Utility of Multiparameter Flow Cytometry Immunophenotypic Studies In Patients with Systemic Light Chain (AL) Amyloidosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4051-4051
Author(s):  
Bruno Paiva ◽  
María-Belén Vidriales ◽  
Jose J. Perez ◽  
Maria-Consuelo López-Berges ◽  
Ramón García-Sanz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Plasma Cell ◽  
Light Chain ◽  
Cell Cycle Analysis ◽  
Disease Assessment ◽  
Multiparameter Flow Cytometry ◽  
Al Amyloidosis

Abstract Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

Multiparameter Flow Cytometry (MFC) for Identification of the Waldenström's Clone in IgM MGUS and Waldenstrom's Macroglobulinemia (WM): New Criteria for Differential Diagnosis and Risk Stratification

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 936-936
Author(s):  
Bruno Paiva ◽  
Maria-Carmen Montes ◽  
Ramón García-Sanz ◽  
Jennifer Alonso ◽  
Natalia de las Heras ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Phenotypic Characterization ◽  
International Prognostic Scoring System ◽  
Multiparameter Flow Cytometry ◽  
Risk Of Progression

Abstract Abstract 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P<.001), as well of light chain restricted B cells (75%, 96% and 99%; P<.001). In contrast, no differences were found for the percentage of PC (median of 0.3%), but light chain restricted PC progressively increased from IgM MGUS to smoldering and symptomatic WM (70%, 85% and 97%; P<.001). Accordingly, only 1% of IgM MGUS patients showed >10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P<.001). Likewise, only 1% of IgM MGUS patients showed 100% light chain restricted B cells, in contrast to 19% and 40% of smoldering and symptomatic WM (P<.001); similar results being also found using a cutoff of 100% light chain restricted PC. Subsequently, we explored whether the percentages of BM and light chain restricted B cells and PC could predict time to progression (TTP) from smoldering into symptomatic WM, as well as overall survival (OS) in symptomatic WM. In smoldering WM, B cells (>10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs <100%: 26m vs 145m; P<.001) but not PC, predicted risk of progression. On the multivariate analysis that included serum M-spike (±3g/dL), BM infiltration (±50% lymphoplasmacytic cells), BM B-cells and light chain restricted B cells (by MFC), only the later retained independent prognostic value (HR: 19.8, P=.001). Upon analyzing factors influencing survival in symptomatic WM patients, cases with >10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs <100% light chain restricted B cells (median OS 44m vs 78m; P=.001). The later marker was independent (HR: 2.6; P=.004) of the International Prognostic Scoring System (HR: 2.2; P=.006). Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P<.001), CD25+ (61%, 88% and 90%; P<.001) and sIgM+ (88%, 95% and 97%; P=.002) B cells from IgM MGUS to smoldering and symptomatic WM. This underlies that the accumulating light chain restricted clonal B cells show a characteristic Waldenstrom's phenotype (CD22dim/CD25+/IgM+). Of note, a bimodal (from - to +) expression for the B cell memory marker CD27 was found in >50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Daratumumab Interferes with Flow Cytometric Evaluation of Multiple Myeloma

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

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

Multiparameter Flow Cytometry Analysis of Peripheral Blood T, NK and Dendritic Cells From High-Risk Smoldering Multiple Myeloma Patients Treated with Lenalidomide and Dexamethasone

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1906-1906
Author(s):  
Bruno Paiva ◽  
Lucía López-Corral ◽  
María-Belén Vidriales ◽  
Luis Ignacio Sánchez Abarca ◽  
Miguel T. Hernandez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
T Cells ◽  
High Risk ◽  
Nk Cells ◽  
Nk Cell ◽  
Multiparameter Flow Cytometry ◽  
Cd8 Cells

Abstract Abstract 1906 Lenalidomide is an immunomodulatory agent that interacts with different components of the immune system by altering cytokine production, regulating T cells and increasing NK cell cytotoxicity. In multiple myeloma (MM), lenalidomide is approved for use in combination with dexamethasone in patients who have received at least one prior therapy. Recent observations have shown that dexamethasone enhances the anti-myeloma effect of lenalidomide; however, dexamethasone may also antagonize the immunomodulatory properties of lenalidomide. In the present study we evaluated by multiparameter flow cytometry (MFC) peripheral blood (PB) T, NK and dendritic (plasmacytoid, myeloid and monocytic) cells (DC) from high-risk smoldering MM (SMM) patients, defined by the presence of at least 2 of the 3 following criteria at diagnosis: bone marrow plasma cell (BMPC) infiltration ≥10%; and/or high M-component (IgG≥30g/L or IgA≥20g/L or B-J Protein>10g/L); and/or ≥95% myelomatous-PC/BMPC and immune paresis. SMM patients were treated according to the QuiReDex trial (NCT 00480363): an induction phase of nine four-week cycles of lenalidomide plus dexamethasone (LenDex) followed by maintenance with lenalidomide until disease progression. In this ongoing study, immunophenotypic data is available in 53 patients at diagnosis (baseline), 30 after 3 cycles of LenDex and 22 at the end of induction therapy (9th cycle). Here we will focus on the 22 cases with information at the 3 time points. For MFC analysis, PB samples were stained using a four-color direct immunofluorescence technique that allowed the quantification and characterization of T, NK and DC cells, including cell cycle analysis. The percentage of PB T cells in total PB cellularity was stable from baseline vs 3 vs 9 cycles of LenDex (22% vs 21% vs 21%; respectively, NS), with similar results also obtained for T CD4 (12% vs 11% vs 9%; respectively, NS) and T CD8 (8% vs 6% vs 8%; respectively, NS) cells. NK cells were slightly increased after 9 cycles of LenDex for both the CD56dim (4.1%, 3.4% and 6%; respectively; NS) and CD56bright (0.05%, 0.04% and 0.15%; respectively; NS) NK cell compartments. Similarly, the percentage of DC slightly increased along treatment, especially for plasmacytoid DC (0.2% at baseline vs 0.4% after 9 cycles; p=0.09). However, when a more detailed immunophenotypic characterization of T and NK cells was carried out significant differences emerged following LenDex treatment (Figure 1A). Accordingly, after 3 and 9 cycles of LenDex both T CD4 and CD8 cells showed increased expression of activation markers such as CD69 (p=.03), CD25 (p=.02 and NS, respectively), CD54 (p<.001), CD28 (p≤.03) and CD120b (p≤.01), together with increased production of IFNγ (p=.03) and IL-2 (p=.1 and p=.008, respectively). Interestingly, after induction therapy an up-regulation of chemokine receptors related to the Th1 (CCR5; p<.001) but also Th2 (CCR4; p≤.002) immune response was detectable in CD4 and CD8 T cells. T CD4 cells displayed a clear maturation into a central memory phenotype following LenDex treatment (38% at baseline vs 50% and 66% at 3 and 9 cycles, respectively; p<.001) while T CD8 cells displayed an increased effector memory phenotype (44% vs 59% vs 62%; p=.004). Further analysis showed increased expression of HLA-DR (p≤.008), the antibody-dependent cell-mediated cytotoxicity associated receptor CD16 (p≤.03), and the adhesion molecules CD11a (p’.006) and CD11b (p≤.004) both on NK (CD56dim and CD56bright) and T cells. No consistent changes were observed in other NK cell receptors, such as CD94 and the immunoglobulin like receptors CD158a, CD161, NKB1 (3DL1) and NKAT2 (2DL3). Concerning cell cycle analysis, the percentage of cells in S-phase was significantly increased from baseline vs 3 vs 9 cycles of LenDex in T CD4 (0.05% vs 0.15% vs 0.16%; p<.001), CD8 (0.05% vs 0.11% vs 0.23%; p<.001) and NK cells (0.09% vs 0.17% vs 0.20%; p=.001). Finally, an unsupervised cluster analysis of the overall immunophenotypic profile obtained after 9 cycles of LenDex (Figure 1B) was able to discriminate two groups of patients (A and B). Interestingly, within the group with higher activation profile (A) 50% of patients achieved ≥VGPR vs 23% in group B (p=.2). In summary, these preliminary results show that in high risk SMM patients the combination of lenalidomide and dexamethasone modulates PB T and NK cells, with increased activation status that may contribute to disease control. Disclosures: Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma. De La Rubia:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Celgene: Honoraria. Oriol:Celgene: Consultancy; Janssen-Cilag: Consultancy; Novartis: Consultancy. Hernández:Celgene: Honoraria. de Arriba:Janssen-Cilag: Honoraria; Celgene: Honoraria. Mateos:Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria, Speakers Bureau.

BrdU incorporation in multiparameter flow cytometry: A new cell cycle assessment approach in multiple myeloma

2018 ◽  
Vol 96 (3) ◽  
pp. 209-214 ◽  
Author(s):  
Guilhem Requirand ◽  
Nicolas Robert ◽  
Stéphanie Boireau ◽  
Laure Vincent ◽  
Anja Seckinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Brdu Incorporation ◽  
Multiparameter Flow Cytometry ◽  
Assessment Approach

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.

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

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

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

scholarly journals Dysphagia unveiling systemic immunoglobulin light-chain amyloidosis with multiple myeloma

2018 ◽  
pp. bcr-2018-226331 ◽  
Author(s):  
Juan Gonzalez ◽  
Ahsan Wahab ◽  
Kavitha Kesari
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cell ◽  
Light Chain ◽  
Amyloid Deposition ◽  
Al Amyloidosis ◽  
Immunoglobulin Light Chain ◽  
Percutaneous Gastrostomy ◽  
Uncommon Presentation ◽  
Immunoglobulin Light Chain Amyloidosis ◽  
Percutaneous Gastrostomy Tube

Dysphagia is an uncommon presentation of systemic immunoglobulin light-chain (AL) amyloidosis with multiple myeloma (MM). Gastrointestinal (GI) involvement usually manifests with altered motility, malabsorption or bleeding. Furthermore, patients identified with GI amyloidosis, without previous diagnosis of a plasma cell disorder, are extremely rare. We report an elderly woman who presented with acute on chronic cardiac dysfunction, sick sinus syndrome and acute renal failure. While admitted, she developed intermittent dysphagia to both solids and liquids. Oesophagogastroduodenoscopy showed ulcerations of oesophagus and duodenum. Biopsies revealed focal amyloid deposition, stained with Congo red. Renal biopsy revealed amyloid deposition in renal arterioles. She underwent a bone marrow biopsy confirming MM, represented by more than 15% plasma cell population. She was started on treatment for heart failure, induction chemotherapy for MM and percutaneous gastrostomy tube for feeding. However, she continued to deteriorate, eventually opting for hospice, and ultimately died 2 days after discharge from hospital.

Genomic Landscape of Immunoglobulin Light Chain (AL) Amyloidosis and Comparative Analyses with Related Malignant Plasma Cell Disorder- Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 809-809
Author(s):  
Vishwanathan Hucthagowder ◽  
Jahangheer Shaik ◽  
Mark Fiala ◽  
Jacob Paasch ◽  
Rakesh Nagarajan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cell ◽  
Light Chain ◽  
Copy Number ◽  
Al Amyloidosis ◽  
Immunoglobulin Light Chain ◽  
Genomic Changes ◽  
Genomic Landscape ◽  
Plasma Cell Disorder ◽  
Cell Disorder

Abstract Abstract 809 Immunoglobulin light chain amlyloidosis (AL) is a rare plasma cell disorder characterized by deposition of misfolded light chains in various organ systems with an average survival of 1–2 years. AL is also the most common form of systemic amyloidosis with 1200–3200 newly diagnosed cases reported annually in the United States. Very little is known regarding specific genomic aberrations associated with AL-amyloidosis. Aside from the light chain selection, no phenotypic or genetic features have been identified that distinguish AL amyloidosis from other plasma cell dyscrasias. Understanding the genetics of AL and the molecular mechanisms involved in amyloid formation may lead to early diagnosis and the identification of novel drug targets and therapies. We therefore have attempted to study the genomic landscape of AL patients and MM for comparison. Genomic copy number and loss of heterozygosity (LOH) analyses were performed on DNA derived from tumor (CD138 sorted cells) and matched germline (skin) from biopsy proven AL patients using Affymetrix single nucleotide polymorphism (SNP) 6.0 arrays. Numerous genomic changes with gains in chromosome 1q, 6, 9, 11q, 15, 19 and 21 and loss on chromosome 1p, 2q, 8, 10, 12, 13, 14, 16, 18, 20 and 22 were observed in more than 10% of the patients. Recurrent genomic changes in about 249 segments involving 457 genes were present in about 1/3 of AL patients. In particular, deletion of IGK, IGH, PIK3CA, FLT3, RB1, PCDH9, GPC6, RASA3, ADAM6 genes and amplification of CFHR1, JAK2, GCNT1, TSC1, PGR genes were observed. Gene network analysis showed five distinct major modules consisting of 51 distinct elements and involving PDGF, TP53, interleukin signaling, TRKA signaling, cell cycle and mitotic pathways were enriched. Allele specific copy number analysis in tumor (ASCAT) profile showed increased ploidy status of the AL genome in 47% of the assessed patients. LOH was observed in chromosomes 4, 5, 6, 8, 9, 12, 13, 18 and 22 in 30% of patients, ranging from 5Mb to entire chromosome. Furthermore, genomic comparisons of AL with multiple myeloma (MM) showed the typical archetype of myeloma's signature with exception of gain of chromosomes 3, 5, 7 and loss of chromosome 6q and 8p. Interestingly deletion of IGH, IGK locus and PIK3CA gene were observed at a higher frequency in AL patients. Categorical analysis using isotype specific classification in AL showed a significantly higher frequency of deletion in chromosome 14, 13, 8 and amplification of chromosome 9q in the kappa type than lambda isotype. To the best of our knowledge, this is the first ultra-high resolution study of the genomic landscape of AL amyloidosis. In this study, we have found several novel genes and pathways associated with this rare disease. The numerous copy number alterations of AL thus reflect the genomic complexity and the heterogeneity of this disease. Additional genome-wide analysis in a larger panel with target organ stratified patients is under way and may further our understanding of genetic changes specifically associated with AL. Disclosures: No relevant conflicts of interest to declare.

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