Immunophenotype Of Plasma Cells Research Articles

A-173 Evaluation of Artificial Intelligence (AI)-assisted Flow Cytometry Analysis for Plasma Cell Neoplasms

Abstract Background Flow cytometry plays a critical role in diagnosing hematologic disorders, yet manual analysis is time-consuming and prone to variability. Significant advancements in artificial intelligence (AI) within healthcare have occurred in recent years. Previously, we developed and evaluated an AI-assisted automated system for diagnosing acute leukemia. This study aims to assess the effectiveness of the AI system in diagnosing plasma cell neoplasms. Methods A total of 76 bone marrow samples were analyzed using a panel of antibodies (CD38, CD56, CD138, Kappa, and Lambda) to determine plasma cell percentages and immunophenotype. All samples were used for initial diagnosis of plasma cell neoplasm or therapeutic monitoring. Manual analysis using Kaluza™ software served as the gold standard. The same flow cytometry data was processed by the AI software (DeepFlow™) for comparison. Pearson correlation coefficients were used to compare AI and manual analysis for diagnosis and plasma cell count. Results Among the 76 cases, manual analysis identified 57 positive cases for monoclonal plasma cells (36 cases of CD38+/CD138+/Kappa+ and 21 cases of CD38+/CD138+/Lambda+), with 89% showing aberrant CD56 expression. AI identified 49 monoclonal cases, missing 8 cases with low plasma cell levels (<0.07%) or brighter fluorescence. AI accurately matched 97% of immunophenotypes with manual analysis. Pearson correlation coefficients demonstrated excellent correlation (r=1.0) between manual and AI plasma cell percentages. Furthermore, AI has also enhanced laboratory efficiency, reducing analysis time per case from 10 minutes with manual methods to just 3 minutes with AI analysis. Conclusions This study shows promise for AI-assisted flow software in diagnosing plasma cell neoplasms. AI has enhanced operational efficiency by reducing the manual processing and analysis time. Further training is needed to improve the identification of variations in fluorescence intensity and low plasma cell levels.

Characteristics and outcomes of plasmablastic lymphoma in a predominantly Hispanic population.

e19079 Background: Plasmablastic lymphoma (PBL) is a rare subtype of diffuse large B-cell lymphoma and is frequently associated with immunosuppression. PBL displays immunoblastic morphology and a plasma cell immunophenotype. Data regarding patient characteristics, outcomes, and optimal treatment for this disease is limited. The reported prognosis of patients with PBL has been historically poor, with median overall survival ranging from 6 to 18 months. A recent publication of the SEER/NCDB database found a significantly improved OS in the Hispanic population (HR 0.7, p=0.014). This study investigates demographic, treatment, and survival outcomes for a primarily Hispanic PBL population. Methods: We identified 42 cases of pathologically confirmed PBL at two high volume tertiary care centers in Southern California between March 2010 and June 2023. Chart review was used to collect data on clinical presentation, treatment, and outcomes. Results: Median age of patients at diagnosis was 46 years (range 17-91 years) and 90% were male. Hispanic patients represented 81% of the total cohort. Predisposing immunosuppression was identified in 60%, and 50% of the total cohort were HIV-positive. Pathologic analysis showed EBV positivity by EBER in situ hybridization in 67%. 76% of patients presented with advanced disease (Stage III/IV). The most common regimens were EPOCH (27%) and V-EPOCH (49%). Response rates and CR rates were 63% and 50% respectively. Median follow-up was 10.5 months. OS was 72% at 1-year and 60% at 2-years. Conclusions: Our cohort demonstrated a 1-year OS of 72% compared to the 56% 1-year OS for PBLs in the SEER/NCDB database. There was no statistically significant difference in survival between the Hispanic and Non-Hispanic cohorts in our study. However, this is likely due to the small sample size in the latter. Notably, our patient population had more advanced disease at presentation (76% Stage III/IV) than the SEER/NCDB cohort (60% Stage III/IV). This improved survival is likely multifactorial, but could represent ethnic differences in PBL biology, high EBV positivity, and the frequent utilization of higher intensity regimens (EPOCH/V-EPOCH = 76%). Further studies are needed to better characterize this disease in the Hispanic population. [Table: see text]

Immune profiles to predict bortezomib-based treatment response for multiple myeloma patients

BackgroundTo evaluate the distribution of bone marrow immune cell subsets and their correlation with treatment efficacy in patients with multiple myeloma (MM). MethodsWe analyzed the bone marrow lymphocyte subsets of 186 newly diagnosed MM patients at diagnosis and their correlation with clinical characteristics. In our study, eight-color flow cytometry, a method commonly used to detect plasma cell phenotypes, was used to analyze seven bone marrow immune cell groups by change gate-strategy. ResultsFirst, for all the 7 immune cell groups, the percentage of immature B cells was significantly lower in stage III patients than in stage I patients, while the trend was reversed in memory B cells in both the International Staging System(p = 0.004) and Revised International Staging System(p = 0.018). Second, the percentage of naïve B cells were significantly lower in patients with severe anemia, while the percentage of memory B cells had reversed trend. The percentage of immature B cells were lower in patients with Cr ≥ 2 mg/dL than in patients with Cr < 2 mg/dL. Then we followed the treatment efficacy of 152 patients who received four cycles of induction therapy (bortezomib + dexamethasone or bortezomib + lenalidomide + dexamethasone) and analyzed the relationship between bone marrow lymphocyte subsets at the initial stage and treatment response datasets. We found that both the percentage of B cells(p＜0.001) and immature B(p = 0.002) were increased in patients who achieved very good partial remission(VGPR) after four cycles of induction therapy. The ROC results indicated the combination of the multiple immune subgroups had predictive values (AUC = 0.802, p<0.001) in the treatment effect after four cycles of induction therapy. ConclusionsOverall, these results suggest that the analysis of lymphocyte subsets along with plasma cell immunophenotyping could be a potential index for determining the prognosis of MM patients.

Investigating the effect of immunomagnetic separation on the immunophenotype and viability of plasma cells in plasma cell disorders.

Plasma cell enrichment plays a pivotal role in the accurate prognosis and molecular characterization of multiple myeloma. The separation is commonly carried out by positive cell selection using CD138 monoclonal antibody conjugated to magnetic beads. Optimally, during the separation procedure, the cells should neither be damaged, nor should their phenotype be significantly altered, as these changes would falsify the results if the isolated cells were subsequently used. For this reason, we investigated the expression patterns of different surface markers by flow cytometry before and after magnetic isolation using bone marrow or peripheral blood samples from 12 patients with plasma cell disorders. The selected markers are not only used as backbone markers in routine diagnostics (CD19, CD38, CD45, CD117, and CD138), but they also play an important role in cell adhesion and connection with microenvironment (CD44, CD49d, CD56, and CD81) or possibly drug resistance (CD69, CD86, and CD184), making them promising targets for myeloma research. Moreover, we examined the effects of separation on cell viability in 8 cases. The intensities of 8 out of the 12 investigated markers were slightly influenced, while CD138, CD38, CD56, and CD184 were changed significantly, however the immunophenotype of the cells was not changed. Positive markers remained positive and negative ones remained negative after the separation procedure. In addition, the number of apoptotic plasma cells was significantly reduced during separation, facilitating further examination of the cells. Our results showed that magnetic isolation can be considered as a reliable option but the immunophenotype of plasma cells should be validated after the separation if the intensities of the markers are important for further experiments.

An alternative processing approach to increase CD138 intensity in flow cytometric analysis of plasma cells.

Surface median immunofluorescence intensity (MFI) of plasma cells antigens, particularly CD138, by flow cytometry underestimates plasma cell populations when compared with that estimated by morphological assessment on Wright's-stained slides. CD138 MFI using traditional sample preparation methods for flow cytometric analysis is often dim and difficult to interpret due to multiple factors. This becomes critical when diagnosing and accurately classifying plasma cell dyscrasias. In this study, we analyzed 280 flow cytometric results collected from 2016 to 2022 for CD38 and CD138 MFI on bone marrow aspirates performed by two different methods of sample processing-traditional method of lyse-wash and the alternative method of lyse-no-wash. Visual examination of histograms showed a clear advantage to CD138 expression intensity with the no-wash method. Although no significant difference was observed in CD38 MFI between the two techniques (p = 0.3), considerable improvement was observed in CD138 MFI with the lyse-no-wash technique of sample processing compared with the conventional method (p = 0.003). We concluded that the method of lyse-no-wash is superior to traditional methods especially when it comes to handling bone marrow aspirate samples for plasma cell immunophenotyping. This alternate technique increases the sensitivity of flow cytometry to detect plasma cells resulting in bright and crisp signal intensity for surface CD138. This technique may be particularly advantageous when analyzing low tumor burden such as minimal residual disease.

Immunophenotypic characterization of normal and abnormal plasma cells in bone marrow of newly diagnosed multiple myeloma patients

Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of multiple myeloma (MM) and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. No single phenotypic marker is sufficient to distinguish NPC from APC. 43 newly diagnosed cases of MM and 13 controls were included in the study. Bone marrow (BM) samples from the 2nd pass were processed on the same day with antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, cytoKappa, and cytoLambda in a 4-color experiment with CD38 and CD138 as gating antibodies. Mean APC% in cases was 96.5%. The expected Immunophenotype (IP) of APC which is CD19-/56+/45-/81-/117+/200+ was found in only 13/43 MM cases. In 30/43 cases, APC revealed deviation from expected IP either for single or a combination of markers. Sensitivity for APC detection was highest for CD19 (95.2%) followed by CD56 (90.4%) and CD81 (83.7%). Specificity was highest for CD19 (100%), CD56 (100%), and CD81 (100%) followed by CD117 (92.3%). Combination of markers with maximum sensitivity to detect APC (97.6%) was CD81- or CD19- and CD200+ or CD56+ (two markers); and for NPC (92.3%) was CD81+ and CD19+ and CD56- (three markers). Plasma cell IP can be highly variable with multiple minor subpopulations in both cases and normal controls. CD 19 and CD56 are highly informative markers for a 4-color experiment. Assessment of multiple markers in an 8-10 color experiment is more informative but the lack of advanced flow cytometers should not limit the use of FC in a 4-color approach. Our results emphasize that even basic equipment with limited fluorochrome can provide meaningful information if used appropriately.

Immunophenotypic Profile of Multiple Myeloma: A Tertiary Care Centre Experience.

Background Immunophenotyping and enumeration of plasma cells (PCs) by flow cytometry are deemed to be prognostically significant. However, PCs enumeration by flow cytometry is challenging owing to discrepancy with morphology and PCs loss during sample processing. Enumeration and differentiation of abnormal plasma cells (APCs) and normal plasma cells (NPCs) is difficult because abnormal antigen expression can be seen in subsets of NPCs. This is particularly true when a limited panel of antibodies are relied upon. Aims and purpose To study the immunophenotypic profile of newly diagnosed multiple myeloma (MM) cases by flow cytometry and evaluate the sensitivities and specificities of individual antigens and combinations. Methods We studied immunophenotype of PCs in newly diagnosed MM cases ( n = 48) and control cases ( n = 10) by a 6-color, 3-tube flow cytometry panel. The sensitivities and specificities of antigens in MM were evaluated and compared with control cases. Results Majority of MM cases ( n = 43) had < 3% NPCs. CD19 was the most sensitive (100%) and CD81 was the most specific marker (100%) for differentiating APCs from NPCs. CD38 MFI came out as a useful marker for APCs identification. In combination, CD19 and CD81 had a higher sensitivity and specificity to detect APCs. Conclusion NPCs may show aberrant antigen expression. A combination of multiple markers including CD81 and CD38 MFI should be used for accurate APC detection.

ALK-positive Large B-cell Lymphoma: A Clinicopathologic Retrospective Descriptive Study from a Tertiary Care Cancer Centre in India

Introduction: Anaplastic Lymphoma Kinase (ALK) positive Large B-Cell Lymphoma (ALK+ LBCL) is a very rare aggressive B-cell lymphoma presenting significant diagnostic challenges due to their rarity and unique immunophenotypic features. ALK is a tyrosine kinase receptor and is expressed by ALK+ LBCL due to ALK rearrangement. Aim: To analyse the histopathological features including morphology and immunophenotype, clinical details, pattern of care, progression-free survival (PFS) and overall survival of cases diagnosed as ALK+ LBCL. Materials and Methods: This clinicopathological retrospective descriptive study was conducted in the Department of Pathology at a Tertiary Care Cancer Centre, Thiruvananthapuram, Kerala, India. The duration of the study was six months, from January 2022 to June 2022. All the cases of ALK+ LBCL were diagnosed over a period of 10 years, from January 1st 2010 to December 31st 2020. The cases of LBCL diagnosed during the 10 year period were reviewed. Clinical details were obtained from the case sheets of the cases diagnosed as ALK+ LBCL and summarised. Data collection variables included age, sex, stage, nodal and extranodal status, bone marrow, Central Nervous System (CNS) involvement, haemoglobin, Total Leukocyte Count (TLC) and platelet count, Lactate Dehydrogenase (LDH) value, performance status, date of diagnosis, date of treatment started, date of progression, date of last follow-up, date of death (if dead). Review of Haematoxylin &amp; Eosin (H&amp;E) sections and immunohistochemical slides were done and observations were recorded. Descriptive statistics was used to summarise the basic features of the dataset and Kaplan-Meier method was used for calculation of survival. Results: The age of study participants was ranged from 16- 56 years. During the 10 year period, LBCL accounted for 2415 cases. Among these, ALK+ LBCL constituted 6 (0.25%) cases. There was a male predilection (n=5). Blood counts were normal except for anaemia in three patients. LDH was raised in all the patients. Advanced stage disease was present in two patients. Histopathologically, tumour cells in all the cases showed plasmablastic morphology. Immunohistochemistry (IHC) revealed plasma cell immunophenotype and positivity for ALK in all the cases. Cytokeratin (CK) and Epithelial Membrane Antigen (EMA) were positive in three cases simulating carcinomas. Six year overall survival and progression free survival in the present study was 50% and 33.3%, respectively. Conclusion: Careful interpretation of the morphology and immunophenotype is essential for diagnosis of ALK+ LBCL, as it can be easily misdiagnosed as a non haematological malignancy thus, affecting the treatment and prognosis in these patients.

Novel 17-Plex Flow Cytometry Assay for Comprehensive Immunophenotyping and Characterization of Plasma Cells (PC) in Relapsed/Refractory (RR) Multiple Myeloma (MM) Patients

Background: Multiple Myeloma (MM) is a hematological malignancy also known as plasma cell myeloma or myeloma, a type of bone marrow cancer characterized by the proliferation of clonal malignant plasma cells (PCs). Characterization of multiple myeloma plasma cells (MMPCs) remains challenging due to their plasticity, especially in response to emerging targeted therapies such as anti-BCMA and anti-CD38. The need for novel options for comprehensive phenotyping remains high due to the evolving target expression profiles and dynamics across MM disease evolution and treatment lines. Recent progress in flow cytometry offers the opportunity to analyze more than 30 surface markers in one panel. This technology advance will help to address the current challenges, answer clinically relevant questions and generate data to enable translational research and identification of new drug targets or combination partners. Methods: We present a novel 17-plex flow cytometry assay for immunophenotyping of PCs in RRMM patients enrolled in a global Phase I multi-center clinical study (NCT04557150). The panel relies on 5 markers for the identification of malignant plasma cells: CD38, CD138, CD45, CD319 and CD229. The inclusion of less commonly used markers (CD229, CD319) allows to overcome some of the challenges in MMPCs identification, such as CD138 instability and CD38 downregulation due to prior therapies. In addition, the assay includes markers commonly used to differentiate normal from malignant PCs (CD19, CD56, CD27, CD81, CD117, CD28). The panel also includes 2 immunotherapy target ligands (PD-L1 and 4-1BBL) and offers the possibility to measure another 3 clinically relevant targets on MMPCs, including BCMA. With this configuration, the assay allows relevant target antigen density quantification via the use of molecules of equivalent soluble fluorochrome (MESF) beads in 3 channels. Results: We present examples of how to apply this strategy for a reliable identification of MMPCs in RRMM patient samples after up to 14 previous lines of therapies. The assay design permits a deep phenotypical characterization of MMPCs and pharmacodynamic changes in markers targeted by immunotherapy drugs, like anti-CD38 or anti-BCMA. As an example, we characterize BCMA and CD38 expression on the surface of MMPCs at the time of enrolment into a Phase I study. Finally, with the goal of discovering new biomarkers that could be overlooked in the traditional manual gating hierarchy applied in flow cytometry, we have visualized the results using dimensionality reduction analysis (Uniform Manifold Approximation and Projection, UMAP). Using this approach, we have observed high inter-patient heterogeneity of MMPC phenotypes. Furthermore, we could compare intra-patient differences in MMPC phenotype in two compartments, whole blood and bone marrow aspirate. Updated data will be presented. Conclusion: The multi parameter flow cytometry assay described here represents a novel tool to reliably identify MMPCs in RRMM patients independently of commonly used unstable surface markers. Furthermore, it represents an important tool in the era of surface antigen targeted therapies (e.g., anti-BCMA) helping to understand drug resistance due to target loss. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Plasmablastic lymphoma: An update.

Plasmablastic lymphoma (PBL) is a highly aggressive B cell non‐Hodgkin lymphoma frequently associated with immunosuppression, particularly human immunodeficiency virus (HIV) infection. Although PBL is rare globally, South Africa has a high burden of HIV infection leading to a higher incidence of PBL in the region. Laboratory features in PBL may overlap with plasmablastic myeloma and other large B cell lymphomas with plasmablastic or immunoblastic morphology leading to diagnostic dilemmas. There are, however, pertinent distinguishing laboratory features in PBL such as a plasma cell immunophenotype with MYC overexpression, expression of Epstein–Barr virus‐encoded small RNAs and lack of anaplastic lymphoma kinase (ALK) expression. This review aims to provide a summary of current knowledge in PBL, focusing on the epidemiology, pathophysiology, laboratory diagnosis and clinical management.

Key markers for diagnosis of minimal residual disease in multiple myeloma

Introduction. Therapeutic advances in recent years, the appearance on the market each time of new drugs that allow patients to achieve complete remission, a long period without progression dictate the need to control treatment by monitoring residual disease in multiple myeloma. Monitoring of multiple myeloma is the detection of a small number of tumor cells after therapy in a patient, which may be the cause of recurrence of the disease – control of minimal residual disease (MRD). This article discusses the key diagnostic markers (CD45, CD56 and CD19) of minimal residual multiple myeloma disease at initial diagnosis and after induction therapy. There are various diagnostic methods of research that can reliably assess the response to therapy and predict the occurrence of relapse. The main methods for detecting MRD are allele-specific polymerase chain reaction, next-generation sequencing and multicolor flow cytometry. The diagnosis of MRD by flow cytometry is the most widely used quite fast, quantitative, sensitive and affordable method, it is used for the primary diagnosis of multiple myeloma, as well as for monitoring MRD. It is important to identify the aberrant immunophenotype during the initial diagnosis for the correct subsequent assessment of MRD. MRD of multiple myeloma is considered an important component in the course of patient therapy before hematopoietic stem cell autotransplantation to assess the effectiveness of therapy, control and prognosis of the disease.The aim of the work MRD-study with the key diagnostic markers in multiple myeloma (CD45, CD56 and CD19).Materials and methods. The study was carried out in 59 patients with multiple myeloma. For all patients, in addition to standard methods of diagnosis and staging of the disease, bone marrow morphology (myelogram) and immunophenotype were examined. Eight-color flow cytometry with a panel of monoclonal antibodies for the diagnosis of plasma cell tumors Euro-Flow 2012 was used to diagnose MRD.Results. MRD was assessed by markers CD45, CD56 and CD19 in patients with multiple myeloma after induction therapy. The incidence of MRD-positivity for CD45 was 87.5 %, for CD56 – 97.5 %, for CD19 – 95.5 %. The incidence of MRD-negative status was for CD45 was 12.5 %, for CD56 – 2.5 % and for CD19 – 4,5 %.Conclusion. The use of a complex of these markers allows the most accurate determination of the MRD-negative status, taking into account the primary immunophenotype of malignant plasma cells.

Flow cytometric immunophenotyping of plasma cells across the spectrum of plasma cell proliferative disorders: A fresh insight with pattern-based recognition.

The expression pattern of common antigens including cytoplasmic kappa/lambda ratio (cyKLR) was evaluated by flow cytometric immunophenotyping (FCMI) to explore their relevance in discriminating normal and aberrant plasma cells (NPC and APC, respectively) across spectrum of plasma cell proliferative disorders (PCPD). In this prospective analysis, 791 samples from PCPD (treatment naive=455; partially treated=336) were evaluated for expression of CD38, CD138, CD45, CD19, CD56, CD27, CD81, CD117, Cy-kappa, and Cy-lambda using FCMI. Amongst the entire cohort, 20.7% (n=164) samples displayed only APC, 21% (n=165) only NPC and 58% (n=462) showed coexistence of NPC and APC. Using pattern-based recognition (PBR) for three common patterns (CD19 vs. CD56; CD27 vs. CD56 and CD19 vs. CD27), APC was separable from NPC in 93% samples. In 6.5% samples, the gating markers contributed in APC-NPC differentiation and in the remaining 0.5% CD117 and CD81 proved useful. Clonality assessment was found to be crucial to label plasma cell compartment as completely normal or aberrant in 42% cases with either all NPC or all APC. Sixty one out of 462 cases (13%) revealed cyKLR within normal reference range and in these cases; abnormal cyKLR was demonstrable only after gating APC separately based on PBR. Fair discrimination between NPC and APC is achievable in all PCPD samples using eight markers (Gating: CD38, CD138, CD45; PBR:CD19, CD56, CD27; clonality: Cy-kappa and Cy-lambda). Thus, combined assessment of clonality and immunophenotypic aberrancies is required for accurate, reliable and precise assessment of NPC and APC compartments in PCPD.

Adhesion molecule immunophenotype of bone marrow multiple myeloma plasma cells impacts the presence of malignant circulating plasma cells in peripheral blood.

Multiple myeloma (MM) patients with malignant plasma cells (MMPCs) in their bone marrow (BM) and malignant circulating plasma cells (MMCPCs) in the peripheral blood (PB) are an independent marker of a clinically aggressive disease, and it reflects a poor prognosis defined by a short time to progression and overall survival. We hypothesized that changes in ADM expression on BM MMPCs might contribute to MMCPC presence in the PB of relapsed/refractory multiple myeloma (RRMM) patients. We assessed the difference in expression of adhesion molecules and receptors related to cell-cell interaction: integrins, hyaluronic acid receptors, chemokine receptors and other proteins on healthy donor PCs, RRMM BM and PB MMPCs. Adhesion immunophenotype showed a significant loss of many adhesion molecules when comparing BM MMPCs of MMCPC- and MMCPC+ MM patients (CD49d, CD49e, CD56, CD138). Further decrease of adhesion molecules was shown in MMCPCs (CD49d, CD49e, CD56, CD138, CD58), suggesting that loss of these molecules may allow cells to leave the BM. Loss of adhesion molecule expression enables MMPCs to leave the BM milieu and enter the PB. These changes can be seen in both the PB and BM of MMCPC+ MM patient.

Effects of storage time of bone marrow specimens on the detection results of plasma cells immunophenotyping by flow cytometry

Objective To explore the effects of different storage time of bone marrow specimens on the expressions of different antigens in normal plasma cells (nPC) and clone plasma cells (cPC) by flow cytometry. Methods The bone marrow samples of 12 patients with multiple myeloma (MM) who were treated in Beijing Chaoyang Hospital from September 2017 to January 2018 were selected as MM group. The minimum residual disease (MRD) level in MM group was 10-3-10-2. The bone marrow samples of 12 patients without plasma cell diseases were used as control group. Bone marrow samples were anticoagulated with ethylenediaminetetraacetic acid dipotassium (EDTA-K2) and stored at 2-8 °C. The fluorescent antibodies CD56, CD138, CD45, CD38, CD117, CD81 and cκ, cλ, CD45, CD38, CD19, CD27 were labeled at 0, 24, 48 and 72 h, respectively. The average fluorescence intensity (MFI) of the above 10 antigens expressed in nPC and cPC was analyzed by Diva software. The proportion and absolute count of nPC in control group and cPC in MM group were analyzed. Results In control group, when stored for 24 h, compared with 0 h, the difference of MFI of antigens in nPC was not statistically significant (P > 0.05). When stored for 48 h, compared with 0 h, the MFI of CD38, CD138, CD27, cκ and cλ in nPC decreased, and the differences were statistically significant (28 943±6 591 vs. 23 569±7 587, P= 0.018; 1 412±399 vs. 817±223, P = 0.014; 12 855±3 734 vs. 9 210±3 660, P = 0.005; 26 712±9 025 vs. 17 247±5 078, P = 0.026; 17 707±8 633 vs. 8 307±3 158, P = 0.049); the MFI of CD45 increased, and the difference was statistically significant (7 694± 2 525 vs. 9 184±1 332, P = 0.037). When stored for 72 h, compared with 0 h, the MFI of cκ and cλ increased, but the differences were not statistically significant (both P > 0.05). In MM group, when stored for 24 h, compared with 0 h, the difference in MFI of antigens in cPC was not statistically significant (P > 0.05). When stored for 48 h, compared with 0 h, the MFI of CD38, CD138, CD81, cκ and cλ decreased, and the differences were statistically significant (16 664±11 744 vs. 10 130±10 026, P = 0.003; 2 041±1 145 vs. 1 371±696, P = 0.047; 2 679±784 vs. 1 524±1 153, P = 0.025; 29 102±18 138 vs. 18 372±10 327, P = 0.038; 16 314±12 728 vs. 9 752±6 271, P = 0.034). When stored for 72 h, compared with 0 h, the MFI of cκ and cλ increased, but the differences were not statistically significant (both P > 0.05). The absolute count of nPC and cPC gradually decreased with the prolongation of the storage time, and the difference was statistically significant (both P 0.05). Conclusion Different storage time of bone marrow samples has effects on the MFI of antigens and absolute count of nPC and cPC, and the detection should be completed within 48 h. Key words: Bone marrow; Plasma cell immunophenotyping; Flow cytometry; Storage time

Multiple myeloma immunophenotyping: method validation

Flow cytometric immunophenotyping has become essential for management of multiple myeloma (assessment of clonality, prognostic information on the risk of progression in gammopathy of undetermined significance, minimal residual disease monitoring). Immunophenotyping of bone marrow plasma cells is routinely used in the haematology laboratory of the University Hospital of Toulouse. To guarantee the reliability of this technique, the laboratory decided to check this method in compliance with the NF ISO EN 15189, standard for medical laboratories requirements. As expected, the method showed good technical performances. However, this initiative has demonstrated the importance of the sample quality and of the control of the preanalytical and analytical conditions. This process led us to maximize our professional pratices.

Efficacy of Maintenance Therapy Following Auto-HSCT Depending on MRD Status in Patients with Multiple Myeloma

Background: The results of international randomized clinical trials emphasize the expediency of maintenance therapy following auto-HSCT. However, these studies did not assess such important issues as the need for maintenance therapy in patients who have achieved complete remission (CR) or stringent CR following auto-HSCT. Probably, the results of studying MRD following auto-HSCT will allow receiving a substantiated answer to this question.Aims: To evaluate the efficacy of maintenance therapy following auto-HSCT depending on MRD in patients with multiple myeloma.Patients and methods: Over the period from January 2012 to April 2018, 70 MM patients (24 males and 46 females) aged 32 to 65 years (median=56) were enrolled into a prospective study. The disease stage according to the International Staging System (ISS) was I, II and III in 28, 19 and 23 patients, respectively. All patients received induction therapy with bortezomib; immunomodulatory drugs were used in 10 cases. After the induction therapy, a single and tandem auto-HSCT were performed in 57 and 13 patients, respectively. On Day 100 following auto-HSCT, bone marrow examination was carried out in order to determine MRD using six-color flow cytometry with a panel of antigens: CD38, CD138, CD45, CD56, CD117, CD19. MRD-negative status was diagnosed in case of detection of <20 clonal plasma cells among 2,000,000 white blood cells (<0.001%; detection limit 10-5). On Day 100 after the auto-HSCT, all patients achieved CR of the disease and were randomized to receive maintenance therapy with lenalidomide 15 mg/day from Day 1 to Day 21 of a 28-day course within a year or no such therapy. The follow-up period since the moment of MRD determination was 2-28 months (median 15). Survival curves were constructed using the Kaplan-Meier method. Statistical analysis was done using Statistica 10.Results: 37 patients were randomized to receive maintenance therapy with lenalidomide following auto-HSCT, including 23 patients in whom plasma cell immunophenotyping showed the lack of MRD and 14 patients in whom MRD-positive status was confirmed. Thirty-three patients were followed-up without further treatment after the auto-HSCT, including 24 cases with MRD-negative status and 9 cases with the presence of abnormal plasma cells in the bone marrow. The compared groups were comparable in respect of such parameters as age and the ISS stage.The differences between two-year PFS rates in MM patients with MRD-negative status following auto-HSCT who received (n=23) or didn't receive (n=24) the maintenance therapy, showed no statistical significance (p=0.3) and were 88% and 74%, respectively (Fig.1a). In patients with MRD-positive status following auto-HSCT who received lenalidomide, two-year PFS rate was significantly (p<0.05) higher and was 92% versus 45% in the group of patients who didn't receive the maintenance therapy (Fig.1b).Conclusion: Achievement of MRD-negative status following auto-HSCT was accompanied by high values of PFS regardless of the use or not use the maintenance therapy with lenalidomide (88% versus 74%, p=0.3). Prescription of the maintenance therapy to patients with MRD-positive status following auto-HSCT improves the PFS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Rare Case of Plasmablastic Lymphoma with Gastric Involvement

Plasmablastic lymphoma (PBL) is a relatively rare, but highly aggressive lymphoma commonly found in the setting of HIV disease.PBL is defined by the World Health Organization as a “diffuse proliferation of large neoplastic cells most of which resemble B-cell immunoblasts, but in which all tumor cells have a plasma cell immunophenotype.”Here we present a unique case of Ann Arbor Stage IV PBL with extranodal gastric involvement diagnosed in an immunocompetent male. An 80-year-old Hispanic male presented to the emergency department with nausea and epigastric pain for four days duration. The patient also endorsed progressive dysphagia to solids and liquids over the past several weeks prior to presentation. Significant findings on physical exam included a pale, well-nourished male with mild epigastric tenderness on light palpation. CT scan revealed diffuse thickening of the gastric wall with a 7.4cm x 6.5cm intraluminal infiltrating mass with luminal narrowing. An EGD was subsequently performed for better visualization and biopsy (Figure 1). Histopathologic studies ultimately identified the mass as malignant plasmablastic lymphoma with aberrant cytokeratin expression. HIV testing was negative but EBV IgG antibody testing was abnormally high. Immunophenotype analysis revealed that CD-138 was strongly positive, CD-20 was negative, CD-45 was weakly positive, and Ki-67 expression was greater than 90% (Figure 2). The patient was started on CHOP chemotherapy regimen.2659_A Figure 1. Large, 5cm, semi-circumferential, easily friable and fungating mass as observed from GE junction during EGD2659_B Figure 2. Histology and Immunophenotype Analysis. (A) High-power magnification showing atypical plasma cells. (B) CD-20 negative. (C) CD-138 positive. (D) Ki-67 Index > 90%.Recent meta-analysis has shown that the median age of diagnosis for HIV-negative PBL is 55 years. Although the pathogenesis of PBL remains incompletely understood, the association with EBV infection and the virus' role in prevention of apoptosis of B cells is well documented.EBV positivity and CD45 expression represent good prognostic indicators for this patient; on the other hand, Ann Arbor Stage IV classification and Ki-67 expression > 80% are typically associated with poor outcome. After the oral mucosa, the GI tract is the most common site of involvement for HIV-negative PBL cases. This case represents a rare phenotypic variant in which the BCL-6 transcription factor is positive, suggesting that perhaps this case is a hybrid of diffuse large B-cell lymphoma (DLBCL) and PBL. Despite the recent advances in the treatment for PBL, prognosis remains poor with the median overall survival of nine months and a 2-year overall survival rate of 10% for HIV-negative PBL.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society.

Plasma Cell Immunophenotyping Improve Prognostic Stratification of Multiple Myeloma Patients

Background: The aims of this study were to establish the clinical value of multi-parametric flow cytomentry (MFC) in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Methods: We analyzed bone marrow aspirates from 112 MM and 17 MGUS patients by MFC, using 3 combinations of 9 color labeling: a, CD38 / CD138 / CD45 / CD56 / CD19 / CD27 / CD117 / CD20 / CD33; b, CD38 / CD138 / CD45 / cytoplasmic Kappa / cytoplasmic Lambda; and c, CD38 / CD138. MFC data were classified based on clinical features and prognosis factors. Results: Myeloma’s patients compared to MGUS group had plasma cells (PCs) with abnormal immunophenotypic patterns, including high CD56 and CD20 expression and weak or negative CD45, CD19, and CD27 expression without significant median differences in expression of CD33 and CD117. Multiple myeloma patient with low expression of CD19, CD27 or CD45, overexpression of CD56 or with a high proportion of PCs at diagnosis demonstrated shorter overall survival times. Moreover, MM patients with combined abnormal expressions of 4 or 5 antigens demonstrated shorter survival times (P = 0.001). These high-risk MFC patients were associated with poor clinical outcomes, including ISS stage III and DS stage III, low hemoglobin levels, and elevated serum beta2-microglobilin (P = 0.01, P = 0.006, P = 0.01 and P = 0.008, respectively). Conclusions: The present study highlights the benefits of assessing abnormal antigen expression for clinical uses. These measures could facilitate proper diagnosis of disorders and improve risk stratification for a targeted early treatment regimen.

Multiple myeloma: Maintenance therapy after autologous hematopoietic stem cell transplantation, depending on minimal residual disease

To determine the efficiency of maintenance therapy with bortezomib in patients with multiple myeloma (MM) who have achieved complete remission (CR) after autologous hematopoietic stem cell (auto-HSCT), depending on the presence of minimal residual disease (MRD). In January 2014 to February 2016, fifty-two MM patients (19 men and 33 women) aged 24 to 66 years (median 54 years), who had achieved CR after auto-HSCT, were randomized to perform maintenance therapy with bortezomib during a year. On day 100 after auto-HSCT, all the patients underwent immunophenotyping of bone marrow plasma cells by 6-color flow cytometry to detect MRD. Relapse-free survival (RFS) was chosen as a criterion for evaluating the efficiency of maintenance therapy. After auto-HSCT, MRD-negative patients had a statistically significantly higher 2-year RFS rate than MRD-positive patients: 52.9% (95% confidence interval (CI), 35.5 to 70.5%) versus 37.2% (95% CI, 25.4 to 49.3%) (p=0.05). The presence of MRD statistically significantly increased the risk of relapse (odds ratio 1.7; 95% CI, 1.2 to 3.4; p=0.05). Two-year cumulative risk of relapse (using the Kaplan-Meier) after auto-HSCT did not statistically significantly differ in MRD-negative patients receiving (n=15) and not receiving (n=10) maintenance therapy with bortezomib (p=0.58). After completion of maintenance treatment, 42% of the MRD-positive patients achieved a negative status. In the MRD-positive patients who had received maintenance therapy, the average time to recurrence was 5 months longer than that in the naïve patients: 17.3 versus 12.3 months. The MRD status determined in MM patients who have achieved CR after auto-HSCT is an important factor for deciding on the use of maintenance therapy.