scholarly journals Inability of Monoclonal Anti-light Chain Antibody to Detect Clonal B-Cells in a Patient with Follicular Lymphoma by Multicolor Flow Cytometry

2014 ◽  
Vol 28 (6) ◽  
pp. 493-495
Author(s):  
Jeroen F. van Velzen ◽  
Dorine van den Blink ◽  
Ingrid E.H. Wiegers ◽  
Andries C. Bloem
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Light Chain ◽  
Multicolor Flow Cytometry ◽  
Light Chain Antibody

Flow Cytometric Expression of bcl-2 by CD10+ B-Cells Reliably Distinguishes Follicular Lymphoma from Follicular Hyperplasia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4551-4551
Author(s):  
Zahid Kaleem ◽  
Ronald L. Leonard
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Light Chain ◽  
Predictive Value ◽  
Expression Patterns ◽  
Follicular Hyperplasia ◽  
Biopsy Specimens ◽  
Normal Expression ◽  
Significant Expression

Abstract The diagnosis of follicular lymphoma (FL) is often straightforward in most biopsy specimens. In a minority of cases, however, a differentiation from follicular hyperplasia (FH) can be challenging, especially in small needle core biopsies. The difficulty can be compounded in cases of FL with non-diagnostic immunoglobulin light chain ratios and cases of lymphoma without any significant surface expression of either immunoglobulin kappa or lambda light chain. The expression of bcl-2 by follicle center B-cells is often used to distinguish FL from FH using immunohistochemistry. The interpretation of bcl-2 expression patterns by immunohistochemistry, however, can be difficult because of normal expression of bcl-2 by T-cells present in reactive lymphoid follicles and by normal expression of bcl-2 by mantle zone B-cells. We have utilized multiparameter flow cytometry to evaluate the expression of bcl-2 in cases of FL and FH with successful results. This study includes consecutive biopsy specimens from well-documented cases of FL (n = 12) and FH (n = 16). Other lymphoma types were not included. Cytoplasmic expression of bcl-2 was evaluated on CD10+CD19+ B-cells using permeabilization techniques on a four-color Beckman-Coulter Epics-XL flow cytometer. Appropriate controls were run in each case. All cases of CD10-expressing FL showed significant expression of bcl-2 by CD10+ B-cells in contrast to no significant expression of bcl-2 by CD10+ follicle center B-cells of reactive follicles (Positive predictive value, 100%; Negative predictive value, 100%). Although our patient population is small, the results indicate that the expression of cytoplasmic bcl-2 on CD10+ B-cells by flow cytometry reliably distingushes FL from FH using CD10 gating. This method is superior to immunohistochemical evaluation of bcl-2 in cases with CD10 expression. A minority of FL, however, do not show bcl-2 expression (not seen in our series) and such cases must be taken into consideration when evaluating a clonal B-cell population with CD10 but no bcl-2 expression. DIAGNOSIS CD45+CD19+(%) CD45+CD19+CD10+(%) bcl2+(%) in CD10+ gate FL grade 1/3 65 65 38 FL grade 1/3 68 44 67 FL grade 1/3 72 72 99 FL grade 1/3 88 62 99 FL grade 2/3 72 68 99 FL grade 1/3 59 59 95 FL grade 1/3 96 84 94 FL grade 1/3 59 59 95 FL grade 1/3 45 38 99 FL grade 2/3 76 74 76 FL grade 1/3 54 47 90 FL grade 2/3 35 34 98

The Tumor Microenvironment Measured by Flow Cytometry Predicts Overall Survival (OS) and Transformation Risk (TR) in Follicular Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2406-2406 ◽  
Author(s):  
Pedro Farinha ◽  
John Han ◽  
Abdulwahab Al-Tourah ◽  
Joseph M. Connors ◽  
Diponkar Banerjee ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Light Chain ◽  
Clinical Event ◽  
Light Chains ◽  
Biological Variables ◽  
The Impact

Abstract Background: FL is an indolent but heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma is a dominant clinical event that is frequently followed by shorter survival. There is no consistent biological prognostic marker for TR. Recent studies have highlighted the role of the microenvironment in helping to determine the prognosis of FL. However, its impact on TR is largely unknown. In this study we used diagnostic flow cytometry (FC) analysis to assess the role of non-malignant cells in determining OS and TR in FL. Methods: We identified 567 patients with FL diagnosed at the BCCA over a 5-year period between 1997 and 2001. Of these, 270 cases had diagnostic FC and histological review, of which 137 were nodal and had complete clinical data in our electronic database. FC results were re-analyzed. The antibodies studied included anti-CD3, CD4, CD5, CD8, CD10, CD14, CD19, CD20, CD23, CD45, FMC-7 and IG kappa and lambda light chains. To ensure that biopsies were representative, the sum of the % gated events for CD3 and CD20 had to equal 100% +/− 20%. Light chain restriction was present in all cases and established clonality. The estimate of neoplastic B cells was determined by examining CD19 and CD20 frequency and the ratio of clonal light chain vs non-clonal light chain. Non-neoplastic B cells were estimated using CD19/20 and the amount of non-clonal light chain × 0.5 (λ clonal) or × 2 (κ clonal). Clinical characteristics, different subsets of T cells and the cell content of reactive, non-neoplastic B cells were evaluated using SPSS® software. Results: The median age of the 137 patients was 57 years, 51.8% were male and 38.6% had a high IPI (4/5). There were 97 grade 1, 27 grade 2 and 13 grade 3a FL. The median ratio of CD4/CD8 was 4.4. Patients were given a variety of treatments, including observation if asymptomatic, precluding an analysis of progression-free survival. The median follow-up of the living patients was 5.8 years and the estimated 5-year OS and TR were 70% and 20%, respectively. The IPI was predictive of OS (p<0.0001). Two biological variables showed a significant impact on survival. Firstly, cases in which CD8+ cells represented more than 25% of the total (CD3+) T cells had shorter OS (p = 0.028) and increased TR (p = 0.013). The CD8 ratio (p = 0.026) affected OS independently of IPI (p = 0.046). Secondly, cases with a low content of reactive, non-neoplastic B cells, defined by the ratio between IG light chains >1/15 had shorter OS (p = 0.003) and increased TR (p=0.002). The impact of a reduction in normal reactive B cells (p = 0.014) on transformation risk was independent of the IPI (p = 0.05). Conclusion: Two features of the FL microenvironment studied by diagnostic FC demonstrated an impact on prognosis. The proportion of CD8+ T cells relative to the total T cells and the number of residual, non-neoplastic B cells were both predictors of OS. Importantly, both predict, independently of the IPI, the risk of transformation. These biomarkers are easily measured and may be used to better stratify patients, choose initial treatment options and predict transformation risk in patients with FL. Microenvironment & Transformation Risk in Follicular Lymphoma Microenvironment & Transformation Risk in Follicular Lymphoma

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.

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.

Signaling Diversity in Human Lymphoma B Cells and in Tumor Infiltrating T Cells Correlates with Follicular Lymphoma Patient Clinical Outcomes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 377-377
Author(s):  
Jonathan M. Irish ◽  
Roch Houot ◽  
June H. Myklebust ◽  
Debra K. Czerwinski ◽  
Garry P. Nolan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Bcr Signaling ◽  
Human Lymphoma ◽  
Group 1

Abstract Introduction: Flow cytometry analysis of live cells from cryopreserved tumor samples provides the opportunity to study simultaneously the biology of both cancer cells and the patient’s immune system cells. Furthermore, single cell based approaches can identify and quantify subsets within mixed populations, such as heterogeneous primary tumors, without the need to physically separate cells. We have previously used flow cytometry to identify differences in signaling mechanism between healthy human B cells from two differentiation stages and to compare signaling kinetics of tumor and non-malignant B cells within primary human lymphoma specimens1. Methods: Here we used barcoded phospho-specific flow cytometry to measure signaling events in live lymphoma B cells and tumor-infiltrating T cells from follicular lymphoma (FL) tumor samples obtained before patients received therapy. Patients were then stratified according to signaling and clinical outcome was examined within the resulting groups. We examined response to initial chemotherapy, which was a combination of cyclophosphamide, vincristine, and prednisone (cvp), and overall survival, measured as the time from diagnosis to last follow up or mortality. Results: Differences in B cell receptor (BCR) signaling strength and kinetics characterized previously unappreciated diversity within the lymphoma B cell population. In some cases, BCR crosslinking triggered robust phosphorylation of AKT and ERK in one subset of lymphoma cells, while in another lymphoma subset within the same tumor sample, BCR crosslinking did not lead to phosphorylation of either protein. In these cases, both lymphoma B cell subsets expressed BCL2 and surface immunoglobulin heavy and light chain restricted to the tumor isotype. Patients whose lymphoma tumor contained a BCR insensitive cell subset (Group 2) had significantly worse responses to cvp therapy (p = 0.001) and lower overall survival (p = 0.003) than patients whose lymphoma cells displayed more homogeneous BCR signaling (Group 1). We next examined tumor infiltrating T cell signaling in the FL cases from Group 1, where no significant BCR insensitive subset was observed. Within Group 1, differences in the magnitude of IL-2, IL-7, and IL-15 mediated STAT5 phosphorylation in tumor infiltrating T cells further distinguished a set of patients with significantly higher overall survival (p = 0.04). Conclusions: These results identify BCR signaling in lymphoma B cells and cytokine signaling in tumor infiltrating T cells as clinically relevant biomarkers for tracking and isolating lymphoma cell subsets and for monitoring immune system activity during therapy. By following patients over time, we can now determine whether cell intrinsic signaling diversity enables the emergence of therapy insensitive cancer cell subsets.

Proliferative Activity Of Neoplastic B Cells - Impact On The Prognosis Of Follicular Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4321-4321
Author(s):  
Dulcineia Pereira ◽  
Sergio Pereira Chacim ◽  
Margarida dantas Brito ◽  
Miguel Abreu ◽  
Edgar Mesquita ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Proliferative Activity ◽  
Cox Regression ◽  
Conflicts Of Interest ◽  
Cox Model ◽  
S Phase ◽  
Cox Regression Analysis ◽  
Cytogenetic Changes

Abstract Introdution Follicular Lymphoma (FL) presents a recurrent clinical behavior. The proliferative activity of B-cells in NHL subtypes varies amongst different subtypes, being higher in high grade NHL. In FL without t(14; 18), high proliferative activity appears to be a better predictor of progression free survival (PFS). Objectives Evaluation of proliferative activity of neoplastic B cells in patients with FL and its association with underlying cytogenetic changes (such as t(14;18)). Determination of the prognostic value of proliferative activity of neoplastic B cells and t(14, 18) in patients with FL. Methods Retrospective analysis of 139 patients with FL, followed in our center from 2006 to 2011. Evaluation of the cytogenetic changes and percentage of cells in S phase at the time of diagnosis, performed on lymph nodes. The cell cycle analysis was evaluated by flow cytometry, resorting to DNA labeling kit B-NHL (Cytognos) ModFit software (Verity). The adopted cut-off of S phase cells was based on the median value. The type of response was defined in accordance with the criteria proposed by the NCCN. The patients´ characteristics were compared with a X2 test for binary variables and a Mann-Whitney test for continuous variables. The survival curves were estimated based Kaplan-Meier curves and the data for the various groups were compared with a log-rank test. The multivariate analysis was carried out using a Cox model, after the proportional hazard assumption was checked. A p value below 0.05 was considered as being statistically significant. Results The median follow-up was 34 months ([8-75]). The S phase was analysed in 45 patients with an median age of 56 years ([31-78]). 89% (n = 40) of the patients presented an Ann Arbor stage III-IV, 86.7% (n = 39) a follicular pattern, 89% (n = 40) a grade 1/2 and 57.8% (n = 26) a FLIPI ≥ 3. The t(14;18) was found in 25 patients (55.6%), in 9 patients cytogenetic analyses was not performed. In this study was given immunochemotherapy to 82.2% (n = 37) patients. Thirty patients (66.7%) achieved CR and 15.6% (n = 7) PR, with a PFS of 22 months in 53.3% of patients. In univariate Cox regression analysis, the histological pattern, the number of S-phase cells and the t(14;18) are independent predictors of PFS (p<0.05). Quantification of S-phase cells was done in 86.7% (n = 39) patients. The PFS was better in patients with S ≥ 2.3%, in comparison to patients with S< 2.3% (median 38 vs. 28 months, p = 0.03), without impact in overall survival (p = 0.67). Patients without t(14;18) presented better PFS (p = 0.008) and higher proliferative activity (54.5% S ≥ 2.3% vs. 36.4% S< 2.3%). Analysing the number of S-phase cells in relation to the presence or absence of t(14; 18), it was noted that patients with S ≥ 2.3% and without t(14; 18) (n = 14) have better PFS (p = 0.004). Conclusion Quantification of cells in S phase in FL, by flow cytometry, arises as a simple, fast and reproducible method with a prognosis impact on PFS. Cells in S phase ≥ 2.3% and absence of t(14;18) at diagnosis are associated with reduced risk of progression (p<0.05). Disclosures: No relevant conflicts of interest to declare.

Immunoglobulin Heavy/Light Chain Immunoassays for Response Evaluation in Multiple Myeloma: Comparison with Immunofixation, Serum Free Light Chain, and Multicolor Flow- Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3366-3366 ◽  
Author(s):  
Yasuhito Suehara ◽  
Keisuke Seike ◽  
Kouta Fukumoto ◽  
Manabu Fujisawa ◽  
Masafumi Fukaya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Free Light Chain ◽  
Multicolor Flow Cytometry ◽  
Serum Free ◽  
Serum Free Light Chain ◽  
Normal Ranges ◽  
Best Response

Abstract BACKGROUND: Monitoring of multiple myeloma (MM) patients is required to assess and determine the treatment response. The serum immunoglobulin (Ig) heavy/light chain immunoassays are a new method that enables separate quantification of the different light chain types of each Ig class, i.e., IgGk, IgGl, IgAk, and IgAl. Although the contribution of these tests to disease evaluation has been assessed, available data are still limited. Here, we compared the serum Ig heavy/light chain immunoassays with the conventional methods for disease evaluation. PATIENTS AND METHODS: Three hundred and three samples were obtained from a total of 101 patients with IgG and IgA type MM recruited at Kameda Medical Center and Kanazawa University Hospital. The median age of the patients was 68 years (range: 44 – 89). The median follow-up was 36 months (range: 2.5 – 189.6). The proportions of patients in International Scoring System (ISS) stages I, II, and III were 25%, 35%, and 40%, respectively. Paraprotein type was IgG in 64 patients (44 Gk, 20 Gl) and IgA in 37 (20 Ak, 17 Al). Samples were taken at various times after treatment and analyzed retrospectively with serum Ig heavy/light chain immunoassays (Hevylite®; Binding Site, Birmingham, UK) on a SPAPLUS® turbidimeter. The heavy/light chain ratio (HLCR) was calculated with the Ig' k (either G or A) as the numerator and compared to normal ranges (IgGk:3.84-12.07g/L; IgGl:1.91-6.74g/L; IgGk/IgGl: 1.12-3.21; IgAk:0.57-2.08g/L; IgAl:0.44-2.04g/L; IgAk/IgAl:0.78-1.94). Results were compared to serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), and serum free light chain immunoassays (FLC). HLC-pair suppression was defined as the uninvolved immunoglobulin of the same isotype 50% below the lower limit of the normal range (IgGk, IgGl, IgAk, IgAl). Residual neoplastic plasma cells (MM-PCs) in bone marrow samples were assessed by multicolor flow cytometry (MFC) simultaneously in 140 samples from 64 patients at very good partial response (VGPR), complete response (CR), and stringent CR (sCR). The MFC negativity was defined at a level of < 10-4 MM-PCs. Overall survival (OS) and progression-free survival (PFS) were estimated by the Kaplan–Meier method, and survival was compared using the log rank test. RESULTS: Myeloma responses were assessed serially after induction therapy and were assigned according to international response criteria. Patients' samples at various responses were: sCR, n = 86 (28%); CR, n = 31 (10%); VGPR, n = 116 (38%); and PR, n = 70 (23%). Normal HLCR was obtained at sCR, CR, and VGPR in 73 (85%), 28 (90%), and 69 (59%) cases, respectively. Discordance in the depth of response between standard electrophoretic methods and HLCR was more commonly seen in IgG compared to IgA MM; possibly reflecting the dose dependent half life of IgG immunoglobulins. In the sera from patients who achieved a CR or better, abnormal HLCR and FLC ratio were seen at rates of 12.8% and 26.5%, respectively. Discordance between normalization of HLCR and FLC ratio was seen in 42% of patients with a CR or better; however, a good correlation between normalization of HLCR and FLC ratio was still observed (Fisher's exact test, P = 0.004). Among the 71 sera from patients with a CR or better in which simultaneous MFC data were available, 16 (22.5%) sera were MFC-negative. Among the patients who achieved a VGPR or better, patients with a normal HLCR had fewer MM-PCs than those with an abnormal HLCR (median 9.8x10-4vs. 46.0x10-4, respectively, P=0.062), although the difference was not statistically significant. Abnormal HLCR in CR samples was seen more frequently in patients with IgA type (19%) than IgG type (7.7%). Longer PFS and OS were observed in patients who achieved HLCR normalization at best response than in those who did not (PFS; 83.8 months vs. 41.8 month, respectively, P = 0.016 and OS; not reached vs. not reached, P = 0.018). When patients at best response were divided into with or without uninvolved HLC pair suppression, the latter group showed significantly better OS compared to the former group (not reached vs. not reached, P=0.019). CONCLUSIONS: The findings presented here suggest the potential usefulness of heavy/light chain immunoassays for monitoring of myeloma response in patients with IgA myeloma and residual MM-PC assessment at VGPR or better. Presence of abnormal HLCR at best response and HLC pair suppression were also associated with poorer survival. Disclosures No relevant conflicts of interest to declare.

scholarly journals Aberrant expression of CD54 detected by flow cytometry is a characteristic of B-lymphoma cells in bone marrow specimens

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wei Wang ◽  
Yan Li ◽  
Haval Ali ◽  
Linjun Zhao ◽  
Di Mei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Differentiation ◽  
B Cells ◽  
Plasma Cell ◽  
Light Chain ◽  
Lymphoma Cells ◽  
Aberrant Expression ◽  
Light Chain Restriction ◽  
The Mean

Abstract Background Flow cytometry (FC) is a popular method to detect bone marrow (BM) involvement in patients with B-cell non-Hodgkin lymphoma (B-NHL). The majority of screen panels of FC still rely on finding monoclonal B-cells, e.g., B-cells with immunoglobin (Ig) light-chain restriction, which has many limitations. Therefore, exploring new markers is warranted. Methods A total of 52 cases of B-NHL with BM involvement were collected. The median age was 60 years. Out of these 52 cases, 34 were male, and 18 were female. A 10-color FC panel was used to detect the expression of CD54 on lymphoma cells. The expression of CD54 was calculated as the mean fluorescence index ratio (MFIR) and was described as the mean ± standard error of the mean (SEM). Results Up to 18/52 (34.62%) of BM specimens abnormally expressed an increased level of CD54, including 1/10 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 9/13 cases of mantle cell lymphoma (MCL), 2/14 cases of follicular lymphoma (FL), 5/9 cases of marginal zone lymphoma (MZL), and 1/3 cases of high-grade B-NHL (HG B-NHL). The expression level of CD54 was significantly increased in MCL cases (53.41 ± 11.04) compared with CLL/SLL cases (11.66 ± 2.79) and FL cases (13.49 ± 2.81). The lowest percentage of CD54-positive B-cells attained 0.13%. In 5/9 cases of MZL and 1/3 cases of HG B-NHL, increased expression of CD54 was the only abnormal immunophenotype detected besides Ig light-chain restriction. No aberrant CD54 expression was identified by FC in lymphoplasmacytic lymphoma (LPL) (0/2) and Burkitt lymphoma (BL) (0/1) cases. Aberrant expression of CD54 was not related to plasma cell differentiation. Conclusion Lymphoma cells, especially in MCL and MZL cases, frequently show increased expression of CD54. Such aberrant expression is not related to plasma cell differentiation. We highly recommend adding CD54 to the FC screening panel to detect BM involvement in patients with B-NHL.

CD5-Negative Monoclonal B-Cell Lymphocytosis (MBL) Is Detectable In 9% of Adults Aged Over 40 but Phenotypes Associated with Clinically Common Non-Hodgkin Lymphomas Are Infrequent (<2%)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2002-2002
Author(s):  
Andy C Rawstron ◽  
Chi Doughty ◽  
Ruth M de Tute ◽  
Fiona Bennett ◽  
Selina Denman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
General Population ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Peripheral Blood ◽  
Marginal Zone ◽  
Residual Disease ◽  
High Sensitivity ◽  
Marginal Zone Lymphoma

Abstract Abstract 2002 Introduction: Monoclonal B-cells with a Chronic Lymphocytic Leukemia (CLL) phenotype are detectable in more than 10% of adults in the general population using high sensitivity flow cytometry assays designed to detect minimal residual disease after treatment. However, the prevalence of MBL with a phenotype corresponding to other B-lymphoproliferative disorders (B-LPD) is less than 2% of the general population even using the most sensitive assays. No studies have reported CD10+ MBL whereas several studies have demonstrated that the t(14;18) is frequently detectable in the general population at a level which should be detectable by flow cytometry. The lack of CD10+ MBL may indicate that the t(14;18) alone rarely results in the expansion of a clonal B-cell population in the blood, or that currently available assays are inadequate for detecting circulating follicular lymphoma. We have previously investigated 66 markers to determine the best candidates for diagnosis and monitoring B-LPD, which were then tested in over 1500 cases. We have developed a single-tube assay to screen for residual disease that can detect lymphoma cells when they represent as few as 1 in 10,000 leucocytes. The aim of this study was to asses the frequency with which lymphoma-phenotype monoclonal B-cells are detected in the general population using a high sensitivity assay. Methods: Cells from 679 individuals (342 male, 337 female, median age 64, range 40–99) with a normal blood count and no current or prior history of cancer were incubated with antibodies to Kappa, Lambda, CD19, CD20, CD5, CD10, LAIR1, CXCR5 and 0.5 million cells were acquired using a BD FACSCanto II cytometer. In cases with detectable MBL further phenotyping was performed and B-cells were selected and stored for FISH and molecular clonality studies. Results: MBL was detected 129/679 cases (19.0%): CLL-type MBL in 86/679 cases (12.7%), non-CLL MBL in 60/679 cases (8.8%) with both CLL-type and non-CLL MBL were present in 17/679 cases (2.5%). Within the non-CLL MBL group, in 21/60 cases the monoclonal B cells had no additional features to confirm a neoplastic population and it was not possible to ascertain whether these were neoplastic cells or a reactive population with a highly skewed kappa/lambda ratio. Of the remaining 39 specimens: none showed evidence of germinal centre differentiation; 12 (1.7% of total) showed a phenotype most consistent with marginal zone lymphoma/lymphoplasmacytic lymphoma; and 27 cases expressed strong levels of LAIR1 coupled with strong CD19 and CD20 and an extended phenotype that is relatively rare in clinical B-LPD, restricted to hairy cell leukemia and a small proportion (<15%) of marginal zone lymphomas. Conclusions: Using an assay designed for detection of residual disease in lymphoma it was possible to detect non-CLL phenotype MBL in 8.8% of individuals over 60 years of age with normal blood counts and no history of cancer. Only a small number of these cases had a phenotype comparable to follicular lymphoma or marginal zone lymphoma, which constitute the majority of clinically significant indolent B-LPD. The complete absence of cases with a germinal centre phenotype contrasts with the high frequency of detection of BCL2-IGH rearrangement by PCR in the peripheral blood of healthy individuals. The flow cytometry assay used in this study readily detects circulating cells with appropriate phenotype in patients known to have follicular lymphoma, diffuse large B-cell lymphoma or marginal zone lymphoma/lymphoplasmacytic lymphoma and the data indicate a high specificity for peripheral blood based diagnosis and monitoring in these conditions. The most clinically prevalent lymphomas are rarely detectable in peripheral blood in individuals without lymphadenopathy but there is a surprisingly frequent development in the general population of both CLL and a restricted subset of marginal zone lymphomas which may indicate a common developmental pathway. Disclosures: No relevant conflicts of interest to declare.

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