Phenotyping Studies of Clonotypic B Lymphocytes From Patients With Multiple Myeloma by Flow Cytometry

2009 ◽  
Vol 133 (10) ◽  
pp. 1594-1599 ◽  
Author(s):  
E. Joseph Conway ◽  
Jianguo Wen ◽  
Yongdong Feng ◽  
Albert Mo ◽  
Wan-Ting Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
B Lymphocytes ◽  
Light Chain ◽  
Peripheral Blood ◽  
Remission Rate ◽  
Light Chains ◽  
Immunoglobulin Light Chain ◽  
Peripheral Blood Stem Cells ◽  
Myeloma Cells

Abstract Context.—Clonotypic B lymphocytes, monoclonal B lymphocytes sharing identical, rearranged IGH-CDR3 sequences with the patient's myeloma cells, have been detected in the peripheral blood of patients with multiple myeloma. These cells have been postulated to act as a therapy-resistant tumor reservoir that drives recurrence. Objective.—To characterize clonotypic B lymphocytes for future investigation of their role in myeloma pathogenesis. Design.—Harvests of cryopreserved peripheral blood stem-cells from 20 myeloma patients were enriched for clonotypic B lymphocytes. Cytoplasmic immunoglobulin light chain and surface immunophenotype were analyzed by flow cytometry. IGH-CDR3 gene-rearrangement pattern was performed to determine clonality. Posttransplant remission rate was compared with the percentage of clonotypic B lymphocytes. Results.—Clonotypic B lymphocytes expressing CD34±, CD38+, CD184+, CD31±, CD50±, CD138−, CD19−, CD20−, and the same immunoglobulin light chain as the patients' known myeloma cells were identified in 12 of 20 patients (60%). Progenitor B lymphocytes expressing similar surface immunophenotype but opposite light chains were identified in the same patients. Polymerase chain reaction for IGH rearrangement showed clonal rearrangement pattern in clonotypic lymphocytes but not in B lymphocytes expressing light chains opposite to myeloma cells. There was no statistically significant correlation between the percentage of clonotypic B lymphocytes and response to autologous transplant. Conclusions.—Clonotypic B lymphocytes expressing CD34, but not CD19, were identified in stem cell harvests from patients with myeloma and could represent progenitor cells of neoplastic plasma cells. However, the same or similar immunophenotyping can be detected in both clonotypic B lymphocytes and benign progenitor B cells, suggesting clonality analysis might be needed to determine clonotypic B lymphocytes in patients with myeloma. Further studies are warranted to study the role of clonotypic B lymphocytes in the pathogenesis of myeloma.

scholarly journals Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

ISRN Oncology ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-6
Author(s):  
Anders Ståhlberg ◽  
Pierre Åman ◽  
Linda Strömbom ◽  
Neven Zoric ◽  
Alfredo Diez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Real Time ◽  
B Lymphocytes ◽  
Light Chain ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Light Chains ◽  
Quantitative Real Time Pcr ◽  
Healthy Humans

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

Prognostic Value Of Quantifying Circulating Plasma Cells By Multiparametric Flow Cytometry In Patients With Relapsed Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 754-754
Author(s):  
Wilson I Gonsalves ◽  
Vinay Gupta ◽  
S. Vincent Rajkumar ◽  
William G Morice ◽  
Michael M Timm ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Research Funding ◽  
Prognostic Value ◽  
Plasma Cells ◽  
Light Chains ◽  
Plateau Phase ◽  
Multiparametric Flow Cytometry ◽  
Relapsed Disease

Abstract Background The presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. Methods We evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. Results There were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs. We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. Conclusion The utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

scholarly journals Human IgA1 half-molecules: clinical and immunologic features in a patient with multiple myeloma

Blood ◽  
1979 ◽  
Vol 53 (2) ◽  
pp. 269-278
Author(s):  
I Sakurabayashi ◽  
K Kin ◽  
T Kawai
Keyword(s):  
Molecular Weight ◽  
Multiple Myeloma ◽  
Light Chain ◽  
Enzymatic Degradation ◽  
Large Deletion ◽  
Light Chains ◽  
Myeloma Cells ◽  
Alpha Chain ◽  
Myeloma Proteins ◽  
Iga Production

Abnormal IgA1 half-molecules consisting of one heavy and one light chain were found in a patient (N.N.) with typical multiple myeloma. The serum and the urine of this patient contained both 7.0S and 3.9S IgA myeloma proteins. The IgA half-molecules (3.9S) were found to have a molecular weight of 59,000 daltons and were composed of one alpha1 chain of about 40,000 daltons and one light chain of 22,000 daltons. Furthermore, enzymatic degradation suggested that the alpha chain of the N.N. half-molecules had a large deletion in its Fc portion. We suggest that its heavy and light chains were probably bound noncovalently, since the interchains connecting the heavy and light chains of these IgA half-molecules were easily dissociated with 1% SDS and 8 M urea. Cytologic studies identified at least two types of myeloma cells, and it is possible that half-molecule IgA production might result from mutation among the myeloma cells producing whole- molecule IgA.

scholarly journals Human IgA1 half-molecules: clinical and immunologic features in a patient with multiple myeloma

Blood ◽  
1979 ◽  
Vol 53 (2) ◽  
pp. 269-278 ◽  
Author(s):  
I Sakurabayashi ◽  
K Kin ◽  
T Kawai
Keyword(s):  
Molecular Weight ◽  
Multiple Myeloma ◽  
Light Chain ◽  
Enzymatic Degradation ◽  
Large Deletion ◽  
Light Chains ◽  
Myeloma Cells ◽  
Alpha Chain ◽  
Myeloma Proteins ◽  
Iga Production

Abstract Abnormal IgA1 half-molecules consisting of one heavy and one light chain were found in a patient (N.N.) with typical multiple myeloma. The serum and the urine of this patient contained both 7.0S and 3.9S IgA myeloma proteins. The IgA half-molecules (3.9S) were found to have a molecular weight of 59,000 daltons and were composed of one alpha1 chain of about 40,000 daltons and one light chain of 22,000 daltons. Furthermore, enzymatic degradation suggested that the alpha chain of the N.N. half-molecules had a large deletion in its Fc portion. We suggest that its heavy and light chains were probably bound noncovalently, since the interchains connecting the heavy and light chains of these IgA half-molecules were easily dissociated with 1% SDS and 8 M urea. Cytologic studies identified at least two types of myeloma cells, and it is possible that half-molecule IgA production might result from mutation among the myeloma cells producing whole- molecule IgA.

Increased Circulating Plasma Cells On Multiparametric Flow Cytometry As An Independent Prognostic Biomarker In Newly Diagnosed Multiple Myeloma: Implications For Redefining High-Risk Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1842-1842
Author(s):  
Wilson I Gonsalves ◽  
Vinay Gupta ◽  
S. Vincent Rajkumar ◽  
William G Morice ◽  
Michael M Timm ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Overall Survival ◽  
High Risk ◽  
Peripheral Blood ◽  
Research Funding ◽  
Light Chains ◽  
Newly Diagnosed ◽  
Risk Status ◽  
Multiparametric Flow Cytometry

Abstract Background Prior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. Methods We evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Results There were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). Conclusion Quantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

scholarly journals Monoclonal anti-light chain idiotype as a tumor-specific probe for human neoplastic B lymphocytes

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 919-923 ◽  
Author(s):  
M Wrightham ◽  
AL Tutt ◽  
MJ Glennie ◽  
TJ Hamblin ◽  
GT Stevenson ◽  
...  
Keyword(s):  
B Cell ◽  
Tumor Cells ◽  
B Lymphocytes ◽  
Light Chain ◽  
Normal Serum ◽  
Lymphocytic Leukemia ◽  
Light Chains ◽  
Specific Probe ◽  
Tonsillar Cells ◽  
Binding Curves

Abstract Tumor cells from patients with B cell neoplasms often secrete small amounts of free monoclonal light chains that can be found in the urine. Such tumor-derived light chains of the lambda type from a patient with typical chronic lymphocytic leukemia have been used to raise mouse monoclonal antibodies (MoAbs). A hybridoma-secreting antibody that recognized the idiotypic lambda chain but not normal lambda chains by a preliminary screen but which also reacted with idiotypic IgM from the patient's tumor cells was selected. This MoAb in fact recognized 1 in 20 X 10(3) molecules of pooled normal lambda chains, thus establishing its specificity for a private idiotypic determinant. It failed to give a detectable reaction with normal IgM, normal serum, or a panel of IgM paraproteins. The antibody bound to the patient's neoplastic B cells but not to normal tonsillar cells. The site of binding of the antibody to idiotypic IgM is clearly separate from that of another MoAb specific for idiotypic determinants on heavy plus light chains, since the two showed additive binding curves. The determinant also appeared to be less available in dimeric lambda chains than in monomeric lambda chains or in idiotypic IgM. Antibodies to idiotypic determinants on light chains show some technical advantages and should be useful for monitoring and possibly treating B cell tumors, either alone or together with the more conventional anti-idiotypic antibodies that usually recognize the heavy and light chain combination.

Immunological phenotype of lymphomas induced by avian leukosis viruses

1983 ◽  
Vol 3 (6) ◽  
pp. 1077-1085
Author(s):  
L C Chen ◽  
S A Courtneidge ◽  
J M Bishop
Keyword(s):  
B Lymphocytes ◽  
Cell Lines ◽  
Light Chain ◽  
Avian Leukosis Virus ◽  
Immunoglobulin M ◽  
Light Chains ◽  
Free Light Chains ◽  
Viral Antigens ◽  
Immunological Phenotype ◽  
Avian Leukosis Viruses

The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that the cell lines established from lymphomas induced by avian leukosis virus represent relatively mature, but possibly abnormal, stages in the development of chicken B-lymphocytes. The immunoglobulin M produced by the cell lines failed to form detectable immune complexes with avian leukosis virus. It therefore appears that the immunoglobulin M is not directed against viral antigens and that autogenous antigenic stimulus cannot account for the sustained growth of the neoplastic B-lymphocytes.

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