expression of CS-1 were positive. 25% were also positive for CD20 and none was positive for CD22, CD30 and CD52. Detection of the plasma cell clone by iFISH was possible in all 21 patients (see Table 1). Conclusion: Flow cytometric analysis of the bone marrow is a very sensitive method to detect and characterize the amyloidogenic clone in AL amyloidosis. B cell lymphomas can easily be distinguished from pure plasma cell clones. Secondly, flow provides useful information to specify immune-chemotherapy in AL amyloidosis and related disorders. Table 1: Patients (n=22) Characteristics and Results Age in yrs (median / range) 67 (41 – 77) Sex: female / male 9 / 13 Type of light chain: kappa / lambda 4 / 18 Median dFLC in mg/l (range) 304 (22 - 6621) Median % of plasma cells in BM cytology (range) 10 (0 – 68) Underlying disease leading to AL amyloidosis“MG” / MM III / B-NHL 20 / 1 / 1 Median % of PC by flow (range) 3.8 (0.2 - 34) Detection of the amyloidogenic clone by flow 21 / 22 Flow analysis of clonal plasma cells (% of pts)CD20+ / CD22+ / CD30+ / CD52+ / CD56+ / CS-1+ 25 / 0 / 0 / 0 / 75 / 100 Detection of a clone by iFISH 21/21 % of pts with t(11;14) / Gain of 1q21 / Hyperdiploidy / High-risk cytogenetic (del 17p13, t(4;14)) 52 / 10 / 14 / 10 Figure 1: Representative flow analysis of one pt. with a lambda+, CD38+, CD138+ plasma cell clone (green). Polyclonal CD19+ B cells in red. Figure 1:. Representative flow analysis of one pt. with a lambda+, CD38+, CD138+ plasma cell clone (green). Polyclonal CD19+ B cells in red. Disclosures Schönland: Janssen: Honoraria; Celgene: Honoraria. Hegenbart:Janssen: Honoraria; Celgene: Honoraria. Hose:Novartis: Research Funding. Hundemer:Celgene: Honoraria, Research Funding.

Humoral Immunity and Plasma Cell Changes in Patients Responding to CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T-Cell Adoptive Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1110-1110
Author(s):  
Vijay Bhoj ◽  
Michael C Milone ◽  
Carl H. June ◽  
David Porter ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Humoral Immunity ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric ◽  
Antibody Titers

Abstract Introduction: T cells engineered to express chimeric antigen receptors (CARs) recognizing CD19 (CART19) can eliminate malignant cells in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We and other groups have shown that persistent tumor eradication by CD19-specific T cell immunotherapy is accompanied by normal B-cell aplasia. It is assumed that responding patients cannot make new antibody responses post-successful CART19 treatment; however, the status of previously established humoral immunity in these patients is currently unknown. Understanding the consequence of successful CART19 therapy on established humoral immunity has implications for both the clinical management of CART19-treated patients as well as the potential application of this therapy to non-malignant diseases such as autoimmunity and transplantation. Methods: We performed a prospective, observational study of adult and pediatric patients with ALL and adults with relapsed/refractory CLL, who were enrolled in clinical trials of CART19 at our institution. Serum antibody titers to previously-generated vaccine or vaccine-related pathogens (Streptococcus pneumoniae, Tetanus toxoid, Hemophilus influenza type-B (HIB), Measles, Mumps, and Rubella) were determined along with a quantitative assessment of B-cell and plasma cell frequencies in blood and bone marrow aspirates. Specimens were collected during pre-established study assessments or additional time points when collected as required for clinical management. Due to the challenges of assessing plasma cells, multiple methods were employed for their quantification in fresh specimens including flow cytometry and immunohistochemistry (IHC). Flow cytometric assessment of plasma cells was performed on freshly obtained marrow samples. Only patients with at least 3 months of B-cell aplasia in the absence of regular intravenous immunoglobulin (IVIg) infusions were included in the study. Results: All patients had no evidence of leukemia or peripheral B cells post-CART19 infusion at the time of this study. Compared to pre-CART19 serum titers, antibodies to S. pneumoniae remained stable or increased in 9 of 12 patients despite lack of circulating B-cells. Antibody titers to Tetanus toxoid were stable or increased in 13 of 14 patients. Anti-HIB levels were stable or increased in 9 of 11 patients and antibodies to Measles, Mumps and Rubella were stable or increased in 12 of 13, 11 of 13, and 12 of 13 patients, respectively. Flow cytometric analysis of bone marrow aspirates after CART19 infusion revealed three patients with persistence of CD38+ CD138+ plasma cells (at 1, 3 and 9 months post infusion, respectively) despite a complete absence of peripheral CD19+ B cells. In 9 patients, CD20 and CD138 IHC analysis of bone marrow core biopsies revealed a decrease in plasma cell (ranges: 1-5% pre-CART19, 0-<1% post-CART19), consistent with our previously published data. Finally, in another subset of patients, neither B cells nor plasma cells were detectable by flow cytometry of aspirate material or IHC of core biopsies collected either pre- or post-CART19 treatment. Conclusions: The stable or increased titers of antibodies to previous vaccines are surprising and may, in part, reflect improved marrow function as a result of leukemia eradication. The demonstration of plasma cells in a subset of patients in the absence of detectable tumor or normal B cells provides strong evidence for the existence of a population of plasma cells that are resistant to lysis by CART19 cells. This is consistent with antibody titers to previously generated vaccine antigens, which remain stable despite effective CART19 treatment. The additional finding of a decrease in CD138+ cells in several patients by IHC suggests that some populations of plasma cells are either targeted directly by CART19 or have a short half-life (e.g. plasmablasts); CD138 is not sufficient to distinguish these populations. Overall, these results indicate that long-lived plasma cells are resistant to CART19, likely due to a loss of CD19 during plasma cell differentiation. Continued analysis of remaining plasma cells in the absence of ongoing B-cell maturation as a result of CART19 persistence may provide important information on turnover rates of these long-lived cells in humans. Disclosures Bhoj: Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties. Porter:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Lacey:Novartis: Research Funding. Mahnke:Novartis: Research Funding.

Cerebrospinal Fluid (CSF) Involvement By Plasma Cell Dyscrasias – Single Center Experience

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5686-5686
Author(s):  
Taiga Nishihori ◽  
Jun Zhou ◽  
Kenneth H. Shain ◽  
Rachid Baz ◽  
Melissa Alsina ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
Cerebrospinal Fluid ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Transplant ◽  
Cns Involvement ◽  
Color Flow ◽  
Tumor Load ◽  
Plasma Cell Dyscrasias

Abstract Introduction: Central nervous system (CNS) involvement by plasma cell dyscrasias (PCD) is uncommon but poses significant clinical challenges and has a dismal prognosis. Lumbar puncture (LP) is typically performed only for patients with neurologic signs or symptoms and data on patients with CNS involvement are rather scarce. Here, we report a retrospective single institution review of clinicopathological features and treatment outcomes in the setting of cerebrospinal fluid (CSF) involvement by PCD. Methods: We identified consecutive patients with plasma cell disorders who had abnormal cytology or flow cytometry results in the CSF in the Department of Hematopathology database at Moffitt Cancer Center from 1997 to 2014. Cytology slides [Wright-Giemsa (WG) and Papanicolaou (Pap) stained preparations] and the corresponding flow cytometry were reviewed to confirm the diagnosis. Four-color flow cytometry was performed using antibodies against CD38, CD138, CD56, CD117, CD19, and cytoplasmic kappa and lambda light chains, withadditional markers added when necessary. Clinical variables were abstracted from the patient medical records. Overall survival was estimated from the time CSF involvement was identified using the Kaplan-Meier method. Results: Sixty-seven Pap-stained cytology smeas/cytospins and WG stained cytospins from 65 patients who underwent LP for clinical suspicion of CSF involvement were reviewed. Flow cytometry was preformed on 48 cases positive for atypical plasma cells by cytology. Sixteen of 67 (23.9%) were suspicious or diagnostic for PCD (median age of 58 years (range 44 – 75), 56% were male). However, only 4 of 16 cases (25%) were diagnosed as PCD by cytology without additional flow cytometry study. Median tumor load of PCD by flow cytometry was 81% (range 4 - 99%). PCD included 14 patients (88%) with multiple myeloma [MM; 1 patient progressed to secondary plasma cell leukemia (PCL)], 1 with primary PCL, and 1 with Waldenström macroglobulinemia (Neel-Bing syndrome). Of the 14 MM patients, 57% had high-risk disease by cytogenetics/FISH, and immunophenotypes were IgA (50%), IgG (29%), and light chain (21%). All MM patients had Durie-Salmon stage 3 disease. Median number of prior therapies was 2 (1-4), and 44% received stem cell transplant prior to CSF involvement. Median time from diagnosis to CSF involvement was 23 (range, 6 – 78) months. Presenting symptoms included diplopia/vision loss (31%), headache (25%), and leg weakness (cauda equina/cord compression) (19%). Two patients presented with gross orbital involvement and new/enlarging scalp lesions. On radiologic imaging, 5 (31%) had leptomeningeal, 4 (25%) had epi- or extra-dural, and 2 (13%) had dural enhancement/lesions. None had CSF involvement at the time of initial PCD diagnosis. Treatment included intrathecal chemotherapy (methotrexate, cytarabine or triple regimen; 86%), radiation therapy (including whole brain, craniospinal, radiosurgery or involved field; 63%) and systemic chemotherapy (65%). One patient did not receive treatment due to poor performance status. For 5 patients who had repeat CSF analyses, only one had no evidence of disease on cytology but flow cytometry remained positive. Six-month overall survival rate was 20.2% (95% CI 4.4 – 43.5). At the time of data review, only 2 patients were alive. Conclusions: CSF involvement by PCD carries extremely limited prognosis and represents advanced stage of the disease. Patients may be treated with systemic therapy as well as CNS-directed therapy, though the outcomes are dismal. Careful assessment of patients’ neurologic symptoms and low threshold for performing LP is required for early detection of CSF involvement. Application of flow cytometry appears to be a useful tool in the diagnosis of CSF involvement by PCD; improving sensitivity and specificity over cytology alone, particularly when the tumor load is low or cytologically equivocal for atypical plasma cells. Further research is needed to improve the outcomes of these patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single Arm, Prospective, Open-Label Phase II Trial to Evaluate the Efficacy of Isatuximab in Patients with Monoclonal Gammopathy of Renal Significance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3161-3161 ◽  
Author(s):  
Vikram Premkumar ◽  
Suzanne Lentzsch ◽  
Divaya Bhutani
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Plasma Cell ◽  
Monoclonal Gammopathy ◽  
Plasma Cells ◽  
Cell Clone ◽  
Advisory Committees ◽  
Renal Response ◽  
Hematologic Response ◽  
Stable Renal Function

Background: Monoclonal gammopathy of renal significance (MGRS) is a monoclonal B cell disorder, not meeting the definition of lymphoma or myeloma, that produces monoclonal proteins which deposit in the kidneys. Permanent renal damage can occur either as a consequence of direct deposition of toxic proteins or by an induced inflammatory response. Due to the low burden of the plasma cell clone, patients do not otherwise qualify for potentially toxic anti-plasma cell treatments and treatment is generally based on consensus opinion. To date there are no clinical trials exploring treatment options. Isatuximab is a chimeric mouse/human IgG1k monoclonal antibody which targets CD38 on both malignant and normal plasma cells and exhibits it antitumor effects primarily by antibody-dependent cellular toxicity. Isatuximab has recently been shown to be an active drug in the treatment of multiple myeloma, with improvements seen in hematologic and renal markers, and has been shown to have manageable toxicity. Given the efficacy of isatuximab in multiple myeloma, we propose a trial evaluating isatuximab monotherapy to treat the small plasma cell clone in MGRS with the hopes of maximizing response and minimizing toxicity. Study Design and Methods: The primary objective of this study is to evaluate efficacy of isatuximab monotherapy in patients with MGRS in order to establish a standard of care treatment for patients with this disease. Adult patients with proteinuria of at least 1 gram in 24 hours and a histopathological diagnosis of MGRS on renal biopsy in the last 24 months will be eligible for the trial. Patients will be excluded if their estimated GFR is below 30 mL/min, they have multiple myeloma, high risk smoldering myeloma, other B cell neoplasm meeting criteria for treatment, concurrent diabetic nephropathy, or require dialysis. Patients will be screened for B cell disorders with bone marrow biopsy and aspirate, serum protein electrophoresis (SPEP) with immunofixation (IFE), 24-hour urine protein electrophoresis (UPEP), free light chain (FLC) testing and screening PET/CT at time of enrollment. Enrolled patients will be administered isatuximab 20 mg/kg IV weekly for 4 weeks and then will receive the same dose every 2 weeks thereafter for a total of 6 months. Patients may be continued on treatment following completion of the 6 months at the discretion of the provider. To reduce the risk of infusion related reactions, patients will receive premedications with corticosteroids, diphenhydramine, H2 blockade and acetaminophen at least 60 minutes prior to infusion. Patients will have repeat SPEP + IFE, 24-hour UPEP + IFE and FLC testing every 4 weeks. There will be an optional repeat kidney biopsy 9-12 months following treatment initiation to assess pathologic response in the kidneys. Statistical Methods: The study will be comprised of 20 patients being treated with isatuximab over a span of 24-30 months. Ten patients will be initiated on the therapy for a period of 6 months. Interim analysis will be done after these patients have completed all the treatment cycles. If 4 out of 10 patients show response in form of improved/stable renal function, the study will proceed to include next 10 patients. If >50% of the first group of 10 patients show doubling of creatinine while on therapy, that would be considered as an indication to discontinue the therapy and the study due to drug toxicity. Endpoints: The primary endpoint will be efficacy as measured by renal response and hematologic response. Renal response will be measured by assessing the amount of proteinuria in a 24 hour urine sample. A sustained reduction in proteinuria by 30% from the patient's baseline amount of proteinuria with stable renal function (serum eGFR within 20% of baseline) will be considered a positive renal response. Hematologic response will be quantified per the 2016 International Myeloma Working Group (IMWG) uniform response criteria for multiple myeloma. An important secondary endpoint will be safety and will be analyzed from all patients who receive any study drug. Adverse events will be characterized and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Other endpoints include time to dialysis and rate of minimal residual disease (MRD) negativity. Disclosures Lentzsch: Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy. Bhutani:Sanofi: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Our trial will be evaluating the efficacy of targeting CD38 on plasma cells with isatuximab in patients with monoclonal gammopathy of renal significance (MGRS). We will evaluate the effects of this drug on 24 hour proteinuria and hematologic response.

scholarly journals Therapeutic Targeting of CCR1 to Prevent Dissemination of Multiple Myeloma Plasma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3099-3099
Author(s):  
Mara N Zeissig ◽  
Duncan R Hewett ◽  
Krzysztof M Mrozik ◽  
Vasilios Panagopoulos ◽  
Monika Engelhardt ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Disease Progression ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Primary Tumour ◽  
Nsg Mice ◽  
Rpmi 8226 ◽  
Retention Signal

Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p<0.0001; EV: 1.39±0.7; CCR1-KO: 0.03±0.046) anda 99.9% reduction in BM tumour dissemination (p<0.0001; EV: 49.5±17; CCR1-KO: 0.019±0.013), compared with controls. In a supportive study, CCR1 was expressed in the murine MM cell line 5TGM1 using lentiviral transduction. 5TGM1-CCR1 cells were confirmed to express CCR1 by qPCR and were able to migrate towards CCL3 in vitro. 5TGM1-CCR1 or EV cells were injected into the tibiae of C57BL/KaLwRij mice and allowed to initiate systemic MM disease for 3.5 weeks. Importantly, while 55% of control mice exhibited disseminated tumours, this increased to 92% with CCR1 expression (p<0.0001; n=12/group). These data suggest that CCR1 expression on MM PC may play an important role in MM PC dissemination. To determine whether therapeutic inhibition of CCR1 prevents dissemination, the effect of a small molecule CCR1 inhibitor, CCR1i, was assessed in vivo. OPM2 EV or RPMI-8226 cells were injected into the tibia of NSG mice and, after 3 days, mice were treated with CCR1i (15mg/kg) or vehicle twice daily by oral gavage for 25 days. OPM2-inoculated CCR1i-treated mice had 66.1% lower PB MM PC (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 23.9±7.2; CCR1i: 8.1±3.8) and a 22.1% reduction in BM dissemination (p=0.0002; vehicle: 78.1±4.8;CCR1i: 60.8±7.1) compared with controls. Similarly, CCR1i treatment reduced BM dissemination by 59.6% in RPMI-8226 bearing mice (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 0.86±0.15; CCR1i: 0.26±0.05). This suggests that CCR1 inhibition can slow tumour dissemination in vivo. Conclusion:This study identified CCR1 as a novel driver of MM PC dissemination in vivo, at least in part by overcoming the CXCL12 retention signal. Importantly, this study demonstrated for the first time that targeting CCR1 can be a viable therapeutic strategy to limit dissemination and potentially slow disease progression. Disclosures Croucher: Trovagene: Employment.

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