Multiparameter Flow Cytometry Immunophenotyping Research Articles

Multiparameter Flowcytometry for Diagnosis and Subtyping of Mature Lymphocytic Neoplasms in Peripheral Blood and Bone Marrow: A Prospective Observational Study

Introduction: Multiparameter Flowcytometry (MFC) is a high throughput, quick, and practical technique for diagnoses of Chronic Lymphoproliferative Disorders (CLPDs). Indian CLPDs cases have distinct distribution and presentation than the developed world. Moreover, limited studies have confirmed the diagnostic utility of MFC in Indian CLPDs cases. Aim: To evaluate the diagnostic utility of MFC in peripheral blood and bone marrow aspirate of CLPDs cases. Materials and Methods: This was a single centre, prospective, observational study involving clinico-morphologically suspected or diagnosed 85 CLPDs cases. It was carried out in the Department of Pathology, Government Medical College and Hospital, Nagpur, Maharashtra, India, from January 2016 to November 2019. The patients were followed up for peripheral smear (PS) and Bone Marrow(BM) MFC and staging in nodal or extranodal Non-Hodgkin’s Lymphoma was done. Results: Clinico-morphological examination led to the diagnosis of 74 CLPD cases, while remaining 11 cases were strongly suspected. MFC immunophenotyping was contributory in diagnosing 74 CLPD cases which on further subtyping consisted of B-cell CLPD (N=70), and T-cell CLPD (N=3), while one case of B-NHL could not be subtyped. The most common B-cell CLPD included multiple myeloma (n=27), chronic lymphocytic leukemia (n=25), diffuse large B-cell lymphoma (n=7). T-cell CLPD included hepatosplenic gamma delta T-cell lymphoma (N=2) and adult T-cell lymphoma, follicular lymphoma (n=3), burkitt’s lymphoma (n=2), mantle cell lymphoma (n=2), prolymphocytic leukemia (n=2), splenic marginal zonal lymphoma (n=2), B Cell Non-Hodgkin’s Lymphoma (n=1). Finally, 11 suspected cases mostly comprised of reactive lymphocytosis (81.8%). Conclusion: MFC immunophenotyping led to diagnoses and determination of CLPD sub-class. It also resulted in rapid diagnoses of reactive hyperplasia and non-hematolymphoid malignancy which may mimic CLPD on morphology and hence, difficult to diagnose based on morphology alone.

Multi-parameter flow cytometry immunophenotyping distinguishes different stages of tuberculosis infection

To identify new potential host biomarkers in blood to discriminate between active TB patients, uninfected (NoTBI) and latently infected contacts (LTBI). A blood cell count was performed to study parent leukocyte populations. Peripheral blood mononuclear cells (PBMCs) were isolated and a multi-parameter flow cytometry assay was conducted to study the distribution of basal and Mycobacterium tuberculosis (Mtb)-stimulated lymphocytes. Differences between groups and the area under the ROC curve (AUC) were investigated to assess the diagnostic accuracy. Active TB patients presented higher Monocyte-to-lymphocyte and Neutrophil-to-lymphocyte ratios than LTBI and NoTBI contacts (p<0.0001; AUC>0.8). Lymphocyte subsets with differences (p >0.05; AUC >0.7) between active TB and both contact groups include the basal distribution of Th1/Th2 ratio, Th1-Th17, CD4+ Central Memory (TCM) or MAIT cells. Expression of CD154 is increased in Mtb-activated CD4+ TCM and Effector Memory T cells in active TB and LTBI compared to NoTBI. In CD4+T cells, expression of CD154 showed a higher accuracy than IFNγ to discriminate Mtb-specific activation. We identified different cell subsets with potential use in tuberculosis diagnosis. Among them, distribution of CD4 TCM cells and their expression of CD154 after Mtb-activation are the most promising candidates.

Value of multi-parameter flow cytometry immunophenotyping in T/NK-cell neoplasms in cytology specimens: A retrospective study in Chinese patients

BackgroundInnate limitations of morphological diagnosis of T/NK-cell neoplasms mean that they can be misdiagnosed or missed, especially when mixed with a variety of benign and reactive conditions. The aim of this study was to investigate the application value of multiparameter flow cytometry immunophenotyping (MFCI) in screening and diagnosing T/NK-cell neoplasms with cytology specimens. Material and methodsThe clinical and pathological characteristics of 1028 newly diagnosed cases from Fudan University Shanghai Cancer Center who provided a cytology specimen between June 2010 and January 2016 with correlated histology diagnosis and clinical confirmation were retrospectively reviewed. MFCI was used for screening, diagnosis and typing. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in diagnosis of T/NK-cell neoplasms were calculated. ResultsThere were 606 males and 422 females in 1028cases, with a mean age of 47.5 years (range 9–86 years). Specimens used for cytologic diagnosis included 996 FNAs, 2 US-FNAs, 13 EUS-FNAs and 17 effusions. Screening for types of lymphoma of MFCI, 139 (13.52 %) cases were T/NK cell lymphoma, 3 (0.29 %) cases were B cell lymphoma T-NHL and B-NHL coexist. A total of 146 suspected T/NK-cell neoplasms were screened out (sensitivity = 94.64 %, specificity = 95.63 % PPV = 72.60 %, NPV = 99.32 %) by MFCI, with 112 (76.71 %) histologically confirmed cases and 6 (4.11 %) false-negative cases identified (3 cases diagnosed as B-cell neoplasms and 1 case as T-cell neoplasm with B-cell neoplasm, which also were confirmed by gene rearrangement. 2 cases were suspicious T-cell–immunophenotypic abnormalities). When used at the diagnostic level, a total of 88 T/NK-cell neoplasms were identified (sensitivity = 68.75 %, specificity = 98.80 %, PPV = 87.50 %, NPV = 96.28 %) with 11 false-positive cases recognized, 9 of which showed typical immunophenotypic T-cell neoplasms features, and 2 exhibited aberrant T immunophenotype. ConclusionsMFCI has high sensitivity and specificity in the screening and diagnosis of T/NK-cell neoplasms and may be useful as an alternative diagnosis method in cytology specimens.

Sensitivity of flow cytometry immunophenotyping compared with bone marrow morphology in diagnosis of multiple myeloma

Background The diagnosis of multiple myeloma (MM) is challenging, as it is based on several factors, including serum and urine protein electrophoresis, immunofixation, bone marrow (BM) aspirate, and biopsy, in addition to immunophenotyping. Moreover, quantification of BM plasma cells (PCs) by conventional morphology is a mandatory test for the diagnosis and response assessment in MM. One alternative would be to use multiparameter flow cytometry immunophenotyping to quantify BMPCs, although this is currently still limited to research studies and the differential diagnosis of unusual cases.Aim The aim was to determine the diagnostic sensitivity of flow cytometry in the detection of abnormal PCs in BM specimens of MM compared with BM morphology.Patients and methods We retrospectively analyzed bone marrow aspiration (BMA), bone marrow biopsy (BMB), and immunophenotypic reports of 50 newly diagnosed patients with MM. Patients’ data were collected and analyzed.Results BMA of 43 (86%) patients showed PCs greater than or equal to 10%; BMB of 35 of them showed PCs greater than or equal to 10%, and five patients were with poor BMB quality. The sensitivity of BMB in detection of PCs was 90% and 86% for BMA. Concerning immunophenotyping, the gate of mononuclear cells was in the range of 4–88%; those gated cells were 100% positive for CD138 and CD38. CD56 was positive in 84% whereas CD19 was negative in 96%. Overall, 46% were κ restricted and 54% were λ restricted, with 100% sensitivity to malignant PCs.Conclusion Unlike morphologic and histologic studies, FC is not dependable for providing quantitative information about MM but provides qualitative information for assessing the immunophenotype and light chain clonality of PCs.

Noninvasive Molecular Monitoring in Multiple Myeloma Patients Using Cell-Free Tumor DNA: A Pilot Study

Novel treatments for multiple myeloma (MM) have increased rates of complete response, raising interest in more accurate methods to evaluate residual disease. Cell-free tumor DNA (cfDNA) analysis may represent a minimally invasive approach complementary to multiparameter flow cytometry (MFC) and molecular methods on bone marrow aspirates. A sequencing approach using the Ion Torrent Personal Genome Machine was applied to identify clonal IGH gene rearrangements in tumor plasma cells (PCs) and in serial plasma samples of 25 patients with MM receiving second-line therapy. The same clonal IGH rearrangement identified in tumor PCs was detected in paired plasma samples, and levels of IGH cfDNA correlated with outcome and mirrored tumor dynamics evaluated using conventional laboratory parameters. In addition, IGH cfDNA levels reflected the number of PCs enumerated by MFC immunophenotyping even in the complete response context. Patients determined by MFC to be free of minimal residual disease were characterized by low frequencies of tumor clonotypes in cfDNA and longer survival. This pilot study supports the clinical applicability of the noninvasive monitoring of tumor levels in plasma samples of patients with MM by IGH sequencing.

The prognostic impact of multiparameter flow cytometry immunophenotyping and cytogenetic aberrancies in patients with multiple myeloma

Objectives: The aim of this study was to evaluate the prognostic impact of immunophenotyping in patients with multiple myeloma (MM), as well as other markers of disease, such as serum hyaluronan and cytogenetic aberrancies.Methods: We have prospectively analyzed the prognostic impact of antigenic markers, assessed by multiparametric flow cytometry (MFC), in a series of newly diagnosed MM patients (n = 79).Results and discussion: Our results show that the expression of CD44, CD45, and CD28 and the absence of CD117 were associated with a significantly shorter progression free-survival (PFS). Clinical characteristics were collected; Cytogenetic aberrancies were assessed in 40 patients. Multivariate survival analyses identified that the CD117−, CD28+, CD45+, and the percentage of bone marrow plasma cells by MFC are survival predictor, along with the International Staging System stage. Interestingly, the CD117− patients were associated with chromosomal aberrancies, including del (17p), +1q21, and IgH translocations.Conclusion: The incorporation of multiparameter flow cytometry immunophenotyping into the routine diagnostic evaluation of MM patients can help to identify patients at a high risk of progression.

Issue Highlights—March 2015

In their review Damuzzo et al. 1 focus on the correct identification of circulating and tumour-associated so-called myeloid-derived suppressor cells (MDSCs) in humans and mouse models. The reason to do this is because MDSCs are endowed with suppressive activity and because their expansion has been associated with disease progression and reduced survival. Because of their heterogeneous composition, accurate phenotyping of these cells will almost invariantly require a multicolour approach in order to allow appreciation of MDSC subsets. Correct minimal residual disease (MRD) detection is crucial for therapy response assessment in a considerable number of hematologic disorders including mantle cell lymphoma (MCL) 2. Multiparameter flow cytometry (MFC) immunophenotyping is currently increasingly used to assess MRD in malignant disorders. Recently, a FACSCantoll (DB Biosciences, San Jose, CA)-based 7-color/8-antbody MFC approach has been published, allowing for the determination of MRD in multiple myeloma patients 3. Chovancova et al.4 found that, although MCL presents high immunophenotypic variability, the combination of CD20/23/5/19/200/62 L/45 is very favourable in flow cytometric MRD measurement in MCL reaching a sensitivity of up to 2×10 −4. In another study, Mathis et al. 5 show that this protocol is transposable between harmonized ≥ 7-color instruments and that a homogeneous rapid MRD evaluation can be performed in most MFC platforms. Although not all immunohistochemical techniques can easily be automated by image analytical systems, in a remarkable study Qin, Y et al. 6 concluded that a computerized delineation of nuclei in re-stained PAP smears with p16/ki67 can result in an automated high-throughput profiling method achieving accurate data. Because cigarette smoke is a powerful producer of reactive oxygen species 7, hypothesized that spermatozoa of smokers would be at increased risk of having DNA fragmentation as compared to spermatozoa of non-smoking men. However, by applying a novel TUNEL assay coupled to a vitality marker (LIVE/DEAD®) the authors found no deleterious effect, i.e., increased DNA fragmentation, of smoking on spermatozoa. More studies concerning the potential mutagenic capacities of cigarette smoke on spermatozoa are mandatory. Eidenschink Brodersen et al. 8, utilizing a NH4Cl lysis protocol have defined various immunophenotypic abnormalities that indicate dyserythropoiesis in myelodysplastic syndromes. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically. It is well-known that the epidermal growth factor receptor (EGFR) is overexpressed in carcinoma and can be used as a therapeutic target. Levi et al. 9 developed a Bio-Ferrography method enabling isolating EGFR overexpressing carcinoma cells from human blood. Recovery values as high of 8% for 1 mL PBS and 53% for 1 mL whole blood, with a limit of detection of 30 and 100 target cells, respectively, were achieved. Further studies are warranted to assess whether this technique allows early diagnosis of EGFR overexpressing tumour types. In MRD flow cytometry results can easily be confounded by (unknown) expression of markers on nearby cells. In their article Soma et al. 10 focuses on the expression of CD19 on NK cells. They conclude that it is an apparent but infrequent finding, not a consequence of technical error. More research to optimizing panels, combining techniques or using other specimens like bronchoalveolar lavage is needed. Perspectives from Didier G. Ebo. Didier G. Ebo* Department of Immunology - AllergologyUniversity of Antwerp,Belgium

Utility of 8-colours multiparameter flow cytometry immunophenotyping of plasma cells for the management of monoclonal gammopathy

Bone marrow flow cytometric analysis is a powerful and rapid tool for evaluating aberrant plasma cell. In this study, we have examined the utility of multiparameter flow cytometry (MFC) in 52 patients with multiple myeloma (MM) and in 45 patients with monoclonal gammopathy with unknown significance (MGUS) into routine evaluation for the management of patients with plasma cell-related disorders. The plasma cells (PC) were identified by their light scatter distribution and reactivity patterns to CD138, CD38, and CD45. The combination of these parameters was helpful for identifying distinct subpopulations of PCs. Moderate to bright expression of CD56, CD20, CD24, CD28, and CD117 was detected in 67%, 26%, 13%, 27%, and 57% of MM cases and in 58%, 20%, 11%, 43% and 44% of MGUS cases, respectively. In MGUS group, the median percentage abnormal PCs/total PCs was 88% with 37 patients out of 45 (82%) with ratio <95%. The median ratio of the MM group was 98.9% and a ratio ≥ 95% was observed in 37 samples out of 44 (84%). In conclusion, MFC immunophenotyping of PCs has obvious clinical relevance in differential diagnosis between MM and others monoclonal gammopathies, identification of high-risk MGUS and smouldering MM, and minimal residual disease monitoring of MM. Our results showed that this tool can be easily applied in haematology laboratories.

Multiparameter flow cytometry for the identification of the Waldenström’s clone in IgM-MGUS and Waldenström’s Macroglobulinemia: new criteria for differential diagnosis and risk stratification

Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenström's Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P<.001), with only 1% of IgM-MGUS patients showing >10% B cells or 100% light-chain-isotype-positive B-cells (P<.001). Complete light-chain restriction of the B-cell compartment was an independent prognostic factor for time-to progression in smoldering WM (median 26 months; HR: 19.8, P=0.001) and overall survival in symptomatic WM (median 44 months; HR: 2.6, P=0.004). The progressive accumulation of light-chain-isotype-positive B-cells accompanied the emergence of a characteristic Waldenstrom's phenotype (CD22(+dim) / CD25+ /CD27+ / IgM+) that differed from other B-NHL by negative expression of CD5, CD10, CD11c or CD103. In contrast to myeloma, light-chain-isotype-positive plasma cells in IgM monoclonal gammopathies show otherwise normal antigenic expression. Our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM.

Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/CD99+), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1+/numyogenin+), carcinomas (CD45−/EpCAM+), germ cell tumors (CD56+/CD45−/NG2+/CD10+) and eventually also hemangiopericytomas (CD45−/CD34+). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

Simultaneous cytomorphological and multiparameter flow cytometric analysis of ALK-positive anaplastic large cell lymphoma in children

The aim of this study was to investigate the pathological features of anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) in children and to establish the effectiveness of screening and diagnosing ALCL with multiparameter flow cytometry immunophenotyping (FCI) of lymphoid tissue samples. A total of 121 lymph node tissue specimens obtained from 121 patients with a suspected diagnosis of lymphoma were analyzed with cytomorphological and FCI analysis. Fifteen cases were diagnosed as ALK-positive ALCL based on the pathological features and immunohistochemical results. Of these, there were 3 different types, common type (10 cases), lymphohistiocytic type (4 cases) and neutrophil-rich type (1 case). Thirteen cases (10 common, 2 lymphohistiocytic and 1 neutrophil-rich type) were diagnosed as ALCL using FCI. These cases were CD30-positive and aberrantly expressed at least two T-cell antigens, including CD4 (84.6%), CD2 (76.9%), CD7 (61.5%), CD3 (53.8%) and CD5 (38.4%). Neoplastic cells accounted for only a small proportion of the total cells in FCI, with a median of 19.3% (range, 7.9–31.8%), which was significantly higher than those in the control groups (all <1.0%). The sensitivity of FCI for diagnosing ALCL in lymph node samples was 86.7% with a specificity of 100%. The majority of neoplastic cells demonstrated high light forward and high light side scatter, similar to monocytes or granulocytes in dot plots. FCI may be used as an adjunct to histopathological examination for rapid and reliable diagnosis of pediatric ALCL. Flexible gating strategies and careful analysis are required to identify neoplastic cells with FCI.

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

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

Case study interpretation-Portland: Case 1

Case study interpretation-Portland: Case 1

Multiparameter Flow Cytometry (MFC) Evaluation of Plasma Cell (PC) DNA Ploidy Status and Proliferative Rate in 595 Multiple Myeloma (MM) Patients (pts) Included in the Spanish GEM2000 and GEM2005

Abstract 3938▪▪This icon denotes a clinically relevant abstractThe incorporation of high-dose therapy/autologous stem cell transplantation (HDT/ASCT) and novel agents has significantly improved survival of MM young pts; however, the molecular heterogeneity of the disease warrants the investigation if such improvement also benefits patients harboring poor prognostic features such as non hyperdiploid (HY) karyotypes and high proliferation index.Herein, we have analyzed by MFC the DNA ploidy status and proliferation of bone marrow (BM) PC in a total of 595 newly diagnosed MM pts included in two consecutive PETHEMA/GEM trials: GEM2000 (VBMCP/VBAD followed by HDT/ASCT; N=319) and GEM2005<65y (randomized induction with the same chemotherapy plus bortezomib in the last two cycles or thalidomide/dexamethasone or bortezomib/thalidomide/dexamethasone followed by HDT/ASCT; N=276). Baseline FISH analysis were performed on separated PC. DNA ploidy and cell cycle were assessed by MFC through simultaneous staining with CD38/CD138 and propidium iodide. Median follow-up of the whole series was 38 months.Of the 595 pts, 295 were classified as non-HY (49.6%) and 300 as HY (50.4%). Patients with non-HY vs HY DNA content showed significantly inferior median PFS (34 vs 44 months, P=.004) and OS (67 vs 84, P=.005). Interestingly, we have detected by MFC the presence of two different PC clones (with different DNA ploidy status) in 34 of the 595 (6%) patients, and this subgroup showed a poor outcome (median PFS and OS of 26 and 52 months respectively, P≤.005). Regarding the proliferative index, the median percentage of PCs in S-phase in the whole series was of 1.14% (0%-13%). Accordingly, pts were stratified using a cutoff of ≥1% vs <1% PCs in S-phase, which translated in significantly different PFS (medians of 34 vs 43 months, P<.001) and OS (medians of 66 vs 93 months, P=.001). Of note, the detection of a proliferative rate ≥3% also identified a subgroup of patients (92 of 595, 15%) with high-risk disease (median PFS and OS of 22 and 45 months respectively, P≤.001). At the multivariate analysis including other baseline prognostic factors, the presence of high-risk cytogenetics (HR=1.7;P=.007), ≥1% PCs in S-phase (HR=2.0;P<.001), >15% PCs by MFC (HR=1.7;P=.008) and >5% normal PCs within the BM PC compartment (HR=6.6;P=.008) emerged as independent prognostic factors for PFS; in turn, for OS the presence of high-risk cytogenetics (HR=2.3;P<.001), non-HY DNA content (HR=1.7;P=.02), ≥1% PCs in S-phase (HR=2.0;P=.002) and the ISS disease stage (HR=1.7;P=.03) were selected.After this analysis of the overall population, we investigated whether the incorporation of novel agents in the induction regimen previous to HDT/ASCT could abrogate the poor prognosis of patients classified as non-HY and with ≥1% PCs in S-phase. Patients with non-HY DNA status included in the GEM2005<65y trial do not show significantly superior PFS (40 vs 34 months, P=.8) nor OS (not reached vs 63 months, P=.5) compared to cases enrolled in the GEM2000 protocol. Focusing on pts with ≥1% PCs in S-phase, no differences were founded for PFS (40 vs 33 months, P=.9), but OS was significantly longer for patients treated with novel agents prior to HDT/ASCT (NR vs 61months, P=.004).Finally, we wanted to gain further insight into the potential association between specific cytogenetic abnormalities and PC proliferation, as well as whether there is a difference in the proliferative rate of PCs between diagnosis and disease progression. Interestingly, pts harboring a t(11;14) showed a significantly decreased percentage of PCs in S-phase (0.7% vs 1.2%, P<.001), while no differences were recorded for t(4;14), t(14;16), del(Rb) and del(17p). To address our final question, we compared the proliferative rate of PCs from 52 pts with paired BM samples at diagnosis and at relapse. Of note, 45 of the 52 (85%) pts showed an increased percentage of PCs in S-phase at relapse, with a median 2-fold difference between the proliferative rate at diagnosis vs. relapse (P<.001).In summary, our results show that the evaluation of PCs DNA content by MFC immunophenotyping provides valuable clinical information, as pts with a non-HY DNA status and high proliferative rate show a poor outcome, which is not yet fully abrogated by the incorporation of novel agents prior to HDT/ASCT. Moreover, pts at relapse show an increased proliferative index; therefore, the precise mechanisms leading to PC proliferation deserves further investigations. Disclosures:Paiva:Celgene: Honoraria; Janssen: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Mateos:Janssen: Honoraria; Celgene: Honoraria. Alegre:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc: Consultancy.

High-Risk Cytogenetics and Persistent Minimal Residual Disease (MRD) by Multiparameter Flow Cytometry (MFC) Predict Unsustained Complete Response (CR) After Autologous Stem Cell Transplantation (ASCT) in Multiple Myeloma (MM)

Abstract 630▪▪This icon denotes a clinically relevant abstractThe achievement of CR after high-dose therapy/ASCT (HDT/ASCT) is a surrogate for prolonged survival in MM. While this is well accepted, the long-term clinical outcome of MM patients achieving CR is still heterogeneous and a small fraction of patients unexpectedly lose their CR status early on after HDT/ASCT and experience a dismal survival. In fact, survival of patients with unsustained CR is even poorer than that of those not achieving CR.Herein we sought to identify prognostic markers predictive of unsustained CR after HDT/ASCT. The study included a total of 241 patients achieving CR at day+100 after HDT/ASCT treated in two consecutive GEM/PETHEMA trials: GEM2000 (VBMCP/VBAD, n=140) and GEM2005<65y (randomized induction with the same chemotherapy plus bortezomib in the last two cycles or thalidomide/dexamethasone or bortezomib/thalidomide/dexamethasone followed by HDT/ASCT; N=101). All cases were referred for MRD assessment by MFC at day+100 after HDT/ASCT; baseline FISH analysis were available in 110 of the 241 patients.We first investigated which of the most relevant disease characteristics had prognostic influence in patients in CR at day+100 after HDT/ASCT. In this cohort some markers with consistent influence in unselected MM populations such as patient age, ISS stage, serum β2-microglobulin, BMPC burden and % of PC in S-phase were not significantly predictive. In contrast, the presence of baseline anemia was a significant prognostic marker for OS (P=.01). Regarding cytogenetics, 16% of CR patients were considered with high-risk disease at presentation and showed a significantly inferior TTP (3-years, 40% vs 79%; P=.001) and borderline OS (3-years, 73% vs 96%; P=.07) compared to cases with standard-risk. In addition, MFC immunophenotyping showed persistent MRD in 87 of the 241 (36%) CR patients, and the failure to achieve an immunophenotypic CR at day+100 after HDT/ASCT resulted in significantly inferior TTP (3-years, 58% vs 85%; P<.001) and OS (3-years, 80% vs 90%; P=.001). In the multivariate analysis, the best combination of independent predictive parameters for TTP were immunophenotypic CR status (P<.001;HR=7.1) and FISH cytogenetics (P<.001;HR=5.6); in turn, for OS immunophenotypic CR status (P=.001;HR=7.7), FISH cytogenetics (P=.01;HR=5.1) and age ≥60 years (P=.03;HR=3.4) were selected.We further explored the clinical impact of the immunophenotypic CR in the context of standard-risk and high-risk disease. The best prognosis was for patients with both standard-risk cytogenetics and in immunophenotypic CR (3y: TTP, 92%; OS, 100%), while the worst outcome occurred in cases with both high-risk disease and persistent MRD (3y: TTP, 0%; OS, 32%). The two other categories had an intermediate prognosis, although it was better for patients with high-risk cytogenetics achieving an immunophenotypic CR (3y: TTP, 69%; OS, 100%) than for patients with standard-risk disease but failing to achieve an immunophenotypic CR after HDT/ASCT (3y: TTP, 57%; OS, 90%).We then investigated the parameters that could help to identify patients in CR at risk of early relapse after HDT/ASCT. Of the 241 patients, 30 (12%) progressed within 1 year after HDT/ASCT (unsustained CR) and this subgroup showed a dismal outcome, with a median OS of only 39 months (P<.001). Patients with unsustained CR showed significantly higher incidence of anemia at baseline (48% vs 24%, P=.01), advanced ISS stage 2 or 3 (86% vs 55%, P=.003), high-risk cytogenetics (40% vs 10%, P=.005) and persistent MRD by MFC (63% vs 32%, P=.001) compared with the remaining cases. At the multivariate analysis, only cytogenetic abnormalities (P=.002;HR=12.5) and the immunophenotypic CR status at day+100 after HDT/ASCT (P=.001;HR=8.8) emerged as independent predictive markers for unsustained CR. Based on these two variables, we established a predictive index by assigning one point for each adverse factor. Accordingly, 3 risk groups of patients in CR after HDT/ASCT were defined, with significantly (P<.001) different rates of disease progression within the first year after HDT/ASCT for patients with none, one, or both risk factors (7%, 20% and 100%, respectively; Figure).Our results show that the baseline evaluation of cytogenetic abnormalities combined with response assessment by MRD after HDT/ASCT can discriminate a subset of CR patients with a dismal outcome who should be candidates for novel treatment strategies after HDT/ASCT. [Display omitted] Disclosures:Paiva:Celgene: Honoraria; Janssen: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Mateos:Janssen: Honoraria; Celgene: Honoraria. Cibeira:Janssen: Honoraria; Celgene: Honoraria. Alegre:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria.

The clinical utility and prognostic value of multiparameter flow cytometry immunophenotyping in light-chain amyloidosis

AbstractThe clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients with AL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL.

Comparison of Immunofixation, Serum Free Light Chain, and Immunophenotyping for Response Evaluation and Prognostication in Multiple Myeloma

To investigate the impact of immunophenotypic response (IR) versus complete response (CR) and CR plus normal serum free light chain (sFLC) ratio (stringent CR) in elderly patients with multiple myeloma (MM) treated with novel agents. From a total of 260 elderly patients newly diagnosed with MM included in the GEM05>65y trial, 102 patients achieving at least a partial response with ≥ 70% reduction in M-component after the six planned induction cycles were simultaneously analyzed by immunofixation, sFLC, and multiparameter flow cytometry (MFC) immunophenotyping; this population is the focus of this study. Forty-three percent of patients achieved CR, 30% achieved stringent CR, and 30% achieved IR. Patients in stringent CR showed no significant survival advantage compared with those in CR, whereas patients in IR showed significantly increased progression-free survival (PFS) and time to progression (TTP) compared with those in stringent CR or CR; this was confirmed by multivariate analysis (hazard ratio, 4.1; P = .01 for PFS). Discrepancies between the three techniques were relatively common. Notably, in all seven patients achieving IR but remaining immunofixation positive, the M-component disappeared in follow-up analysis. In contrast, MFC-positive patients who were immunofixation negative (n = 20) showed a tendency toward early reappearance of the M-component (median, 3 months). Similarly, in five of 11 stringent CR but MFC-positive patients, symptomatic disease progression was recorded at a median of 13 months after induction. Achieving an IR translates into superior PFS and TTP compared with conventional CR or stringent CR. These techniques provide complementary information and thus, an effort should be made to refine response criteria in MM.

Utility of flow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell‐related disorders

In recent years, multiparameter flow cytometry (MFC) immunophenotyping has become mandatory in the clinical management of hematological malignancies, both for diagnostic and monitoring purposes. Multiple myeloma (MM) and other clonal plasma cell-related (PC) disorders should be no exception to this paradigm, but incorporation of immunophenotypic studies in the management of patients with PC disorders is still far from being routinely established in many diagnostic flow cytometry laboratories. For clonal PC disorders, MFC is of clear and established clinical relevance in: (1) the differential diagnosis between MM and other PC-related disorders; (2) the identification of high-risk MGUS and smoldering MM; (3) minimal residual disease investigation after therapy; additionally it may also be useful for (4) the definition of prognosis-associated antigenic profiles; and (5) the identification of new therapeutic targets. In this article, we review the clinical value of MFC in the study of PC disorders, with specific emphasis in those areas where consensus exists on the need to incorporate MFC into routine evaluation of MM and other clonal PC-related disorders.

Multiparameter flow cytometry quantification of bone marrow plasma cells at diagnosis provides more prognostic information than morphological assessment in myeloma patients

Quantification of bone marrow plasma cells in multiple myeloma patients using conventional morphology is of limited prognostic value, while the merit of multiparameter flow cytometry immunophenotyping is still considered unproven. Here we compare the bone marrow plasma cell counts obtained by morphology and multiparameter flow cytometry and explore the potential prognostic impact of both techniques in 765 newly diagnosed, uniformly treated multiple myeloma patients. Although multiparameter flow cytometry generally yields lower plasma cell counts (median percentage of 11% vs. 40%, respectively; p<0.001), there is a significant positive correlation between the two techniques (R =0.46, p<0.001). Regarding prognosis, multivariate analysis selected the bone marrow plasma cell counts obtained by multiparameter flow cytometry as an independent prognostic factor for overall survival (p=0.007), supporting the incorporation of multiparameter flow cytometry immunophenotyping into the routine diagnostic evaluation of multiple myeloma patients and validating the clinical utility of bone marrow plasma cell counting by multiparameter flow cytometry approaches. (clinicaltrials.gov identifier: NCT00560053).

Megakaryocytic and Platelet Dysplasia in MDS Patients as Detected by Multiparameter Flow Cytometry Immunophenotyping

Introduction: Multiparameter flow cytometry immunophenotyping (FC) has shown the presence of phenotypic abnormalities in both CD34 precursors and maturing myeloid and erythorid cells in patients with myelodysplastic syndromes (MDS). However, detection of megakaryocytic dysplasia by FC still remains a challenge.Aims: To evaluate the potential utility of FC analysis of megakaryopoesis and platelet immunophenotype in MDS patients for the diagnosis and prognostic evaluation of the disease.Methods: A total of 54 peripheral blood (PB) samples corresponding to 39 patients with MDS and 15 controls (6 healthy subjects, 3 reactive cytopenias, 3 patients with May-Heglin syndrome and 3 myeloproliferative disorders) were collected in sodium citrate. In all cases, the median amount of expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b, CD61, CD62P, CD63, PAC-1 and Fibrinogen receptor was evaluated on circulating platelets as refleted by the median fluorescence intensity (MFI). Morphologic megakaryocytic dysplasia was evaluated in parallel in bone marrow samples from the same MDS individuals.Results: Morphological megakaryocytic dysplasia was detected in 49% of MDS patients who showed a shorter median overall survival (50 ± 8 vs 107 ± 12 months; p = 0.04). The following FC abnormalities were detected in circulating PB platelets of MDS patients: increased FSC and SSC (36%), overexpression of CD36 (18%), CD31 (36%), CD41b (20%), CD42b (2.5%) and CD61 (20%) and underexpression of CD36 (10%) and CD61 (5%). One case of aberrant CD34 expression was detected. A total of 27 (69.2%) patients presented at least one of these abnormalities by FC. Eighty percent of patients with morphological dysplasia and 64% without showed immunophenotypic abnormalities. According to IPSS, 6 patients constituted a high risk group (HRG) (Int-2 and High) and 28 a low risk group (LRG) (Int-1 and Low). Platelet phenotypic aberrations were detected in 83% of the HRG and in 75% of the LRG. Immunophenotypic abnormalities were not detected in myeloproliferative disorders. All May-Heglin cases and 2 reactive cytopenias presented overexpression of at least one of the following: FSC, SSC, CD31, CD36, CD41a, CD41b, CD42b and CD61. Deficient expression of CD36, CD61 and aberrant expression of CD34 were unique to MDS.Conclusion: Our results show the presence of immunophenotypic abnormalities in PB platelets from MDS patients, the abnormally decreased reactivity for CD36 and CD61 together with aberrant CD34 expression being of potential diagnostic utility in MDS.