5 Multiple myeloma flow cytometry panel validated for clinical monitoring of patients

BackgroundMultiple myeloma (MM) is an incurable plasma cell malignancy with significant heterogeneity in clinical presentation. Plasma cells are antibody-producing cells of lymphoid origin that are resident in secondary lymphoid organs and in the bone marrow (BM). The detection of circulating malignant plasma cells using flow cytometry has also been described in patients with MM. Enumerating and phenotyping malignant plasma cells in the BM and peripheral blood (PB) may be of value when evaluating the presence of MM antigens targeted by therapies before and during treatment and at relapse. To this end, a flow cytometric panel was developed to enumerate and characterize malignant plasma cells and additional immune subsets.MethodsPB and BM aspirates (BMA) were obtained from healthy donors and MM donors who consented to research testing. MM cell lines were also used to spike into donor samples to detect specific antigens (collected in Cyto-Chex® blood collection tubes). Samples were then transferred to TruCount tubes to enumerate immune populations. Fluorescently labeled antibodies directed against CD38, CD138, CD56, CD45, BCMA were evaluated to assess parameters such as time and temperature stability of the reportable immune populations by monitoring the frequencies of the populations. In addition, the limit of quantitation, intra- and inter-assay precision were determined.ResultsThe MM Counting Panel was optimized to leverage antigen expression and fluorophore combinations. A gating strategy enabled enumeration of MM cells based on antigens that can be further subdivided based on BCMA expression. Further testing showed that the precision in frequencies and absolute counts of key reportable populations was deemed acceptable (%CV of <30%). The precision was within the acceptance criteria of%CV <30% for populations with =100 cells. Stability testing revealed that samples were more stable at ambient temperature relative to 4oC, with stability being maintained for 48 h post-collection, where at least 85% of reportable immune readouts were stable (%change <30% relative to baseline), for BMA and PB from various donors (healthy and MM).The panel was ultimately deployed for use with clinical samples from MM clinical trials. Clinical data generated from the MM Counting Panel allowed the identification of malignant plasma cell populations in BMA of patients from trial assessing a BCMAxCD3 bi-specific antibody (NCT03761108).ConclusionsA flow cytometric assay to enumerate and identify normal and malignant plasma cells in MM patients was successfully developed. The approach used can be applied to develop assays for other indications in which patients are treated with therapies.

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Flow Cytometric Disease Monitoring in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation: A Retrospective Study.

Blood ◽

10.1182/blood.v108.11.5014.5014 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5014-5014

Author(s):

Hong Liu ◽

Constance M. Yuan ◽

Raul C. Braylan ◽

Myron N. Chang ◽

John R. Wingard ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Plasma Cells ◽

Flow Cytometric ◽

Before And After

Abstract The persistence of abnormal neoplastic plasma cells (APC) detectable in the bone marrow by flow cytometry at more than 3 months after autografting for multiple myeloma (MM) has been reported to predict early disease progression. In this study, we retrospectively reviewed the flow cytometric data from bone marrow aspirates of MM patients before and after autologous stem cell transplantation (ASCT). Light scatter properties and CD38 expression were used to identify plasma cells, and CD19/CD45/CD56 further distinguished normal plasma cells (NPC) from APC. Conventional response criteria (Blade criteria) and survival data were also collected. Forty-seven (47) patients treated with the same conditioning regimen were screened. Median follow up from ASCT was 19 months. After ASCT, 66% (31/47) patients achieved complete remission (CR)/very good partial remission (VGPR), as compared to only 36% (17/47) prior to ASCT. In 39 patients with data before and after ASCT, all 39 (100%) had a detectable abnormal plasma cell population identified phenotypically by flow cytometry prior to ASCT. Of these patient, 18/39 (46%) had greater than 30 APC and these patients had significantly shorter PFS independent of other covariates (1-sided P=0.036, 2 sided P=0.072, logrank). Twenty-six out of 39 patients (67%) also had detectable NPC. Following ASCT, the number of patients with detectable NPC increased to 35/39 (89%), while 3/39 (8%) had no detectable NPC and 1/39 (3%) had neither NPC or APC. The proportion of APC decreased significantly after transplant (81% prior to transplant vs. 59% post-transplant, P=0.008, 2 tailed t-test). Patients with a APC to NPC ratio < 1 post transplant has higher PFS rate at 2 year (54%) when compared to patients with higher APC/NPC ratio (29% PFS at 2 year), however, the difference is not statistically significant. In addition to the presence of APC, the ratio of APC to NPC, age, beta-2 microglobulin levels, and the presence of normal immunoglobulin levels were analyzed. Patients who achieved CR/VGPR after transplant had significantly longer PFS (23 months vs. 11 months, P=0.03). All other covariates were not found to be significant. Because only 10 deaths were observed, covariate analysis for OS was not feasible. In conclusion, the recovery of NPC after ASCT is seen in a substantial propotion of patients with a trend towards better PFS in patients with low APC/NPC ratio. On the other hand, the presence of a significant population of APC (> 30) prior to transplant appears to correlate with poorer PFS in MM patients.

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

Blood ◽

10.1182/blood.v108.11.5036.5036 ◽

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.

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Daratumumab Interferes with Flow Cytometric Evaluation of Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.5630.5630 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5630-5630 ◽

Cited By ~ 2

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.

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The importance of flowcytometry study with the first aspirate taken during bone marrow aspiration in the diagnosis of multiple myeloma and follow-up of minimal residual disease

Medical Science and Discovery ◽

10.36472/msd.v8i4.520 ◽

2021 ◽

Vol 8 (4) ◽

pp. 219-224

Author(s):

Ali Eser

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cells ◽

Residual Disease ◽

Bone Marrow Aspiration ◽

Morphological Examination ◽

Flow Cytometric ◽

Flow Cytometric Study

Objective: Flow cytometry (FC) is a diagnostic method supporting traditional morphological examination in disease follow-up and the diagnosis of Multiple myeloma (MM). Normal and atypical plasma cells (PCs) can be told apart from each other by means of FC method. The plasma cell rate is the highest in the blood obtained in the first aspirate during bone marrow aspiration in MM. Material and methods: A total of 60 patients that have been diagnosed with MM between 2018 and 2020, including 30 patients whom flow cytometry was studied with the first aspirate during bone marrow aspiration, and 30 patients whom FC was studied with the second aspirate were included in our study. The characteristics of the patients were analyzed retrospectively from their files. Results: The median ratio of plasma cells (PCs) detected by FC and bone marrow biopsy was 17,5% and 44%, respectively. While this rate was median 37,5% in patients that flow cytometric study was performed with the first aspirate, the rate was found to be median 7% in patients that FC was performed with the second sample. The PCs rates were statistically significantly higher with the flow cytometric study with the first aspirate than the second one (p=0.000). Conclusion: Flow cytometric study with the first aspirate during bone marrow aspiration in patients with MM is diagnostically important.

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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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High Frequency of Aberrant Phenotype in Multiple Myeloma Patients Detected by Multiparametric Flow Cytometry.

Blood ◽

10.1182/blood.v104.11.4862.4862 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4862-4862

Author(s):

Luiz Arthur Calheiros ◽

Eliza Y.S. Kimura ◽

Manuella S.S. Almeida ◽

Maria de Lourdes L.F. Chauffaille ◽

Jandey G. Bigonha ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Plasma Cell ◽

High Frequency ◽

Plasma Cells ◽

Residual Disease ◽

Lymphoproliferative Disease ◽

Multiparametric Flow Cytometry ◽

Normal Plasma ◽

Aberrant Phenotype

Abstract Multiple Myeloma (MM) is a B cell lymphoproliferative disease with clonal plasma cell accumulation in bone marrow. Multiparametric flow cytometry (MFC) is an usefull tool to distinguish MM cells from normal plasma cells. Normal plasma cells are characterized by the expression of CD19+, CD45++, CD38++, CD138++, cytoplasmic immunoglobulin light chains (κ and λ) and CD56- while most MM plasma cells lose CD19, CD45 and gain CD56. In addition, many other antigens may be expressed by myeloma cells such as myeloid or lymphoid lineage associated antigens and these abnormal antigen expression is known as aberrant phenotype (AP). We studied 29 MM patients at diagnosis, in attempt to evaluate AP, it’s frequency and relation to prognostic parameters. The following monoclonal antibodies were used: CD45, CD38, CD138, CD56, CD19, CD20, CD22, CD10, CD13, CD14, CD33, CD117, CD28 and CD40, conjugated to FITC, PE, PerCP and APC) and acquisition / analysis were done through flow cytometer (FACS calibur, BD, San Jose) using CELL QUEST software (BD). Plasma cells were identified by the expression of CD38, CD138 and CD45 and the monoclonality confirmed by immunoglobulin light chain restriction. Our results showed presence of at least 2 AP in all cases : 2 AP (7 patients), 3 AP (12 patients), 4 AP( 5 cases), 5 AP (4 cases) and 8 AP in one case. The most frequent APs were CD45−, CD56+, CD117+, CD13+, CD33+, and were observed in 88% of patients. The most frequent AP association was CD56+/CD45− (40%), followed by myeloid antigen associated phenotypes (CD117, CD33, CD13). The lymphoid antigens expression was more observed in patients with large number of AP (>4 AP). CD56- patients presented serum β2-microglobulin and ionic calcium labeling levels higher than CD56+ patients (p=0,02) showing the usefulness of this antigen as prognostic marker. Morphological analysis showed that the majority (55%) of plasmablastic cases expressed >2 myeloid antigens against 18% of mature plasma cell morphology cases. These results allow us to conclude that MM express high frequency of AP, highlighting the importance of CD56 as a prognostic factor. MFC may be useful to the immunological detection of minimal residual disease in a great majority of MM patients and we suggest the panel CD45, CD56, CD117, CD33 and CD13 for this purpose, in addition to CD38 and CD138.

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CD 138 Immunostaining of Bone Marrow Trephine Specimens Is the Most Sensitive Method for Quantifying Marrow Involvement in Patients with Plasma Cell Dyscrasias.

Blood ◽

10.1182/blood.v106.11.5071.5071 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5071-5071

Author(s):

Ashley P. Ng ◽

Andrew Wei ◽

Dinesh Bhurani ◽

Peter Chappell ◽

Frank Feleppa ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cell ◽

Plasma Cells ◽

Flow Cytometric Analysis ◽

T Test ◽

P Value ◽

Flow Cytometric ◽

Paired T Test ◽

Sensitive Method

Abstract AIM In this study plasma cell quantitation comparing four modalities comprising morphology of the aspirate, flow cytometry, HEtrephine section examination and bone marrow immunohistology was undertaken to determine the most sensitive technique. METHODS 70 patients with a plasma-cell dyscrasia were retrospectively analysed. Plasma-cell quantitation was performed on Romanowsky-stained aspirate slides by a 200-cell differential. Flow cytometric quantitation of plasma-cell burden was performed by identifying CD38/138 co-expressing cells. Trephine specimens were analysed after HEand CD138 staining. Statistical analysis was performed using a two-tailed paired t-test. RESULTS See Table 1. DISCUSSION Significant discrepancy was noted between the four methods for determining plasma-cell infiltration. CD138 immunohistochemical staining of paraffin embedded trephine specimens was the most sensitive modality. Flow-cytometric analysis underestimated the degree of marrow infiltration. Morphology of the aspirate sample may also underestimate plasma-cell burden as sampling may be affected by patchy marrow infiltration as well hypoplastic, fibrotic marrows or clotted specimens. Quantification of plasma cells based on HEstained trephine specimens is less sensitive, and is dependent on the technical quality of the specimen and observer experience. Immunostaining has the advantages of improved plasma-cell identification compared to HEsections and avoids the problems inherent in the analysis of aspirate samples. In conclusion, CD138 immunostaining is the most sensitive method for quantifying plasma-cell burden in the bone marrow. Two tailed Paired t-test analysis of Quantitation of Plasma Cell burden by various modalities Flow Cytometry Aspirate Hematoxylin & Eosin Immunostaining Mean % of plasma cells 5.6 18.9 23.6 29.7 Paired t-test (two tailed) Mean Difference compared to CD138 immunostaining −24.12 −11 −6.14 - 95% C.I. −30.5 to −17.8 −15.8 to −6.1 −8.34 to −3.9 - p-value p<0.0001 p<0.0001 p<0.0001 -

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The Location of Multiple Myeloma Adhesion Variants in Diverse Niches within the Bone Marrow Affects Plasma Cells Enumeration.

Blood ◽

10.1182/blood.v108.11.5063.5063 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5063-5063

Author(s):

Liat Nadav ◽

Ben-Zion Katz ◽

Shoshana Baron ◽

Lydia Lydia ◽

Aaron Polliack ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Gene Expression Profile ◽

Plasma Cell ◽

Expression Profile ◽

Plasma Cells ◽

Response To Therapy ◽

Morphological Evaluation

Abstract Background - The diagnosis of multiple myeloma (MM) is based on clinical and laboratory criteria combined with bone marrow (BM) plasmocytosis, estimated by inspection of bone marrow aspirates. Recent advances in flow-cytometry (FCM) have provided an additional tool for the diagnosis of MM and for monitoring response to therapy. However, significant discrepancy has been reported regarding the enumeration of plasma cells in marrow samples of MM patients using these two methods. Aims - In this study we compared the bone marrow plasmocytosis by microscopic examination of BM aspirates, to the flow cytometry results in samples obtained form MM patients. We tested whether the noted discrepancy between these two methods applies only to MM, or represents a trend in other hematopoietic malignancies as well. We defined this discrepancy and explained it. Methods - The number of plasma cells or blasts from BM aspirates of 41 MM or seven acute myeloid leukemia (AML) patients respectively were analyzed simultaneously by morphological evaluation and by FCM. Each sample was assessed independently by two qualified laboratory specialists and/or hemato-pathologist. In MM we found plasma cell fractions that were characterized by FCM and gene expression profile. Results - In MM it was evident that FCM under-estimated the number of BM plasma cells samples by an average of 60%, compared with conventional morphological evaluation. On the other hand in AML there was a good correlation between the morphological and FCM assessments of the blast cell population, indicating that the discrepancy observed in the MM BM samples may be related to unique characteristics of the malignant plasma cells. Since flow cytometry is performed on the bone marrow fluid which is depleted of fat tissue-adhesive plasma cells, we disrupted spicules from MM BM samples (by repeated passages through 21g needle) and found a 40% increase in plasma cell compared with the fluid of the same BM samples. In order to determine the FCM profile of the cells in these two fractions, we isolated BM derived spicules from aspirates of MM patients and treated them with extracellular matrix (ECM) degrading enzymes followed by mechanical shearing. This combination released the highly adhesive plasma cells from the spicules. The released myeloma cells displayed a different FCM profile and in particular had a higher level of CD138 expression. Gene expression profile, which was performed on similar adhesion variants of cultured MM cells, demonstrated distinct oncogenic and transcriptional programs. Summary - We have shown a major discrepancy between the percentage of MM cells obtained by routine BM morphology and flow cytometry counts. It is possible that this discrepancy is partially attributable to the two distinct microenvironmental components occupied by MM cells in the BM sample - the lipid spicules, and the fluid phase. MM cells located in different niches of the BM also differ in their FCM and gene expression profile. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells. These sub-populations may play distinct roles in the biological and clinical manifestations of the disease and differ in their response to anti-myeloma therapy.

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

Blood ◽

10.1182/blood.v122.21.4647.4647 ◽

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.

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

Blood ◽

10.1182/blood.v124.21.1110.1110 ◽

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.

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