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

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 -

Download Full-text

Quantitation of Plasma Cells in Bone Marrow Aspirates by Flow Cytometric Analysis Compared With Morphologic Assessment

Archives of Pathology & Laboratory Medicine ◽

10.5858/2007-131-951-qopcib ◽

2007 ◽

Vol 131 (6) ◽

pp. 951-955

Author(s):

Kristi J. Smock ◽

Sherrie L. Perkins ◽

David W. Bahler

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cell ◽

Gold Standard ◽

Plasma Cells ◽

Flow Cytometric Analysis ◽

Flow Cytometric ◽

Bone Marrow Plasma ◽

Plasma Cell Dyscrasias ◽

Cellular Processing

Abstract Context.—Accurate quantitation of bone marrow plasma cells is an important component in the diagnosis and posttreatment assessment of plasma cell dyscrasias. Although flow cytometry is sometimes used for this purpose and can rapidly evaluate many cells, the accuracy of flow-based plasma cell quantitation compared with morphologic assessment (currently the gold standard) is uncertain as direct comparison studies have not been previously reported. Objective.—To determine how percentages of plasma cells in diagnostic aspirate smears quantitated by morphologic assessment relate to percentages of plasma cells quantified by flow cytometry. Design.—Thirty bone marrow cases with 10% or more plasma cells and leukemia/lymphoma flow cytometry immunophenotyping studies were identified from our hematopathology database. The Wright-stained aspirate smears, marrow biopsy sections, and flow cytometry histograms were reviewed. Results.—Morphologically determined plasma cell percentages from the diagnostic aspirate smears were consistently higher than those determined by flow cytometry. Much of this difference appeared to be related to differences in sample quality. However, the cellular processing involved in performing flow cytometry also appeared to reduce plasma cell percentages in many cases. Conclusions.—This study helps define the limitations of flow cytometry for quantitating plasma cell loads in marrow aspirate specimens that may significantly affect the diagnosis or assessment of treatment response.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Expression of VEGF Receptors on Plasma Cells: Evidence of Heterogeneity.

Blood ◽

10.1182/blood.v104.11.3361.3361 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3361-3361

Author(s):

Teresa K. Kimlinger ◽

Thomas E. Witzig ◽

S. Vincent Rajkumar

Keyword(s):

Bone Marrow ◽

Plasma Cell ◽

Plasma Cells ◽

Receptor Expression ◽

Vegf Receptors ◽

Flow Cytometric Assay ◽

Blocking Antibody ◽

R And D ◽

Flow Cytometric ◽

Indirect Assay

Abstract Background: In previous studies quantitating VEGF receptors we have found no significant differences in expression between plasma cells from normal controls, multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), or smoldering myeloma (SMM). (Kumar S, Blood, May 2004; 10.1182/blood-2003-11-3811 ePub). These studies were done using immunohistochemistry or Western Blotting (on CD138+ plasma cells) and may have been limited by low levels of receptor expression and by heterogeneity of expression. We measured expression of VEGF receptors (VEGFR1, VEGFR2 and VEGFR3) on the surface of plasma cells and in plasma cell subsets using direct and indirect flow cytometric assays to determine if significant differences in VEGF receptor expression existed between MGUS, SMM and MM. Methods: In the indirect flow cytometric assay, 32 bone marrow samples (3 amyloid, 9 MGUS, 12 MM, 8 SMM) were ACK lysed and tested using the FLUOROKINE TM rhVEGF biotin kit (R and D Systems, Minneapolis, MN) according to manufacturer’s instructions. In brief, in one tube (tube 1) cells were incubated with VEGF biotin. In tube 2, VEGF biotin preincubated with a blocking antibody was added to the cells (specificity control), while in a third tube a non-specific biotinylated protein (negative control) was added. After incubation, FITC-avidin, CD38 APC and CD45 Percp was added to each tube. Gates were drawn around the cells of interest and fitc staining was evaluated for the % positive cells. The % of signal blocked was calculated by comparing the fitc intensity (channel number) of the blocked VEGF peak (tube 2) to FITC intensity of tube 1. This system does not determine the identity of the receptor, but indicates the presence of VEGF receptors. In the direct flow cytometric assay, bone marrow from 25 individuals (2 amyloid, 5 MGUS, 7 MM, 7 SMM, and 4 normals) were lysed and blocked with mouse Ig and stained with CD38/CD45. In individual tubes, PE labeled VEGF R1, R2, R3 antibodies (R and D Systems, Minneapolis, MN) or isotype control were added. Plasma cells were identified, divided according to CD45 expression, and analyzed for % and intensity of receptor staining. Results: In the indirect assay, plasma cells in all groups bound VEGF ( 96% positive) at high intensity. There was also no difference in VEGF binding between CD45+ and CD45- plasma cell fractions (93 and 98% respectively).The specificity of VEGF binding (to one of the VEGF specific receptors) was confirmed by a significant drop in peak channel numbers of FITC intensity in the presence of blocking antibody. Specific VEGF binding at a similar intensity was seen in monocytes (95%) and at a lower intensity in lymphocytes (66%) and granulocytes (28%). Staining for VEGFR1, 2, and 3 in plasma cells using the direct assay revealed that except for 2 patients (1 amyloid and 1 SMM) none had >20% cells staining for any of the 3 receptors. The same results were seen in the CD45− fraction as well. In contrast, the CD45+ plasma cell fraction was highly positive for all 3 of the receptors in nearly all cases, with no significant differences between MGUS, SMM, amyloid, or MM. Conclusions: Plasma cells in MM and related disorders have specific VEGF receptors on the cell surface. The expression of VEGFR1, 2, and 3 seems to be primarily restricted to the CD 45+ subset of plasma cells. The finding of specific VEGF binding in CD45− plasma cells seen in the indirect assay may reflect the higher sensitivity of this assay due to the inbuilt amplification process or the presence of additional VEGF receptors such as neuropilin 1.

Download Full-text

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.

Download Full-text

Evaluation of intensive chemotherapy in AL amyloidosis: usefulness of flow cytometric analysis of plasma cells in bone marrow

Amyloid ◽

10.3109/13506120309041742 ◽

2003 ◽

Vol 10 (4) ◽

pp. 250-256 ◽

Cited By ~ 5

Author(s):

Masayuki Matsuda ◽

Takahisa Gono ◽

Yasuhiro Shimojima ◽

Kazuo Sakashita ◽

Kenichi Koike ◽

...

Keyword(s):

Bone Marrow ◽

Plasma Cells ◽

Flow Cytometric Analysis ◽

Al Amyloidosis ◽

Intensive Chemotherapy ◽

Flow Cytometric

Download Full-text

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0005 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A5-A5

Author(s):

Bevan Gang ◽

Vicky Sgouroudis ◽

Virginia Litwin ◽

Anita Boyapati

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Plasma Cell ◽

Plasma Cells ◽

Temperature Stability ◽

Clinical Samples ◽

Blood Collection ◽

Significant Heterogeneity ◽

Flow Cytometric ◽

Limit Of Quantitation

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.

Download Full-text

Flow cytometry of bone marrow aspirates from neuroblastoma patients is a highly sensitive technique for quantification of low-level neuroblastoma

F1000Research ◽

10.12688/f1000research.53133.1 ◽

2021 ◽

Vol 10 ◽

pp. 947

Author(s):

Neha Jain ◽

Shaista Sattar ◽

Sarah Inglott ◽

Susan Burchill ◽

Jonathan Fisher ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Bone Marrow Involvement ◽

Flow Cytometric Analysis ◽

Standard Of Care ◽

Tumour Cells ◽

Street Hospital ◽

Low Level ◽

Bone Marrow Trephine ◽

Flow Cytometric

Background: Bone marrow involvement is an important aspect of determining staging of disease and treatment for childhood neuroblastoma. Current standard of care relies on microscopic examination of bone marrow trephine biopsies and aspirates respectively, to define involvement. Flow cytometric analysis of disaggregated tumour cells, when using a panel of neuroblastoma specific markers, allows for potentially less subjective determination of the presence of tumour cells. Methods: A retrospective review of sequential bone marrow trephine biopsies and aspirates, performed at Great Ormond Street Hospital, London, between the years 2015 and 2018, was performed to assess whether the addition of flow cytometric analysis to these standard of care methods provided concordant or additional information. Results: There was good concurrence between all three methods for negative results 216/302 (72%). Positive results had a concordance of 52/86 (61%), comparing samples positive by flow cytometry and positive by either or both cytology and histology. Of the remaining samples, 20/86 (23%) were positive by either or both cytology and histology, but negative by flow cytometry. Whereas 14/86 (16%) of samples were positive only by flow cytometry. Conclusions: Our review highlights the ongoing importance of expert cytological and histological assessment of bone marrow results. Flow cytometry is an objective, quantitative method to assess the level of bone marrow disease in aspirates. In this study, flow cytometry identified low-level residual disease that was not detected by cytology or histology. The clinical significance of this low-level disease warrants further investigation.

Download Full-text

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.

Download Full-text

Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-150484 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4339-4339

Author(s):

Valentina Giudice ◽

Idalucia Ferrara ◽

Marisa Gorrese ◽

Francesca D'Alto ◽

Roberto Guariglia ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myeloid Leukemia ◽

Plasma Cells ◽

Immune Cell ◽

Nk Cell ◽

Case Series ◽

Elderly Aml ◽

Paired T Test ◽

Acute Myeloid

Abstract Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients. A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines. In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment. Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. Disclosures No relevant conflicts of interest to declare.

Download Full-text