Gene Expression Profile of Circulating Myeloma Cells Reveals CD44 and CD97 (ADGRE5) Overexpression

Abstract Introduction: Release of the aberrant plasma cells (PC) from the bone marrow (BM) and their presence in the peripheral blood (PB) is a maker of disease progression and worse survival in multiple myeloma (MM) (Nowakowski et al., 2005). Circulating plasma cells (cPCs) are able to survive without homing microenvironment, evade the original tumor and colonize other bone marrow niche. Detailed analysis of various surface proteins showed that cPCs display decreased levels of integrins, adhesion molecules N-CAM (CD56) and the stem cell factor receptor (Paiva et al., 2013). Comprehensive analysis of the genome-wide gene expression profiling that could provide deeper insight into the expression patterns of cPCs of MM is still lacking. Aims: To identify differentially expressed genes in paired samples of aberrant plasma cells from BM and PB and to describe potential biomarkers of cPCs in MM. Material and methods: Ten patients with multiple myeloma (seven new diagnoses and three relapses) have been included in the study after signing the informed consent form. Paired samples of aberrant plasma cells from bone marrow and peripheral blood were obtained from each patient. Aberrant plasma cells (aPCs) were sorted according to the immunophenotype as CD45dim/CD38+/CD19-/CD56-/+ cells. Gene expression profiling (GEP) was performed on paired samples using Affymetrix GeneChip Human Gene ST 1.0 array. RMA normalized data at gene level were analyzed using Wilcoxon paired test with Benjamini-Hochberg multiple testing correction. Results: The median infiltration of aberrant PC in the BM was 27.5% (range 1.1 - 93%) and 1.2% (range 0.19 - 2.8%) for cPCs in the PB. The median level of M-protein was 32.35 g/l (range 18.6 - 62.2 g/l). GEP analysis of paired BM and PB samples revealed 1001 significantly changed genes in cPCs (adjusted p-value<0.05). Gene ontology analysis did not reveal any significantly affected pathways. Nevertheless, two genes upregulated in cPCs, ADGRE5 and CD44, can be suggested as biologically relevant potential biomarkers of cPCs (Figure 1). Conclusion: The infiltration of aPCs in the bone marrow does not correlate with the amount of cPCs (p=0.16). Among differentially expressed genes, two surface markers upregulated in cPCs are of particular interest: CD44 and ADGRE5 (CD97). The CD44 antigen is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. Moreover, CD44 contribute to lenalidomide resistance in multiple myeloma (Bjorklund et al., 2014). CD97 is encoded by ADGRE5 gene and belongs to the EGF-TM7 subgroup of adhesion G-protein-coupled receptors. The expression of CD97 has been linked to invasive behavior in thyroid and colorectal cancer. Moreover, higher CD97 expression levels have been detected in 54% (208/385) of primary AML samples based on flow cytometric analysis (Wobus et al., 2015). Nevertheless, neither ADGRE5 nor CD97 expression were described in plasma cell dyscrasia previously. Thus, despite non-systemic changes of gene expression at the whole transcriptome level, cPCs in MM likely represent distinct biological entity with specific expression profile underlying advanced PC malignant transformation. To confirm the results, flow cytometric analysis on the bigger cohort will be performed. Acknowledgment: This study was supported by Institutional Development Plan of University of Ostrava (IRP201550) and The Ministry of Education, Youth and Sports (Specific university research of the Faculty of Medicine, University of Ostrava) project no. SGS03/LF/2015-2016, Ministry of Health Czech Republic RVO-FNOs/2014/17P and RVO-FNOs/2016/21. Figure 1 Genes of interest differentially expressed in the bone marrow (BM) versus peripheral blood (PB) aberrant plasma cells. Figure 1. Genes of interest differentially expressed in the bone marrow (BM) versus peripheral blood (PB) aberrant plasma cells. Disclosures Hajek: BMS: Honoraria; Onyx: Consultancy; Novartis: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

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Mir-485-3p and Mir-654-3p Expression in Bone Marrow Mesenchymal Stromal Cells in Patients with Monoclonal Gammopathies Is Related to the Status of the Disease

Blood ◽

10.1182/blood-2018-99-112222 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3155-3155

Author(s):

Carlos Fernandez de Larrea ◽

Tania Diaz ◽

Alfons Navarro ◽

Ester Lozano ◽

Mari-Pau Mena ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Mirna Expression ◽

Stromal Cells ◽

Plasma Cells ◽

Differentially Expressed ◽

Monoclonal Gammopathies ◽

The Status

Abstract Background: Crosstalk between malignant plasma cells and surrounding cells in the bone marrow (BM), such as mesenchymal stromal cells (MSCs), endothelial cells and immune cells, is crucial for pathogenesis of multiple myeloma (MM) and in asymptomatic monoclonal gammopathies. In these diseases, microRNAs (miRNAs) could be useful as biomarkers for diagnosis, prognosis and evaluation of treatment response. miRNAs can be released to the serum and transferred among MM cells and BM-MSCs as cell-cell communication. Previously, we have showed a serum 14-miRNA signature associated with complete remission (CR) after autologous stem-cell transplantation (ASCT). In this sense, patients in CR with partial recovery of two normal serum miRNA levels, similar to those with monoclonal gammopathy of undetermined significance (MGUS), was associated with better prognosis. The aim of this study was to analyze the miRNAs profile in mesenchymal stromal cells derived from bone marrow of patients with multiple myeloma in different status of the disease, comparing with MGUS controls. Methods: We analyzed samples from 95 patients with MGUS (N=23), MM at diagnosis (N=14), relapsed/refractory MM (N=14), MM in partial response (PR) or very good partial response (VGPR) (N=15), MM in CR (N=24) and healthy donors (N=5). Mononuclear cells from BM samples were cultured in DMEM containing 10% FBS. After a week, non-adherent cells were removed, whereas BM-MSCs were selected by their adherence to the plastic and their phenotype was confirmed by multiparametric flow cytometry. In a first screening phase, we analyzed 670 microRNAs in 20 primary BM-MSC from patients with MGUS (N=4), symptomatic MM (N=8) and MM in CR (N=8). miRNAs differentially expressed were identified according to a supervised analysis using significance analysis of microarrays (SAM) and Student's t-test based on multivariate permutation (with random variance model). miRNAs differentially expressed between groups of patients were validated in the whole cohort of BM-MSC from patients. Paired malignant plasma cells (CD38+) miRNA expression from patients with symptomatic MM as well as miRNA in serum samples paired with BM-MSC samples were also compared. RmiR package was used to identify miRNA targets, cross-correlating the miRNA expression data from the present study with our findings on the gene expression signature (Affymetrix Human Genome U219 array) in 12 BM-MSCs from patients (4 MGUS, 4 symptomatic MM and 4 in CR), based on the predicted targets from TargetScan and miRBase databases. Results: In the screening phase, we identified a miRNA profile of 10 miRNAs (miR-663b, miR-654-3p, miR-206, miR-411*, miR-885-5p, miR-668, miR-638, miR-485-3p, miR-744* and miR-199a) differentially expressed between patients with symptomatic MM and MM in CR (adjusted p-value <0.0001). In the validation phase, miR-485-3p and miR-654-3p resulted differentially expressed in the three groups of patients: MGUS, symptomatic MM and patients in CR (ANOVA test: p=0.0101 and p=0.0228, respectively). The levels of these miRNAs were significantly decreased in patients with MM than in those with MGUS, and these levels seemed to recover when patients achieved CR. These two miRNAs (miR-485-3p and miR-654-3p) were also correlated with all degrees of response in MM and with asymptomatic gammopathies (ANOVA test: p=0.0154 and p=0.0487, respectively). Moreover, paired cross-correlation among these two miRNAs expression with our results in mRNA gene expression profile data showed 324 for miR-485-3p and 265 for miR-654-3p genes (correlation index < -0.8) (Figure 1A and 1B). miR-485-3p and miR-654-3p showed a higher expression in BM-MSC than in MM CD38+ cells, suggesting MSC as cell of origin for these miRNAs. Serum expression of these two miRNAs was concordant with the observed in BM-MSC, with higher in patients in CR and MGUS than in those with symptomatic MM (Figure 1C and 1D). miRNA expression in BM-MSC supernatant as well as the identification of the biological role and validation of the miRNA targets are ongoing. Conclusion: miR-485-3p and miR-654-3p expression in mesenchymal stromal cells from bone marrow in patients with multiple myeloma and asymptomatic monoclonal gammopathies is related to the status of the disease and the response to treatment. These miRNAs are also expressed in serum, resulting in potential biomarkers for disease activity and risk of progression. Disclosures Rosinol: Janssen, Celgene, Amgen, Takeda: Honoraria. Bladé:Janssen: Honoraria.

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A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

Blood ◽

10.1182/blood.v122.21.1907.1907 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1907-1907

Author(s):

Eva Sahakian ◽

Jason B. Brayer ◽

John Powers ◽

Mark Meads ◽

Allison Distler ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

Plasma Cell ◽

Research Funding ◽

Plasma Cells ◽

Myeloma Cells ◽

Peripheral Blood Plasma

Abstract The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures: Alsina: Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

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Characterization of Mesenchymal Stem Cells Abnormalities in Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.511.511 ◽

2006 ◽

Vol 108 (11) ◽

pp. 511-511 ◽

Cited By ~ 2

Author(s):

Philippe Bourin ◽

Jill Corre ◽

Karène Mahtouk ◽

Mélanie Gadelorge ◽

Patrick Laharrague ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Multiple Myeloma ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Plasma Cells ◽

Osteoblastic Differentiation ◽

Differentially Expressed ◽

Matrix Mineralization ◽

Protein Levels

Abstract Introduction: The bone marrow microenvironnement (BMMe) play a significant role in the physiopathology of the multiple myeloma (MM). However, its abnormality still remains controversial. To address this question, we studied bone marrow mesenchymal stem cells (MSCs), the only long-lived cells of the BMMe. We compared, at a genomic and functional level, the MSCs isolated from patients with MM, to MSCs isolated from healthy subjects and those with monoclonal gammopathy of unknown significance (MGUS). Material and methods : Bone marrow samples from 26 MM patients, 7 MGUS patients and 11 healthy individuals were compared. The MSCs were selected by their adherence on plastic and were cultured in alpha-MEM medium + 10% SVF and antibiotics during 2 passages (primo-culture = P0 and first passage = P1). The gene expression profiling was carried out by Affymetrix GeneChip microarrays (U133 plus 2.0). The expression of interesting differentially expressed genes was validated by ELISA or qRT-PCR. The phenotype was studied by flow cytometry (CD45, CD90, CD73, CD13, CD14). The CFU-F frequencies in BM samples and in cell suspensions after P0 and P1 were studied as well as the cell productions after P0 and P1. The osteoblastic differentiation was evaluated both by alkaline phosphatase dosing and matrix mineralization quantification. We also carried out co-cultures of the MSCs with CD34+ cells to quantify their hematopoietic supportive potential. Finally XG1 and Molp-6, respectively stroma independent and stroma dependent cell lines, were co-cultured with MSCs to check the capacity of the MSCs to support malignant plasma cell growth. Results: Gene expression profile independently classified the MSCs in a normal and in a MM group. MGUS MSCs were interspersed between those 2 groups. 145 distinct genes were differentially expressed in MM and normal MSCs. Among them, 46% could be involved in tumor-microenvironment cross-talk. Known soluble factors involved in MM physiopathologic features, such as IL-6, IL-1ß, DKK1 and amphiregulin, were identified and new ones found. In particular growth and differentiation factor-15 (GDF-15), already described as a accurate biomarker of numerous tumours, was significantly overexpressed (p<0.001) in MM MSCs both at mRNA and protein levels (183.5 ± 64.9 vs 749 ± 90.9 for mRNA, 1 10−4 pg/cell ± 1.9 10−5 pg/cell vs 4.3 10−4 pg/cell ± 1.4 10−4 pg/cell for protein respectively for normal and MM MSCs). It was also able to induce dose-dependant growth of Molp-6, in the absence of a supportive stroma. The phenotype and the CFU-F frequencies and the cell productions were similar in the 3 groups of MSCs and their hematopoietic supportive capacity was maintained. The MM MSCs complete differentiation towards the osteoblastic lineage, evaluated quantitatively, was faded. And very importantly, MM MSCs constituted a better supportive feeder layer for the Molp-6 cell line as compared to normal MSCs (cell expansion after 7 days : 2.1 ± 0.3 vs 3.3 ± 0.4, p = 0.04, respectively for normal and MM MSCs). On the other hand, the growth of XG1 was not influence by the subject origin of the MSCs. Conclusion: Our results show that the MSCs, like the malignant plasma cells, are abnormal in MM. This confirms the place of microenvironnement in the physiopathology of the MM and makes it possible to identify new potential therapeutic targets.

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miRNA in Serum and Bone Marrow Plasma Cells From Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v120.21.2921.2921 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2921-2921

Author(s):

Andrew Stiff ◽

Alberto Rocci ◽

Craig C. Hofmeister ◽

Paola Omedè ◽

Susan Geyer ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Peripheral Blood ◽

Plasma Cells ◽

Immunoglobulin M ◽

Differentially Expressed ◽

Healthy Controls ◽

Circulating Mirnas ◽

Serum Samples ◽

Screening Analysis

Abstract Abstract 2921 Background: Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the aberrant expansion of clonal plasma cells (PCs) within the bone marrow. Malignant PCs produce intact or partial monoclonal immunoglobulin (M protein) and cause organ damage. More than 20,000 new cases of multiple myeloma (MM) are diagnosed every year in the US with approximately 10,700 deaths occurring. The pathogenesis of MM is still largely unclear, but several reports suggest that interaction of tumor cells with the bone marrow microenvironment and microRNAs (miRNAs) deregulation may play a role in the etiology and progression of MM. miRNAs are small non-coding RNAs capable of regulating protein expression by binding to mRNA, and have been implicated in the development of MM. First identified inside cells, miRNAs can also be detected in body fluids, including serum and plasma, and may be a valid biomarker. Few studies have investigated the agreement between circulating miRNAs and intracellular myeloma PC miRNAs at diagnosis. Methods: Using Nano-String nCounter technology we first performed a screening analysis on serum samples obtained from MM patients and healthy controls. We identified a candidate set of miRNAs differentially expressed in the serum of MM patients. The levels of these miRNA markers were validated by RT-PCR in both serum and bone marrow PCs from the same cohort. Agreement of the quantitative miRNA marker levels between sample types was evaluated using intraclass correlation coefficients (ICC) (both for normalized and log2 measures). Results: Thirty-nine MM patients (21 male, 18 female) with a median age of 72 years (range: 65 – 83) were included in the analysis. Most were ISS stage I or II (59% vs. 41% ISS stage III) and 39% were high risk according to FISH abnormalities – 21% of patients carried del17p, 24% t(4;14) and 5% t(14;16). Medians and ranges for lab markers were as follows: hemoglobin 10.0 g/dl (7.2 to 15.1), beta2-microglobulin 5.18 ug/ml (1.38 – 12.1), creatinine 0.94 mg/dl (0.65 – 2.49), CRP = 1.6 mg/dl (0.02 – 116.0). Nine age-matched healthy controls were also used for the analysis. After the screening analysis, the following miRNAs were differentially expressed between healthy subjects and MM patients in serum samples: miR-92a, miR-451, miR-19b, miR-21, miR-16, miR-25, miR-30a, and miR-126. There was no significant agreement or correlation between serum and myeloma cell samples using either untransformed as well as log2measures (all p>0.40) (Table 1). Conclusion: Our preliminary results suggest a difference between circulating miRNAs in myeloma patients from controls. This indicates that future studies are needed to better define the role of miRNAs in the peripheral blood as a prognostic and even diagnostic biomarker in myeloma. From our preliminary data it also appears that circulating miRNAs are not simply secreted into the peripheral blood by myeloma PCs as it seems that circulating miRNAs do not reflect those of myeloma PCs. Differential expression could be determined by other cells that can release and or modify their miRNA expression in response to MM. Ongoing studies are examining the origin and function of miRNAs in the peripheral blood. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2019-130697 ◽

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.

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Tumor Circulating Plasma Cells Detected By Flow Cytometric Single Platform Method Correlate with Clinical Response to Therapy and Unfavorable Patients' Characteristics

Blood ◽

10.1182/blood-2019-122657 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4357-4357

Author(s):

Vittorio Emanuele Muccio ◽

Milena Gilestro ◽

Elona Saraci ◽

Andrea Capra ◽

Alessandro Costa ◽

...

Keyword(s):

Multiple Myeloma ◽

Clinical Response ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Plasma Cells ◽

Response To Therapy ◽

Advisory Committees ◽

Flow Cytometric ◽

Baseline Characteristics

Background: In multiple myeloma (MM), different clinical parameters and molecular prognostic factors can predict disease course and response to therapy. The classification of myeloma patients includes laboratory parameters associated with higher tumor activity, resistance to therapy and proliferative competence. Tumor circulating plasma cells (TCPC) in MM patients showed a strong correlation with a more aggressive disease. Aim: For the first time, we quantified the amounts of TCPC with single platform flow cytometric method and evaluated their relationship with patients' baseline characteristics and response to therapy before maintenance. Methods: Whole peripheral blood samples from 413 newly diagnosed MM patients ≤65 years enrolled in the UNITO-MM-01/FORTE trial were collected. Patients were randomized [1:1:1; stratification: International Staging System (ISS) and age] to ARM A: carfilzomib-cyclophosphamide-dexamethasone (KCyd) followed by melphalan 200 mg/m2 and autologous stem-cell transplantation (MEL200-ASCT) and consolidation with 4 KCyd; ARM B: carfilzomib-lenalidomide-dexamethasone (KRd) followed by MEL200-ASCT and 4 KRd; ARM C: 12 KRd cycles. Enrollment was completed in March 2017; data cut-off was November 30, 2018. For the single platform tube, the antibody combination CD38PC7/CD138PC5.5/ CD45KO/CD56PE/CD19PB was mixed with 100µL of EDTA peripheral blood, dispensed with reverse pipetting, and incubated for 15 min, added with 500µL of lysing solution and, after 15 min, 100µL of flow count fluorospheres were dispensed with reverse pipetting and cells acquired with Navios flow cytometer. Intracytoplasmic tube was set up to confirm the clonality of CPC. Results: Circulating plasma cells (CPC) were quantified in 413 samples, with median values of 0.03% (range: 0-51%) and 2.37/mm3 (range: 0-6272/mm3). White blood cells were 5710/mm3 (range: 1752-26102/mm3); total events acquired 1285000 (range: 40000-2000000); median CPC events were 58 (range: 0-441000); cellular events acquired were 190000 (range: 4428-1300000). In 390 out of 413 samples (94.4%), CPC were detected; 272 samples (66%) showed TCPC with a median of 1.24/mm3 (range 0.06- 6272/mm3). Patients were sorted according to different baseline characteristics and the medians of absolute TCPC were compared. The most statistically significant differences (p<0.001) were: haemoglobin (Hb) <10 (12.9/mm3) vs. ≥10 (0.81/mm3); ISS I (0.30/mm3) vs. ISS II (2.85/mm3) vs. ISS III (5.14/mm3); R-ISS I (0.25/mm3) vs. II (2.76/mm3) vs. III (7.45/mm3); albumin <3.5g/dL (2.76/mm3) vs. ≥3.5g/dL (1.05/mm3); β2-microglobulin <3.5mg/dL (0.67/mm3) vs. 3.5mg/dL-5.5mg/dL (3.88/mm3) vs. >5.5mg/dL (16.47/mm3); lactate dehydrogenase (LDH) ≤upper limit of normal (ULN, 1.14/mm3) vs. >ULN (7.36/mm3); plasma cells (PC) in biopsy <60% (0.60/mm3) vs. ≥60%(2.76/mm3); with (3.18/mm3) vs. without amp1q (1.18/mm3); Morgan risk standard (1.21/mm3) vs. high (3.00/mm3). Finally, we compared the absolute number of TCPC and the quality of response at the end of consolidation therapy. Higher values of TCPC were related to worst response: <partial response (PR, 4.23/mm3) vs. ≥PR (1.23/mm3); <very good PR (VGPR, 2.91/mm3) vs. ≥VGPR (1.20/mm3); <complete response (CR, 1.95/mm3) vs. ≥CR (1.09/mm3); <stringent CR (sCR, 1.71/mm3) vs. ≥sCR (1.00/mm3), p<0.05. Conclusions: Single-platform flow cytometry is a simple method to quantify TCPC, present in almost all peripheral blood from MM patients; a high number is related to poor clinical response to therapy and helps in identifying high-risk patients. Moreover, it allows the discrimination between normal and pathological plasma cell population in peripheral blood. However, a longer follow up is needed to evaluate how TCPC can affect survival in patients with MM. Disclosures Musto: Amgen: Honoraria; Celgene: Honoraria. Gay:Janssen: 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; AbbVie: Membership on an entity's Board of Directors or advisory committees; Amgen: 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding. Omedé:Janssen: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: This presentation includes discussion of off-label use of a drug or drugs for the treatment of multiple myeloma.

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Enumeration of Plasmacytoid Dendritic Cells in Peripheral Blood and Bone Marrow by Flow-Cytometric Analysis

Methods in Molecular Biology - Immunophenotyping ◽

10.1007/978-1-4939-9650-6_11 ◽

2019 ◽

pp. 193-201

Author(s):

Abdullah Alsuwaidan ◽

Franklin Fuda ◽

Weina Chen ◽

Mingyi Chen

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Plasmacytoid Dendritic Cells ◽

Flow Cytometric

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Multiple myeloma: circulating lymphocytes that express plasma cell antigens

Blood ◽

10.1182/blood.v64.2.352.bloodjournal642352 ◽

1984 ◽

Vol 64 (2) ◽

pp. 352-356

Author(s):

GJ Ruiz-Arguelles ◽

JA Katzmann ◽

PR Greipp ◽

NJ Gonchoroff ◽

JP Garton ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Peripheral Blood Lymphocytes ◽

Tumor Burden ◽

B Cell Differentiation ◽

Blood Lymphocytes ◽

Bone Marrow Plasma

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

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Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Blood ◽

10.1182/blood-2019-123277 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 865-865 ◽

Cited By ~ 1

Author(s):

Reona Sakemura ◽

Michelle J. Cox ◽

Michael J. Hansen ◽

Mehrdad Hefazi ◽

Claudia Manriquez Roman ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Therapy ◽

Cell Line ◽

Research Funding ◽

Plasma Cells ◽

Flow Cytometric Analysis ◽

Data Safety Monitoring Board ◽

Nsg Mice ◽

Dual Targeting ◽

The Impact

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding; Morphosys: Research Funding.

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