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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 936-936
Author(s):  
Bruno Paiva ◽  
Maria-Carmen Montes ◽  
Ramón García-Sanz ◽  
Jennifer Alonso ◽  
Natalia de las Heras ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Phenotypic Characterization ◽  
International Prognostic Scoring System ◽  
Multiparameter Flow Cytometry ◽  
Risk Of Progression

Abstract Abstract 936 Demonstration 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.

Utility of Multiparameter Flow Cytometry Immunophenotypic Studies In Patients with Systemic Light Chain (AL) Amyloidosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4051-4051
Author(s):  
Bruno Paiva ◽  
María-Belén Vidriales ◽  
Jose J. Perez ◽  
Maria-Consuelo López-Berges ◽  
Ramón García-Sanz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Plasma Cell ◽  
Light Chain ◽  
Cell Cycle Analysis ◽  
Disease Assessment ◽  
Multiparameter Flow Cytometry ◽  
Al Amyloidosis

Abstract Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4943-4943
Author(s):  
Charles Repetti ◽  
Hsueh-Hua Chen ◽  
Yongbao Wang ◽  
Vanessa A Jones ◽  
Albert K Ho ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Somatic Mutation ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Sequence Variant ◽  
Low Grade ◽  
Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results  Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15).  Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

Multicentric Analyses of the CD148, CD180, and CD200 Combination for the Diagnosis of Mature B-Cell Neoplasm Using Flow Cytometry

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2662-2662 ◽  
Author(s):  
Laurent Miguet ◽  
Luc Fornecker ◽  
Marie Wyrwas ◽  
Sarah Cianferani ◽  
Raoul Herbrecht ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cell ◽  
Expression Profile ◽  
Standard Error ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Strong Expression ◽  
Group A ◽  
Weak Expression

Abstract Introduction Diagnosis of mature B-cells proliferations, especially those involving the spleen, do not always falls into any of the WHO types of B-cell neoplasms using standart diagnosis tools. This situation in notably encountered in the case of the differential diagnosis of marginal zone lymphoma (MZL), atypical chronic lymphocytic leukemia (aCLL), mantle cell lymphoma (MCL), and lymphoplasmacytic lymphoma (LPL), mostly due to the lack of immunological positive markers. In order to find new markers to discriminate between these different malignancies, we have previously developed a proteomic strategy based on the analyses of plasma membrane microparticles and proposed two new specific markers: CD148 and CD1801,2 for MCL and MZL respectively. The simultaneous use of these two markers, together with the CD200 that is positive in most cases of CLL and negative in MCL could be of great interest to better assess the differential diagnosis. Methods Flow cytometry analyses have been realized in Nancy and Strasbourg hospitals by combining these three markers: CD148 (Clone 143-41 FITC); CD180 (Clone G28.8 PE) and CD200 (Clone OX104 APC). Expression profile of these proteins was established on a well characterized set of patients (N=287): CLL with a Matutes score > 3 (N=81); MCL harboring t(11;14) translocation or CCND1 overexpression (N=44); LPL (N=58) classified following cytological morphology, IgM peak and the positivity of CD38 and/or Myd88 mutation, MZL (N=84), displaying a CD5- CD23- immunophenotype associated to a splenomegaly and 20 controls. For each group the mean of fluorescence intensity and Standard Error have been determined. Results MCL exhibited a strong expression of CD148 combined with a weak expression of CD180 and CD200. A weak expression of CD148 and CD180 coupled to a strong expression of CD200 was typical of the CLL group and a weak expression of CD148 and CD200 coupled to a strong expression of CD180 was observed in the MZL group. A moderate expression of these three markers was observed in the LPL group. A threshold corresponding to MFI +/- 4 standard error was then calculated for each group, and patients were categorized following the expression profile of these 3 markers (see figures). In this cohort, the above described profiles correctly identified MCL cases with a specificity of 92% and a sensitivity of 64%, aCLL cases with a specificity of 100% and a sensitivity of 47%, LPL cases with a specificity of 90% and a sensitivity of 54% and MZL cases with a specificity of 99% and a sensitivity of 60%. Conclusion These results strongly suggest that the incorporation of these three markers CD148 CD180 and CD200 in addition of the routinely used flow cytometry panel can be helpful in a number of cases for which the diagnosis is difficult. References: 1) Miguet et al leukemia 2013 2) Miguet et al journal of proteome research 2009 Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Proposal For a Novel Scoring System For The Diagnosis Of CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4150-4150 ◽  
Author(s):  
Thomas Köhnke ◽  
Veronika K Wittmann ◽  
Daniela Sauter ◽  
Veit Bücklein ◽  
Zlatana Pasalic ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
B Cell ◽  
Sensitivity And Specificity ◽  
Validation Cohort ◽  
Diagnostic Value ◽  
Internal Validation ◽  
Expression Intensity ◽  
Higher Sensitivity

Abstract Background Immunophenotyping is essential for the diagnosis of chronic lymphocytic leukemia (CLL). The scoring proposed in the modified Matutes score has been the basis of diagnosis for the past 15 years and is defined by strong expression of CD5 and CD23, low or absent expression of CD79b, sIgM and FMC7. However, some markers within the current score such as sIgM display a high variability in staining patterns and thus the interpretation of expression intensity is not easily reproducible. Furthermore, the newly identified marker CD200 is not included in the current score in spite of its highly informative value in the differential diagnosis of B-cell disorders. In the study presented here we aimed to improve the current score through the addition of highly informative markers such as CD200 and the omission of sIgM as a less informative, error-prone marker. Methods Between February 2011 and May 2013, peripheral blood or bone marrow aspirates of patients with suspected B-cell lymphoproliferative disorders were subjected to evaluation by flow cytometry. Immunophenotyping was performed using a Navios flow cytometer (Beckman Coulter) and samples were stained by monoclonal antibodies targeting the antigens CD45, CD19, CD5, CD10, CD23, CD79b, CD200, FMC7, sIgM, kappa and lambda. Corresponding isotype controls were used. The modified Matutes score was calculated as described previously (Moreau et al., Am J Clin Pathol 1997) with positivity defined as ≥20% positive cells. Mean Fluorescence Intensity (MFI) ratio (MFI sample/MFI isotype) was calculated as a measure of expression intensity. For our new score, optimized cut-offs for positivity vs. negativity (CD5, CD23, CD200, FMC7) and low or absent expression (CD79b) as well as sensitivity and specificity were calculated by receiver operating characteristics (ROC). The final clinical diagnosis was defined as the diagnosis established by the treating physician taking into account clinical symptoms as well as all results from diagnostic procedures, including cytomorphology, flow cytometry, cytogenetics, molecular genetics and immunohistochemistry, if available. In order to perform an internal validation of our proposed score, we divided the patient cohort into an exploratory and a validation cohort by a 2:1 ratio based on the date of receipt of the samples. Result Flow cytometry data of 371 patients with B-cell disorders were available for analysis. 247 patients were assigned to the exploratory cohort and 124 patients were assigned to the validation cohort. 84.2% and 82.1% of patients, respectively, were diagnosed with CLL. In the exploratory cohort, sIgM-expression intensity on CD19+ B-cells (as measured by MFI ratio) was significantly lower in CLL versus non-CLL cases (p=0.001). However, low or absent sIgM-expression displayed poor specificity in distinguishing CLL from non-CLL cases (51,3%; sensitivity 83,7%). Absent or low CD79b-expression on CD19+ B-cells showed a higher sensitivity and specificity (94.2% and 71.8%, respectively). Positivity for CD200 as well as lack of FMC7-expression showed high diagnostic value (sensitivity and specificity all above 80%). Interestingly, positivity for CD5 on CD19+ B-cells did not have a strong diagnostic value (sensitivity and specificity 69.7% and 76.9%, respectively), but double positivity for CD5 and CD23 on CD19+ B-cells showed higher sensitivity and specificity (79.8% and 87.2%, respectively). Therefore, CD200+, CD23+/CD5+, FMC7- and low or absent CD79b on CD19+ B-cells were included in a new diagnostic score. The resulting score showed comparable sensitivity (97.1% for our score versus 98.6% for the Matutes score, McNemar’s test p=0.38), but markedly increased specificity (87.2% versus 53.8%, p<0.001). These results were confirmed in the internal validation cohort (sensitivity 97.0% versus 100%, p=N/A; specificity 86.4% versus 59.1%, p=0.03). Conclusion The data support the use of the improved score for the differential diagnosis of CLL. This novel scoring system exhibits significantly higher specificity while maintaining very high sensitivity and might therefore contribute to less false positive results. Finally, the surface markers contained in the novel score show more consistent staining patterns, which might further improve reproducibility. External validation of the proposed score will be pursued. Disclosures: No relevant conflicts of interest to declare.

Demylinating Peripheral Neuropathy and Lymphoplasmacytic Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5090-5090
Author(s):  
Carol Ann Huff ◽  
Vinay Chaudhry ◽  
Charlotte Sumner ◽  
David Cornblath
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Neuropathy ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Demyelinating Polyneuropathy ◽  
Lymphoplasmacytic Lymphoma ◽  
Modest Improvement ◽  
Bone Marrow Biopsies

Abstract Abstract 5090 Lymphoplasmacytic lymphoma (LPL) is a neoplasm of small B lymphocytes, plasmacytoid lymphocytes, and plasma cells, which does not fulfill criteria for any of the other small B-cell lymphoid neoplasms (WHO 2008). Neuropathy has been described in association with Waldenstrom's macroglobulinemia, but less so with LPL. We present 7 cases of neuropathy and LPL to highlight the variable presentations. 1) A 56 year old man developed sensory ataxia with an IgM kappa non-MAG paraprotein. CSF protein was 97. Stabilized on MMF, he slowly worsened. Plasma exchange (PE) was given with improvement. Bone marrow (BM) 4 years into his course revealed LPL. Rituximab was given, and his PE reduced. 2) A 49 year old woman developed progressive weakness with rapid decline in January 2010. NCS showed demyelinating polyneuropathy, and CSF protein was 179. An IgM kappa non-MAG paraprotein was found. BM was normal. PE was given with improvement but later prednisone, IVIg and rituximab twice did not help. Repeat BM revealed 2% clonal CD20+ CD5negCD10neg B cells by flow cytometry. PE was given with modest improvement. Cyclophosphamide 1 gm/m2 monthly was given with improvement. 3) A 53 year old man noted imbalance and distal weakness. NCS showed absent SAPs and prolonged distal and F wave latencies. An anti-MAG positive, IgM kappa paraprotein was found. In 2002 BM was normal. He received rituximab weekly × 4 doses. 7 years later, he developed anemia and worsening neuropathy. Repeat BM showed 0.5% CD20+CD5negCD10neg kappa-restricted B cells by flow cytometry and weekly rituximab was reinitiated. 4) A 62 year old man developed weakness and areflexia. NCS showed asymmetric demyelinating polyneuropathy. Biclonal gammopathy of IgM kappa and IgG kappa was found. BM showed LPL by histopathology. Prednisone was given with improvement. Later two courses of weekly rituximab were given. 5) A 55 year old woman developed asymmetric weakness. NCS showed asymmetric demyelinating polyneuropathy. MRI showed enlargement, abnormal signal intensity, and abnormal enhancement of bilateral radial, median, and ulnar nerves. She was found to have an IgG kappa paraprotein and LPL on BM biopsy. She was treated with rituximab, cyclophosphamide, fludarabine, and PE. In each case, the primary feature driving the need for therapy was the neuropathy and not the underlying hematologic process. Further, worsening neuropathy in 3 cases led to repeat bone marrow biopsies revealing a clonal B cell process and a diagnosis of lymphoplasmacytic lymphoma. Thus, in the presence of an IgM monoclonal gammopathy and peripheral neuropathy, we suggest bone marrow examination for LPL and then consideration of therapy directed toward the abnormal B cell clone. Disclosures: No relevant conflicts of interest to declare.

CD200 has an important role in the differential diagnosis of mature B-cell neoplasms by multiparameter flow cytometry

2013 ◽  
pp. n/a-n/a ◽  
Author(s):  
Alex F. Sandes ◽  
Maria de Lourdes Chauffaille ◽  
Cláudia Regina M.C. Oliveira ◽  
Yumi Maekawa ◽  
Nair Tamashiro ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cell ◽  
Multiparameter Flow Cytometry

Junctional Adhesion Molecule C (JAM-C) Constitutes a New Marker for the Differential Diagnosis of B Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3940-3940
Author(s):  
Thomas Matthes ◽  
Christiane Ody ◽  
Beat Imhof ◽  
Carmen Donate ◽  
Dominique Cossali ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Marginal Zone ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
B Cell Lymphomas

Abstract Abstract 3940 Poster Board III-876 Introduction Differentiation of naïve B cells into plasma cells or memory cells occurs in the germinal centres (GC) of lymph follicles or alternatively in the marginal zone via a GC- and T cell independent pathway. It is currently assumed that B cell lymphomas correspond to normal B cell differentiation stages, but the precise correlation of several B cell lymphomas to these two pathways remains controversial. We have previously shown that junctional adhesion molecule C (JAM-C) originally identified at the cell-cell border of endothelial cells, constitutes also a marker of B lymphocytes with a tightly regulated expression during B cell differentiation: immature B cells, GC-B cells and plasma cells stain negatively, whereas mature, memory and marginal zone derived B cells stain strongly positive. Here we test the expression of JAM-C on a series of patients with B cell lymphomas. Methods B lymphocytes from the peripheral blood of 158 untreated patients were analyzed using flow cytometry with standard antibody panels (CD5, CD10, CD11c, CD22, CD23, CD25, CD38, CD103, FMC7, sIg). Diagnosis of a B cell lymphoma was established according to WHO guidelines, using additionally RT-PCR, karyotyping, or FISH, if necessary. Expression of JAM-C was studied by flow cytometry with a polyclonal antibody obtained from a rabbit immunized with the soluble JAM-C molecule. Results MCL, HCL and MZBL with a supposed origin in the marginal zone stained mostly positive, whereas CLL and FL with a supposed origin in the germinal centre showed mostly a negative staining. No correlation was found in CLL between JAM-C expression and staining for ZAP70 or CD38. In 12 cases routine work-up was not able to precisely establish a diagnosis of CLL or MZBL, and CLL or MCL. In these cases the presence of JAM-C was considered a strong argument against a GC-origin of the malignant B cells. Addition of JAM-C to antibodies used in the Matutes score increased the sensitivity and specificity of this score for the diagnosis of CLL. Furthermore, it may help differentiating MZBL from LPL which otherwise display overlapping immunophenotypes. Conclusion JAM-C constitutes a new diagnostic marker for the differential diagnosis of B cell lymphomas, and is particularly useful for the distinction between CLL and LPL (negative staining) on the one hand and mantle cell and marginal zone B cell lymphomas (positive staining) on the other hand. Disclosures: No relevant conflicts of interest to declare.

Dominance of Non-CLL Phenotype of Monoclonal B-Cell Lymphocytosis (MBL) in the Middle East and An Overall MBL Prevalence Comparable to Western Countries

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4586-4586
Author(s):  
Maher Albitar ◽  
Faisal Rawas ◽  
Randa Nounou ◽  
Nasir Bakshi ◽  
Fahed Almhareb ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Middle East ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Light Chain ◽  
Peripheral Blood ◽  
Color Flow ◽  
Western Countries ◽  
Two Populations

Abstract Abstract 4586 Chronic lymphocytic leukemia (CLL) is considered more common in Western countries and its incidence is believed to decrease moving east across the globe. CLL is believed to be less common in the Middle East compared to Western countries, although reliable statistical data is not available. Therefore, monoclonal B-cell lymphocytosis (MBL), a pre-CLL condition, is expected to be less common in non-Western countries. In Western countries, MBL prevalence as detected by flow cytometry varies from 0.6% to 14% in healthy individuals older than 40 years, dependent on the level of sensitivity and number of parameters applied. Using 4 to 6 color flow cytometry, most studies report a prevalence of approximately 5 % in the Western population with the CLL-phenotype about 5 times more prevalent than the non-CLL phenotype. MBL incidence and relative proportions of CLL-phenotype versus non-CLL phenotype have not been adequately studied in non-Western countries. We investigated the prevalence and phenotype of MBL in a population sample in the Middle East. Method: 365 individuals, mostly Saudi Arabian nationals and a smaller number of individuals from neighboring countries, aged over 50 years with normal peripheral blood counts and no evidence of hematologic disease. Peripheral blood samples were immunophenotyped by 8-color flow cytometry detecting CD45, CD19, CD20, CD5, CD10, CD3, kappa and lambda light chains, based on acquiring approximately 1 million cells each. Result: Monoclonal B-cells were detected in 21 (6%) individuals (14 male, 7 female, median age 70, range 64–91). However, only 10 cases (48%) displayed the typical CD19+/CD5+ CLL-phenotype. Two cases (9.5%) were CD5-negative clonal B-cells, and 2 (9.5%) were CD10+ clonal B-cells. The remaining 7 cases (33%) showed concomitantly a CD5+ and a CD10+ clonal population, both expressing the same light chain. While we cannot be certain if these CD5+ and CD10+ cell populations represent the same or different clones, the finding that the two populations in all 7 cases showed the same light chain restriction supports the assumption that the two populations represent the same clone. Conclusion: MBL in the Middle Eastern region observed in this study is as common as reported in the Western world. In contrast to Western countries, however, it is the non-CLL phenotype which is more prevelant, comprising 52% of our MBL group and most of these cases show cells expressing CD5 and cells expressing CD10. The exact classification of these cases is difficult. It seems likely that these cases represent marginal zone phenotype, but the possibility of a coexisting follicular lymphoma clone cannot be excluded. Pure follicular lymphoma phenotype is seen in 10% of our MBL cases. Further studies with long follow-up are needed. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Surface Light Chain Expression in Primary Mediastinal Large B-Cell Lymphomas by Multiparameter Flow Cytometry

2015 ◽  
Vol 144 (4) ◽  
pp. 635-641 ◽  
Author(s):  
Olga K. Weinberg ◽  
Scott J. Rodig ◽  
Olga Pozdnyakova ◽  
Li Ren ◽  
Daniel A. Arber ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Light Chain ◽  
Multiparameter Flow Cytometry ◽  
B Cell Lymphomas ◽  
Large B Cell
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