A B S T R A C T PurposeDiffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. Patients and MethodsWe determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. ResultsIn the training cohort (n ϭ 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P Ͻ .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P Ͻ .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P Ͻ .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. ConclusionAssessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.

BackgroundThe eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section.MethodsThe HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays.ResultsHER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively.ConclusionsWe have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297

Loss of major histocompatibility complex class II (MHC II)

The major aim of this study was to evaluate the performance of anti-BRAF V600E (VE1) antibody in colorectal tumors with and without KRAS mutation. KRAS and BRAF are two major oncogenic drivers of colorectal cancer (CRC) that have been frequently described as mutually exclusive, thus the BRAF V600E mutation is not expected to be present in the cases with KRAS mutation. In addition, a review of 25 studies comparing immunohistochemistry (IHC) using the anti-BRAF V600E (VE1) antibody with BRAF V600E molecular testing in 4041 patient samples was included.One-hundred and twenty cases with/without KRAS or BRAF mutations were acquired. The tissue were immunostained with anti-BRAF V600E (VE1) antibody with OptiView DAB IHC detection kit. The KRAS mutated cases with equivocal immunostaining were further evaluated by Sanger sequencing for BRAF V600E mutation. Thirty cases with BRAF V600E mutation showed unequivocal, diffuse, uniform, positive cytoplasmic staining and 30 cases with wild-type KRAS and BRAF showed negative staining with anti-BRAF V600E (VE1) antibody. Out of 60 cases with KRAS mutation, 56 cases (93.3%) were negative for BRAF V600E mutation by IHC. Four cases showed weak, equivocal, heterogeneous, cytoplasmic staining along with nuclear staining in 25-90% of tumor cells. These cases were confirmed to be negative for BRAF V600E mutation by Sanger sequencing. Overall, IHC with anti-BRAF V600E (VE1) antibody using recommended protocol with OptiView detection is optimal for detection of BRAF V600E mutation in CRC. Our data are consistent with previous reports indicating that KRAS and BRAF V600E mutation are mutually exclusive.

The major histocompatibility complex (MHC) class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4+ T cells. Multiple cancers express MHC class II, which may influence the anti-tumor immune response and patient outcome. Low MHC class II expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL), the most common form of aggressive non-Hodgkin lymphoma. Therefore, we investigated whether gamma-interferon-inducible lysosomal thiol reductase (GILT), an upstream component of the MHC class II-restricted antigen processing pathway that is not regulated by the transcription factor class II transactivator, may be important in DLBCL biology. GILT reduces protein disulfide bonds in the endocytic compartment, exposing additional epitopes for binding to MHC class II and facilitating antigen presentation. In each of four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of a classical MHC class II gene, HLA-DRA, was associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival was independent of established clinical and molecular prognostic factors, the International Prognostic Index and the cell of origin classification, respectively. Immunohistochemical analysis of GILT expression in 96 DLBCL cases demonstrated variation in GILT protein expression within tumor cells which correlated strongly with GILT mRNA expression. These studies identify a novel association between GILT expression and clinical outcome in lymphoma. Our findings underscore the role of antigen processing in DLBCL and suggest that molecules targeting this pathway warrant investigation as potential therapeutics.