CD79b Expression Research Articles

Downregulation of the B‐cell receptor signaling component CD79b in plasma cell myeloma: A possible post transcriptional regulation

The CD79 molecule, encoded by the CD79a and CD79b genes, is a signaling unit of the B-cell receptor complex, which transmits signals of B-cell activation, growth, and differentiation. They are B-cell-specific and expressed at most stages of B-cell development. Although plasma cells have been believed to lack these gene products, the regulation of CD79 expression in plasma cells is still controversial. In particular, the regulation of CD79b expression remains unclear. We sought to examine CD79b expression in normal and neoplastic plasma cells by immunohistochemical analysis. Out of the 23 clinical samples and 11 cell lines of plasma cell myeloma (PCM), none of the clinical samples and only 1 of 11 cell lines expressed CD79b immunohistologically, whereas non-neoplastic plasma cells in reactive hyperplastic lymph nodes exhibited loss of CD79b protein expression. This finding is quite different from our previous report on CD79a. Not only immunocytochemistry, but also RT-PCR and Western blot analysis of PCM cell lines gave identical results. Interestingly, we detected mRNA transcripts of CD79b in PCM cell lines, although protein translation was lacking. These findings suggest that expression of CD79b is downregulated in both plasma cells and plasma cell myeloma, and this process is possibly under post transcriptional regulation.

High CD79b Expression Predicts a Poor Outcome in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

[Display omitted] The immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form the signaling component of the B-cell antigen receptor complex (BCR). Given the critical role of Ig beta in BCR signaling, it has been suggested that its variable expression in CLL may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong expression of surface Ig, advanced clinical stage and short overall survival in B-CLL (Garcia Vela, 1999; Zucchetto, 2006). Moreover, high CD38 expression was correlated with high CD40, CD69 and CD79b which are consistent with an activation phenotype (Damle, 2002). Furthermore, we have demonstrated that the ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). Since it has been described that BCR engagement has significant effects on B-CLL cell survival, activation and cell cycle progression (Deglesne, 2006), we decided to evaluate the real impact of CD79b expression on B-CLL prognosis. The primary aims of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD79b expression; 2) whether CD79b could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally 3) whether CD79b was an independent prognostic factor. Therefore, we investigated 401 patients (pts), median age 65 years (range 33–89), 213 males and 188 females. With regard to modified Rai stages, 123 pts had a low stage, 261 an intermediate stage and 17 a high stage. CD79b was determined by multicolor flow cytometry, using the monoclonal antibody anti-CD79 beta-FITC (clone SN8, Dako) and fixing a cut-off value of 30%. CD79b+ B-CLL pts were 207/401 (52%). CD79b>30% was significantly associated with the intermediate/high Rai stage (p=0.00001), beta-2 microglobulin >2.2 mg/dl (p<0.0001) and with multiple intrathoracic/abdominal lymphadenopathies and/or splenomegaly (p<0.0001). Low CD79b was significantly correlated either with IgVH mutated status (>2%) (86/105; p<0.0001) or lower soluble CD23 levels (125/161; p<0.00001). Significant associations were found either between CD38<30% and lower CD79b (172/194; p<0.0001) or ZAP- 70<20% and CD79b<30% (145/193; p<0.0001). With regard to cytogenetics, there were strict correlations either between high CD79b and trisomy 12 (25/33; p=0.001) or high CD79b and del17p (16/20; p=0.001). Median follow up duration from diagnosis was 68 months (range 6–230). Concerning clinical outcome, both a shorter PFS (Figure) and OS were observed in CD79b+ pts (12% vs 58% at 16 years; p<0.0001 and 49% vs 85% at 16 years; p<0.0001) as well as in ZAP-70+ pts (5% vs 52% at 11 years; p<0.0001 and 33% vs 87% at 18 years; p<0.0001). To further explore the prognostic value of CD79b, we investigated its expression within ZAP-70+ (159 pts) and ZAP-70 negative (242 pts) subsets. As a matter of fact, CD79b+ pts showed a shorter PFS and OS both within the ZAP-70+ subset (7% vs 19% at 10 years; p=0.00006 and 25% vs 65% at 16 years; p=0.006) and within the ZAP-70 negative subset (29% vs 67% at 14 years, p<0.00001 and 86% vs 100% at 14 years; p=0.01). In multivariate analysis of PFS and OS, in which age, Rai stages, CD38, CD69, CD79b and ZAP-70 entered, ZAP-70 (p=0.001 and p=0.001), CD69 (p=0.001 and p=0.01) and CD79b (p=0.0007 and p=0.03) resulted to be independent prognostic factors. Therefore CD79b, easily determined by flow cytometry, should be considered another important prognostic parameter and could be used to early identify and stratify B-CLL progressive pts.

Development and Therapeutic Potential of an Anti-CD79b Antibody-Drug Conjugate, Anti-CD79b-Vc-MMAE, for the Treatment of Non-Hodgkin's Lymphoma

Antibody-drug conjugates (ADCs), antibodies linked to potent cytotoxic drugs via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. We have previously shown that ADCs targeted to CD79b are highly effective in xenograft models of non-Hodgkin's lymphoma. Here we report the development of an ADC consisting of a humanized anti-CD79b antibody (hu-anti-CD79b) conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABS) by a maleimidocaproyl-valinecitrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (hu-anti-CD79b-thioMAb-MC-vc-PAB-MMAE) that is designed to be cleaved by cathepsins. To determine the potential of this ADC, hu-anti-CD79b-thioMAb-MC-vc-PAB-MMAE (referred to as anti-CD79b-vcMMAE henceforth), as a therapeutic for NHL we interrogated its potency across a large panel of NHL cell lines. Strikingly, anti-CD79b-vcMMAE has very potent activity across a large panel of NHL cell lines in vitro with 68% (23 out of 34) cell lines having greater than 50% reduction in cell viability. Quantitative FACS across the cell line panel revealed that of the 11 insensitive cell lines, 9 had negligible surface CD79b, suggesting a threshold effect in that below a specific level of antigen on the cell surface resulted in cells being insensitive to anti-CD79b-vcMMAE. Within the sensitive cell lines, there was not a direct correlation per se with anti-CD79b-vcMMAE IC50 values and cell surface expression levels and this prompted us to investigate other potential molecular parameters. Gene expression profiling and gene set enrichment analysis revealed that genes predominantly involved in antigen processing and presentation and genes induced by IFN-gamma were significantly enriched in the less sensitive cell lines to CD79b-vcMMAE. Classifying our NHL cell lines as GCB or ABC subtypes by gene expression revealed that both ABC and GCB were responsive. In addition, the activity of anti-CD79b-vcMMAE was very potent in p53 mutant as well as p53 wild-type cell lines. Since the pre-clinical evidence suggests that anti-CD79b-vcMMAE will be a very promising drug candidate for NHL and we have established that cell surface expression is perhaps the best predictor of response, we wished to determine the prevalence of expression of CD79b on the cell surface of primary human lymphoma and CLL samples to estimate a patient population that may gain benefit. Strikingly, CD79b was detected in all cases of CLL, MZL, HCL, DLBCL, FL, and MCL. Furthermore, CD79b expression was detected in all cases that had relapsed from prior chemotherapy regimens, highlighting the clinical relevance of this target and potential therapeutic utility of anti-CD79b-vcMMAE. To assess the potential of anti-CD79b-vcMMAE in vivo we compared its efficacy to R-CHOP in three xenograft models of NHL. In all three models, a single dose of anti-CD79b-vcMMAE resulted in complete sustained tumor remission and was more effective than R-CHOP. These data suggest that anti-CD79b-vcMMAE could be broadly efficacious as a treatment for NHL.

CD21 Expression Is Inversely Correlated with the Ability of CLL Cells To Induce Tyrosine Phosphorylation in Response to Anti-IgM.

B-cell chronic lymphocytic leukaemia (CLL) cells have a differential capacity to signal through the B-cell receptor (BCR) following ligation with anti-IgM. This difference in signaling capacity has been suggested to contribute to the clinical disparity between the two subsets. However, as there appears to be no obvious correlation between response to IgM ligation and surface IgM expression, CD20 expression or CD79b expression it may be that the differential signaling response in these two distinct subsets is not caused by the BCR itself. In this study we investigated the expression of the BCR co-receptor complex (CD19, CD21 and CD81) in a cohort of CLL patients (n = 60) in order to determine whether expression of the co-receptor was associated with known CLL prognostic markers; IgVH gene mutational status, ZAP-70 and CD38 expression. In addition, we examined the relationship between co-receptor expression and the capacity of CLL cells to respond to anti-IgM cross-linking of the BCR. CLL samples showed significantly lower expression of both CD19 and CD21 when compared to normal B-lymphocytes (P <0.0001 and P<0.0001 respectively). In addition, samples derived from patients with unmutated IgVH genes showed significantly lower expression of CD19 and CD21 but not CD81 when compared to samples derived from patients with mutated VH genes (P = 0.04, P = 0.005 and P = 0.5 respectively). In contrast, none of the co-receptor molecules (CD19, CD21, CD81) were associated with CD38 expression (P = 0.82, P = 0.6, P = 0.64 respectively) or ZAP-70 expression (P = 0.2, P = 0.13 and P = 0.4 respectively). We next compared CD21 expression with levels of tyrosine phosphorylation (pre- and post-IgM ligation of the BCR). The results showed that there was no correlation between CD21 expression with either basal or IgM-stimulated tyrosine phosphorylation (P = 0.84 and P = 0.24 respectively). However, there was a significant inverse correlation with the percentage change in tyrosine phosphorylation following BCR cross-linking (P = 0.01). These data suggest that CLL cells with higher CD21 expression have a diminished magnitude of response to BCR ligation supporting the hypothesis that CLL cells with higher CD21 expression undergo receptor densitisation. Conversely, patients with lower CD21 expression may avoid receptor densitisation and consequently have a higher capacity for signaling following ligation of the BCR. Taken together, our data indicate a role for the BCR co-receptor in the signaling capacity of CLL cells and hence in the clinical course of the disease.

Heterogeneous intracellular expression of B‐cell receptor components in B‐cell chronic lymphocytic leukaemia (B‐CLL) cells and effects of CD79b gene transfer on surface immunoglobulin levels in a B‐CLL‐derived cell line

B-cell chronic lymphocytic leukaemia (B-CLL) cells display low amounts of surface immunoglobulins (sIg). To investigate the mechanisms underlying this phenomenon, we performed a thorough study of surface and intracellular expression of the B-cell receptor (BCR) components in B-CLL cells using flow cytometry. There was an heterogeneous pattern of expression. Overall, 20 of 22 samples showed reduced sIgM levels, compared with normal B cells. Among them, three (15%) had very low to undetectable intracellular IgM levels and variable amounts of CD79a and CD79b; nine (45%) had low intracellular CD79b levels but appreciable levels of IgM and CD79a; and eight (40%) had relatively normal intracellular levels of all BCR components. To investigate whether surface BCR levels could be controlled by the rate of CD79b synthesis, adenoviral vectors encoding CD79b were generated and used for gene transfer experiments. Delivery of CD79b to non-B cells transfected with IgM and CD79a lead to high-level expression of a functional BCR. Moreover, CD79b gene transfer in a B cell line derived from a B-CLL patient and characterised by low intracellular levels of endogenous CD79b consistently increased sIgM levels. These findings indicate that the phenotype of B-CLL cells in a subset of patients may depend primarily on poor CD79b expression, and suggest that upregulation of CD79b expression may correct the phenotype of these cells.

Gamma radiation-induced human B cell defects: Model for space flight

Rationale One of the most feared space flight conditions is radiation due to the potential to weaken the immune system. Leukocyte studies in space demonstrated a reduction in proliferative responses, alterations in signal transduction and cytokine production, along with reactivation of latent viruses. Utilizing our previously developed human B cell in vitro model, we present evidence of space-like gamma radiation-induced B cell defects. Methods CD40-activated purified human peripheral blood B cells were exposed to varying levels of gamma radiation (1-6 Gy) delivered by a 137Cs source. Determination of B cell responses and apoptotic processes were made by flow cytometric analysis, ELISA, colorometric, protein, and proliferative assays. Results Human B cells were significantly altered by 3 Gy gamma radiation (estimated radiation absorbed by humans on a two-year space journey). There was significant reduction in cell viability (40%), proliferation (70%), and CD79b (B cell receptor) expression (50%) ( p<0.01). B cell apoptosis was induced as evidenced by increased Annexin V binding (95%), pro-apoptotic receptor expression (Fas/CD95) (70%) and the anti-apoptotic oncogene (Bcl-2) (120%) ( p<0.01). At 6 Gy radiation, only 50% viable cells remained. As a first attempt countermeasure, the general caspase inhibitor, z-VAD-fmk, reversed CD95 expression while exerting no significant effect upon Bcl-2. Conclusion Exposure to gamma radiation significantly altered normal human B cell responses to defined ligands and induced enhanced apoptosis in B cells. Thus, these data will aid in the evaluation of the extremes of radiation that humans are likely to be exposed to in space exploration.

CD79b expression in chronic lymphocytic leukemia. Association with trisomy 12 and atypical immunophenotype

○ Context.-CD79b is a relatively newly characterized B-cell marker that is expressed in a minority of chronic lymphocytic leukemia (CLL) cases. Objective.-To systematically correlate CD79b expression with specific morphologic and immunophenotypic findings and trisomy 12. Design.-We assessed CD79b expression in 100 consecutively accrued CLL cases that were also analyzed by conventional cytogenetics. Based on the association between trisomy 12 and CD79b expression, we then assessed 43 additional CLL cases with trisomy 12. CD79b expression was correlated with morphology and expression of other immunophenotypic markers. Results.-Eighteen (18%) of 100 consecutively accrued cases were CD79b positive. No significant association was found between CD79b expression and atypical morphology. CD79b expression correlated with CD22 and FMC7 positivity. Eight (8%) cases had trisomy 12; 4 (50%) of these were CD79b positive, suggesting an association with trisomy 12. Examination of a second group of 51 CLL cases with trisomy 12 (including 8 cases from the initial study group) showed that CD79b was positive in 26 cases (49%), a frequency significantly higher than that of the consecutively accrued CLL cases without trisomy 12 (P <.05). Conclusions.-We conclude that CD79b immunoreactivity is positive in approximately 20% of CLL cases and that expression correlates with trisomy 12 and atypical immunophenotypic findings.

Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy

AbstractPrevious studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 104 to 105leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 × 109/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P = .0001) and overall survival (P = .007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.

CD79: a review.

CD79 is composed of CD79a and CD79b components expressed almost exclusively on B cells and B-cell neoplasms. CD79a and CD79b expression precedes immunoglobulin (Ig) heavy-chain gene rearrangement and CD20 expression during B-cell ontogeny and disappears later than CD20 in the late (plasma cell) stage of B-cell differentiation. Therefore, antibodies to CD79a and CD79b are useful in the differential diagnosis of B-cell neoplasms from T-cell neoplasms or myeloid neoplasms, or L and H lymphocyte predominance Hodgkin's lymphoma from classic Hodgkin's lymphoma. In addition, CD79a and CD79b antibodies are useful markers in the diagnosis of precursor B-acute lymphoblastic leukemia (pre-B-ALL) because many of these tumors are negative for other B-cell markers, such as CD20 and CD45RA. Furthermore, for B-cell neoplasms, wherein CD20 expression is aberrantly lost, such as in diffuse large B-cell lymphoma, or for B-cell neoplasms after CD20-antibody therapy, CD79a may be used as a first-line B-cell marker for the diagnosis. In this review, the authors discuss the molecular biology of CD79 and the frequency and usefulness of CD79 expression in these neoplasms.

Morphologically Typical and Atypical B-Cell Chronic Lymphocytic Leukemias Display a Different Pattern of Surface Antigenic Density

Recent evidences suggest that B-cell chronic lymphocytic leukemia (B-CLL) may have heterogeneous biological and clinical features. Immunological phenotype may be useful for distinguishing these different forms of disease.We used a quantitative flow cytometric approach to analyze the expression of several membrane molecules (CD19, CD20, CD22, CD23, CD11c, CD5, CD79b) commonly used to diagnose and characterize B-CLL in a choort of 84 consecutive B-CLL patients diagnosed according to morphological and immunological findings. We found that morphologically so-called “atypical” B-CLL displayed a significantly higher number of CD20 and CD22 molecules than typical forms. On the other hand, CD19 was found to be more expressed in typical B-CLL, although without reaching statistical significance. Finally, no difference was detected with respect to CD23, CD79b, CD11c and CD5 number of molecules/per cell between typical and atypical B-CLL. Other clinico-biological features, such as surface membrane immunoglobulin density, percentage of CD79b and FMC7 expression, peripheral blood lymphocytosis, trisomy 12 and advanced clinical stages were also found to be more frequent in atypical B-CLL. In conclusion, our data confirm the hypothesis that atypical B-CLL is a disease sustained by more mature B-cells, closely related but, at the same time, clearly distincted from neoplastic cells of typical B-CLL.

Usefulness of CD79b expression in the diagnosis of B-cell chronic lymphoproliferative disorders.

We evaluated anti-CD79b for its usefulness in the diagnosis of B-cell chronic lymphoproliferative disorders (BCLPDs), particularly chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). We analyzed 100 BCLPDs for CD5, CD19, CD20, CD23, CD79b, and surface immunoglobulin light chain (sIg) expression by 4-color flow cytometry. CD20, CD79b, and sIg expression were quantified. Correlational analysis and univariable and multivariable logistic regression models were used to determine the best combination of antigens for the immunophenotypic classification of CLL vs other BCLPDs. Positive and statistically significant Spearman pairwise correlations between CD20, CD79b, and sIg fluorescence intensity were demonstrated. In the simplest models in which a single variable was considered, cutoff points were chosen that gave misclassification rates for CLL of 16% for CD79b, 19% for sIg, and 18% for CD20. Low-intensity CD79b, CD20, and sIg are associated highly with CLL. A panel containing CD5, CD19, CD23, and sIg allowed correct classification of most cases. Addition of CD20 or CD79b improved diagnostic accuracy; CD79b was slightly better than CD20. CD79b seems to be a useful addition to a standard flow cytometry panel for the evaluation of BCLPDs.

CD79b expression in B cell chronic lymphocytic leukemia: its implication for minimal residual disease detection.

The surface expression of CD79b, using the monoclonal antibody (Mab) CB3-1, on B lymphocytes from normal individuals and patients with B cell chronic lymphocytic leukemia (CLL) has been analyzed using triple-staining cells for flow cytometry. In addition, the clinical significance of CD79b expression in CLL patients and its possible value for the evaluation of minimal residual disease (MRD) was explored. A total of 15 peripheral blood (PB) samples from healthy blood donors, five bone marrow (BM) samples from normal donors and 40 PB samples from CLL untreated patients were included in the study. In addition we studied the expression of CD79b in B lymphocytes from five CLL patients after fludarabine treatment in order to support our method. The expression of CD79b in B lymphocytes from PB was analyzed by flow cytometry, using simultaneous staining with the Mabs CD22, CD79b, CD19 and CD5, CD79b and CD19. Since normal immature bone marrow B cells are CD79b-/dim+ on their surface, in BM samples we used the combination CD45, CD79b and CD19 selecting mature B lymphocytes according to their bright CD45 intensity. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD19+ cells. For data analysis, the PAINT-A-GATE PRO software (Becton Dickinson) was used. Dilution experiments of CD79b- CLL cells and CD79bdim+ CLL cells with normal PB and BM cells were performed in order to assess the sensitivity level of the technique for detection of CD79b-/dim+residual CLL cells. All B lymphocytes from normal samples showed reactivity for the CD79b antigen. In contrast, CD79b was absent in 18/40 CLL patients (42.5%) and 20/40 CLL cases (50%) exhibited a low CD79b expression. Therefore, CD79b- B lymphocytes would be restricted to the CLL population and thus could be considered a 'tumor phenotype' for monitoring MRD in CLL patients. Dilution experiments indicate that the detection limit with this marker almost reaches the levels obtained by molecular biology methods as the PCR technique. All cases studied after fludarabine presented leukemic cells in their PB or BM samples detected by flow cytometry. Upon comparing the clinical and morphological characteristics of CD79b- and CD79b+ cases, all atypical CLL cases included in the present study were CD79b+ and advanced clinical stage (B and C Binet stage) was most frequently observed in CD79b+ cases than in CD79b- cases.