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Background: Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies., Methods: The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator., Results: 39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant., Conclusions: Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856.

Background: During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of μ heavy chain complexed with VpreB and λ5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma., Design and Methods: Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas., Results: We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%)., Conclusions: We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.

Context: Diffuse large B-cell lymphoma (DLBCL) can be assigned to prognostic subgroups, including germinal center B-cell (GCB) and activated B-cell subgroups, by using gene expression profiling and, reportedly, immunohistochemistry for CD10, Bcl-6, and multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF4)., Objective: To compare 2 commercial MUM1/IRF4 antibody formulations for accuracy in subtyping DLBCL against gene expression profiling, compare subtyping to patient survival, and evaluate the usefulness of GCB and non-GCB subtyping in relapsed and transformed DLBCL., Design: Evaluation of 2 commercial MUM1/IRF4 antibodies, ICSTAT/M17 and Mum-1p, by using 40 cases of de novo, relapsed, and transformed DLBCL; and comparison of the results obtained with gene expression profiling and survival., Results: Immunohistochemistry predicted the gene expression profiling subtype 71.8% and 69.2% of the time overall with use of the Mum-1p and ICSTAT/M17 antibodies, respectively, and 100% and 91.7% of the time when MUM1/IRF4 expression determined subtype. Gene expression profiling and immunohistochemistry revealed nearly identical 5-year overall survival rates for the GCB vs non-GCB subtypes (68.0% for GCB vs 24.7% for non-GCB with use of gene expression profiling [P = .03] and 70.2% vs 18.4%, respectively, with use of immunohistochemistry [P < .001]). When de novo, transformed, and relapsed cases were analyzed separately, 5-year overall survival rates were also significantly different., Conclusions: Immunohistochemistry can be used to subclassify DLBCL, including a very small series of transformed and relapsed cases, into GCB and non-GCB subtypes and predict survival rates similar to those predicted by use of gene expression profiling. The 2 MUM1/IRF4 antibodies performed similarly.

Loss of major histocompatibility class II (MHC II) expression in diffuse large B-cell lymphoma (DLBCL) correlates with worse outcome, possibly from decreased immunosurveillance. Primary mediastinal B-cell lymphoma (PMBCL) is a subtype of DLBCL which reportedly has frequent loss of MHC II proteins; however, PM-BCL has better survival than DLBCL. To investigate this paradox, we used geneexpression profiling (GEP) data and immunohistochemistry to study expression of MHC II and its regulatory genes and to determine their relationship to PMBCL survival. We found that GEP levels correlated between MHC II genes and the transcriptional regulator MHC2TA but not other adjacent genes, implying that transcriptional regulation of MHC II in PMBCL was intact and that MHC II gene deletion was unlikely. MHC II average expression was lower than in certain subtypes of DLBCL; however, only 12% had complete loss of MHC II expression. Poor patient survival in PMBCL correlated with incremental decreases in MHC II expression. Although overall survival was better, survival of the lowest 10% of MHC II expressers was similarly poor in DLBCL and PMBCL. MHC II expression may define a therapeutic target in both these diseases.

Background: The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is crucial because these two types of lymphoma require different treatments. We examined whether gene-expression profiling could reliably distinguish Burkitt's lymphoma from diffuse large-B-cell lymphoma., Methods: Tumor-biopsy specimens from 303 patients with aggressive lymphomas were profiled for gene expression and were also classified according to morphology, immunohistochemistry, and detection of the t(8;14) c-myc translocation., Results: A classifier based on gene expression correctly identified all 25 pathologically verified cases of classic Burkitt's lymphoma. Burkitt's lymphoma was readily distinguished from diffuse large-B-cell lymphoma by the high level of expression of c-myc target genes, the expression of a subgroup of germinal-center B-cell genes, and the low level of expression of major-histocompatibility-complex class I genes and nuclear factor-kappaB target genes. Eight specimens with a pathological diagnosis of diffuse large-B-cell lymphoma had the typical gene-expression profile of Burkitt's lymphoma, suggesting they represent cases of Burkitt's lymphoma that are difficult to diagnose by current methods. Among 28 of the patients with a molecular diagnosis of Burkitt's lymphoma, the overall survival was superior among those who had received intensive chemotherapy regimens instead of lower-dose regimens., Conclusions: Gene-expression profiling is an accurate, quantitative method for distinguishing Burkitt's lymphoma from diffuse large-B-cell lymphoma., (Copyright 2006 Massachusetts Medical Society.)

Background: The role of BCL2 as a predictor of survival in diffuse large B-cell lymphoma (DLBCL) is controversial. DLBCL is heterogeneous, and the expression of BCL2 is variable within the two major subgroups of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, as well as primary mediastinal DLBCL., Patients and Methods: In this study, we investigated the correlation of BCL2 expression with survival in the two major subgroups of DLBCL, as well as the mechanisms of BCL2 expression., Results: There was no significant correlation between BCL2 protein expression and overall survival within the GCB subgroup, but BCL2 expression had a significant adverse effect on overall survival within the ABC subgroup (P = .008). This correlation was also observed at the mRNA level (P < .04). The difference remained significant when the analyses were performed at different cutoff values. The t(14;18) was frequently observed in the GCB subgroup and was highly associated with BCL2 expression. Patients with ABC DLBCL did not exhibit t(14;18) but had a markedly higher frequency of chromosome 18q21 amplification, on which BCL2 resides. Thus, alternative mechanisms such as 18q21 amplification or activation of the nuclear factor-kappa B pathway, as reported previously, seem to be mainly responsible for the upregulation of BCL2 expression in the ABC subgroup., Conclusion: Treating all DLBCL as a single entity ignores the mechanistic differences in BCL2 upregulation and obscures the prognostic significance of BCL2 expression. Hence, the significance of BCL2 and other biomarkers should be assessed in the context of DLBCL subgroups in future studies.

Decreased major histocompatibility class II (MHCII) expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL). Immune-privileged site DLBCL (IP-DLBCL) patients reportedly have frequent large deletions at the MHCII locus whereas the mechanism of decreased expression in non-IP-DLBCL is unknown. Gene expression profiling data were used for correlation analyses between expression levels of MHCII genes with each other and their transcriptional regulator, CIITA. Comparative genomic hybridization (CGH) assessed chromosomal alterations at MHCII-related loci. Finally, a map was created of expression of genes that are telomeric, within, or centromeric to the MHCII locus. Correlation coefficients among MHCII genes ranged from 0.73 to 0.92, whereas those between adjacent and intervening genes were lower (-0.12 to 0.49). Correlations between MHCII and CIITA expression were higher (0.53 to 0.60) than between CIITA and neighboring genes (-0.05 to 0.22). In 23 MHCII(-) cases, CGH detected 2 losses and 2 gains at MHCII loci. Expression of genes telomeric, within, and centromeric to MHCII loci were near normal in most MHCII(-) cases. Large deletions of the MHCII locus are uncommon in non-IP-DLBCL, implicating altered transcription as the operative mechanism for decreased expression.

To glean biological differences and similarities of peripheral T-cell lymphoma-not otherwise specified [PTCL-NOS] to diffuse large B-cell lymphoma (DLBCL), a transcriptosome analysis was done on five PTCL-NOS and four DLBCL patients and validated by quantitative real-time reverse transcription-PCR on 10 selected genes. Normal peripheral blood T cells, peripheral blood B cells, and lymph node were used as controls. The resultant gene expression profile delineated distinct "tumor profile signatures" for PTCL-NOS and DLBCL. Several highly overexpressed genes in both PTCL-NOS and DLBCL involve the immune network, stroma, angiogenesis, and cell survival cascades that make important contributions to lymphomagenesis. Inflammatory chemokines and their receptors likely play a central role in these complex interrelated pathways: CCL2 and CXCR4 in PTCL-NOS and CCL5 and CCR1 in DLBCL. Highly overexpressed oncogenes unique to PTCL-NOS are SPI1, STK6, alpha-PDGFR, and SH2D1A, whereas in DLBCL they are PIM1, PIM2, LYN, BCL2A1, and RAB13. Oncogenes common to both lymphomas are MAFB, MET, NF-kappaB2, LCK, and LYN. Several tumor suppressors are also down-regulated (TPTE, MGC154, PTCH, ST5, and SUI1). This study illustrates the relevance of tumor-stroma immune trafficking and identified potential novel prognostic markers and targets for therapeutic intervention.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which approximately 40% of the patients respond well to current chemotherapy, but the prognosis for the other 60% is poor. The Leukemia/Lymphoma Molecular Profiling Project (LLMPP) used microarray technology to define a molecular profile for each of 240 patients with DLBCL and develop a molecular outcome predictor score that accurately predicted patient survival. Data from our laboratory and others suggest that alterations in antioxidant defense enzyme levels and redox environment can be oncogenic and affect the response to glucocorticoid treatment, one of the components of combination chemotherapy regimens for lymphoma. The goal of the current study was to reanalyze the LLMPP microarray data to determine whether the levels of antioxidant defense enzymes and redox proteins were correlated with prognosis in DLBCL. We found that patients with DLBCL with the worst prognosis, according to the outcome predictor score, had decreased expression of catalase, glutathione peroxidase, manganese superoxide dismutase, and VDUP1, a protein that inhibits thioredoxin activity. The data suggest that the patients with the worst prognosis combine a decrease in antioxidant defense enzyme expression with an increase in thioredoxin system function (the redox signature score).

Gene-expression profiling has identified 3 major subgroups of diffuse large B-cell lymphoma (DLBCL): germinal center B-cell-like (GCB), activated B-cell-like (ABC), and primary mediastinal DLBCL (PMBCL). Using comparative genomic hybridization (CGH), we investigated the genetic alterations of 224 cases of untreated DLBCL (87 GCB-DLBCL, 77 ABC-DLBCL, 19 PMBCL, and 41 unclassified DLBCL) previously characterized by gene-expression profiling. The DLBCL subgroups differed significantly in the frequency of particular chromosomal aberrations. ABC-DLBCL had frequent trisomy 3, gains of 3q and 18q21-q22, and losses of 6q21-q22, whereas GCB-DLBCL had frequent gains of 12q12, and PMBCL had gains of 9p21-pter and 2p14-p16. Parallel analysis of CGH alterations, locus-specific gene-expression profiles, and global gene-expression signatures revealed that DNA amplifications and gains had a substantial impact on the expression of genes in the involved chromosomal regions, and some genes were overexpressed in a DLBCL subgroup-specific fashion. Unexpectedly, specific chromosomal alterations were associated with significant changes in gene-expression signatures that reflect various aspects of lymphoma cell biology as well as the host response to the lymphoma. In addition, gains involving the chromosomal region 3p11-p12 provided prognostic information that was statistically independent of the previously defined gene-expression-based survival model, thereby improving its predictive power.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup.

The Leukemia and Lymphoma Molecular Profiling Project recently published results from DNA microarray analyses of 240 diffuse large B-cell lymphomas (DLBCLs). Four gene expression "signatures" were identified as correlated with patient outcome, including the major histocompatibility complex (MHC) class II genes (eg, HLA-DRA) which correlated with better survival. We further analyzed the effects of HLA-DRA on survival and correlated gene expression with protein status and tumor-infiltrating lymphocytes. The 5-year overall survival was 24% in the lowest 10% of HLA-DRA expression, 37% in the 10% to 25% group, 50% in the 25% to 50% group, and 55% for patients in the highest 50%. Further analysis demonstrated that the hazard ratio of death was a nonlinear function of HLA-DRA expression. Adjustment for the International Prognostic Index did not alter the impact of HLA-DRA on survival. Other MHC class II genes were found to predict survival similarly. Microarray HLA-DRA expression correlated with the presence or absence of human leukocyte antigen-DR (HLA-DR) protein in 20 of 22 cases assessed. Fewer tumor-infiltrating CD8(+) T cells were detected in MHC class II-negative cases compared with positive cases (2.8% versus 11.0%; P =.001), supporting the hypothesis that loss of tumor immunosurveillance has a devastating effect on patient outcome in DLBCL.

Using current diagnostic criteria, primary mediastinal B cell lymphoma (PMBL) cannot be distinguished from other types of diffuse large B cell lymphoma (DLBCL) reliably. We used gene expression profiling to develop a more precise molecular diagnosis of PMBL. PMBL patients were considerably younger than other DLBCL patients, and their lymphomas frequently involved other thoracic structures but not extrathoracic sites typical of other DLBCLs. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients. Gene expression profiling strongly supported a relationship between PMBL and Hodgkin lymphoma: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in Hodgkin lymphoma cells. PDL2, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in Hodgkin lymphoma cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in Hodgkin lymphoma cell lines. The molecular diagnosis of PMBL should significantly aid in the development of therapies tailored to this clinically and pathogenetically distinctive subgroup of DLBCL.

Follicular lymphoma (FL) is characterized by a continuous rate of relapse and transformation to a high-grade lymphoma, usually diffuse large B-cell lymphoma (DLBCL), associated with a dismal prognosis and a poor response to conventional chemotherapy. The progression of indolent to aggressive FL is accompanied by the successive accumulation of recurrent chromosomal defects, but the resultant alterations of gene expression are largely unknown. To expand the understanding of the pathogenesis of FL transformation, we initially performed oligonucleotide microarray analyses using Affymetrix HuFL chips on five cases with matched snap-frozen lymph nodes before and after transformation. Expression data were analyzed using the Affymetrix Microarray Suite 4.0 and Genespring 4.0. Thirty-six genes with increased expression and 66 genes with decreased expression associated with transformation were identified and functionally classified. The expression of differentially expressed genes was confirmed by real-time quantitative RT-PCR (QRT-PCR) using a total of seven matched pairs and an additional five FL and five unrelated DLBCL. In addition, selected genes were further analyzed by QRT-PCR or immunohistochemistry using a large, unrelated series of FL (grades 1 to 3) as well as transformed and de novo DLBCL (total of 51 samples). The microarray results correlated with the protein expression data obtained from samples at the time of initial diagnosis and transformation. Furthermore, the expression of 25 candidate genes was evaluated by QRT-PCR with a 78% confirmation rate. Some of the identified genes, such as nucleobindin, interferon regulatory factor 4, and tissue inhibitor of metalloproteinases 1, are already known to be associated with high-grade non-Hodgkin's lymphoma. Novel candidate genes with confirmed increased and decreased expression in transformed DLBCL include ABL2 and NEK2, and PDCD1 and VDUP1, respectively. In summary, this study shows that transformation of FL to DLBCL is associated with a distinct set of differentially expressed genes of potential functional importance.

Background: The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival., Methods: Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index., Results: Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators., Conclusions: DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.

Tumor-infiltrating CD8+ T-lymphocytes (T-TILs) are thought to be relevant to immunosurveillance of several tumor types including B-cell non-Hodgkin's lymphoma. B- and T-lymphocyte interactions via cellular adhesion molecules (CAMs), recognition molecules (HLAs), and costimulatory molecules (CSMs) are necessary for optimal antigen-specific T-cell activation to occur and may be important in generating effective host T-TIL responses. We previously found that low T-TIL response (CD8+ T cells < 6%) correlates with statistically shorter relapse-free survival in patients with diffuse large-cell lymphoma (DLCL). We now extend our observations in 71 DLCL patients by analyzing malignant B-cell expression of the following molecules important in T-cell activation: (a) recognition molecules [MHC I (MAS and MCA) and MHC II (HLA-DR, -DP, -DQ)]; (b) CAMs [leukocyte function antigen 1 (CD11a and CD18) and intracellular adhesion molecule 1 (CD54)]; and (c) CSMs [B7.1 (CD80) and B7.2 (CD86)]. Eighteen patients (25%) had low a T-TIL response, and 53 patients (75%) had a high T-TIL response. Overall, expression of the MHC class H molecules HLA-DR and HLA-DQ was most conserved. The loss of B7.2 (P = 0.04), intracellular adhesion molecule 1 (P = 0.0004), MAS (P = 0.02), and HLA-DR (P = 0.0004) expression was significantly associated with decreased T-TIL response. In 100% of patients with low T-TIL responses, at least one HLA, CAM, or CSM was undetectable on the malignant B cells by immunohistochemical staining (mean number of molecules lost = 2.67). In contrast, 49% of patients with high T-TIL responses had no losses in HLA, CAM, or CSM expression (mean number of molecules lost = 0.89). The mean number of absent molecules (HLA, CAM, or CSM) was significantly associated with T-TIL response (P = 0.0001). We conclude that loss of HLA, CAM, or CSM expression on malignant B cells is associated with a poor host T-cell immune response. In addition, because patients with low T-TIL response had lost expression of multiple cellular adhesion, recognition, and costimulatory molecules, our results suggest that a combination of immunorestorative therapies may be required to generate effective antitumor T-cell responses in B-cell DLCL.