Archived Hematoxylin and Eosin (H&E) stained pathology slides are routinely stored to index formalin-fixed paraffin-embedded (FFPE) sample tissue blocks. FFPE blocks are clinically annotated human tumor specimens that can be valuable in studies decades after the tissue is collected. If stored properly, they have the potential to yield a valuable number of serial sectioned slides for diagnostic or research purposes. However, some retrospective studies are limited in scope because the tissue samples have been depleted or not enough material is available in stored blocks for serial sections. The goal of these studies was to determine if archived H&E-stained slides can be directly reutilized by optimizing methods to de-stain and then re-stain the H&E stained slides to allow the detection of several biomarkers of interest using a conjugated antibody with chromogen multiplex immunohistochemistry procedure. This simple but innovative procedure, combined with image analysis techniques, demonstrates the ability to perform precise detection of relevant markers correlated to disease progression in initially identified tumor regions in tissue. This may add clinical value in retaining H&E slides for further use.

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.

Background and Aims: Multiple laboratory methods are used to screen patients with colorectal cancer (CRC) for mismatch repair (MMR) protein deficiency to identify possible Lynch syndrome patients. The goal of this study was to compare the agreement between ready-to-use immunohistochemistry (IHC) assays for MLH-1, PMS-2, MSH-2, MSH-6, and mutated BRAF at V600E and molecular methods in CRC cases. The inclusion of the BRAF V600E mutation testing is important for the identification of patients with sporadic CRC, as the BRAF V600E mutation is very rarely observed in patients with Lynch syndrome tumors. Methods: CRC cases were analyzed by ColoSeq^TM tumor sequencing assay and VENTANA MMR IHC Panel that included anti-MLH1, anti-PMS2, anti-MSH2, anti-MSH6, and anti-BRAF V600E antibodies. Additionally, CRC cases with MLH1 IHC loss were evaluated for MLH1 promoter hypermethylation. Results: One hundred and eighteen cases were analyzed. The overall percent agreement (OPA) for each evaluated marker status compared to next-generation sequencing (NGS) exceeded 96%. Twenty-three cases were positive for the BRAF V600E mutation by IHC and NGS, and twenty cases showed loss of MLH1 protein and were positive for MLH1 hypermethylation. Samples with loss of MMR protein expression by IHC demonstrated genetic and/or epigenetic alterations that were consistent with the observed protein expression patterns. Conclusions: The results of this study indicate that ready-to-use IHC assays can correctly identify the loss of MMR proteins and the presence of mutated BRAF V600E protein, supporting the utility of the VENTANA MMR IHC Panel as an aid to stratify patients with sporadic CRC vs. potential Lynch syndrome.

About 15–20% of lung neoplasms are of neuroendocrine origin. In addition to evaluation of morphological features, these neoplasms can be identified by immunohistochemistry (IHC) using antibodies against neuroendocrine markers such as synaptophysin, chromogranin A, and neural cell adhesion molecule, CD56. Recent studies have shown that insulinoma-associated protein 1 (INSM1) is increasingly used as a diagnostic marker for neoplasms of neuroendocrine origin, as it is highly sensitive and specific. The primary focus of this study was to evaluate the sensitivity and specificity of a recently developed assay that uses a new INSM1 (SP493) antibody for detecting primary neuroendocrine lung neoplasms and to compare this IHC assay with currently used neuroendocrine markers. Tissue microarrays containing 391 lung neoplasms were stained with INSM1 (SP493) antibody, INSM1 (MRQ-70) antibody, CONFIRM anti-Synaptophysin (SP11) antibody, anti-Chromogranin A (LK2H10) antibody, and CD56 (MRQ-42) antibody on BenchMark ULTRA instruments using OptiView IHC DAB Detection Kit or ultraView Universal DAB Detection Kit using manufacturer’s recommended protocols. Sensitivity, specificity, and accuracy were calculated for each biomarker. The sensitivity and specificity of the new assay using INSM1 (SP493) antibody for detecting primary lung neoplasms with neuroendocrine differentiation were 96.8% and 92.3%, respectively. Comparable results were seen for the lung neoplastic cases stained with INSM1 (MRQ-70) antibody, yielding a sensitivity of 94.9% and specificity of 94.1%. In contrast, the anti-Chromogranin A (LK2H10) antibody and CD56 (MRQ-42) antibody showed considerably lower sensitivities. The sensitivity was 78.7% for the anti-Chromogranin A (LK2H10) antibody and 85.4% for the CD56 (MRQ-42) antibody. However, the specificities of anti-Chromogranin A (LK2H10) antibody and CD56 (MRQ-42) antibody were comparable with the specificity of the INSM1 (SP493) antibody [91.7% for anti-Chromogranin A (LK2H10) antibody, 90.4% for CD56 (MRQ-42) antibody]. The performance of the anti-Synaptophysin (SP11) antibody was suboptimal, with a sensitivity of 82.9% and a specificity of 87.6%. Overall, our data demonstrate that the IHC assay using the newly developed INSM1 (SP493) antibody is highly specific and sensitive for identifying primary neuroendocrine lung neoplasms.