Search

Mucinous adenocarcinoma (MAC) is conventionally diagnosed by WHO definition when the extracellular mucin is >50% of the tumor area, while tumors with <50% mucin are designated as having a mucinous component. The study is aimed at analyzing the clinicopathologic characteristics, mutation spectrum, and prognosis of colorectal adenocarcinoma with mucinous component (CAWMC). Mutation analyses for exon 2 to 4 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing. Expression of DNA mismatch repairs and P53 proteins were evaluated by immunohistochemistry. Density of tumor-infiltrating lymphocyte (TIL) status was scored. We also evaluated the percentage of glands producing mucin and the morphology of the different tumor cell types in mucin pools. We retrospectively analyzed the prognosis of 43 patients with stage II/III. The overall frequencies of KRAS and BRAF mutations were 36% and 8%, respectively. Patients with MAC exhibiting high levels of mucin were related to the increase of tumor diameter (P=0.038) but were not associated with any of the other clinicopathologic parameters. The proportion or variable morphology of mucinous component did not stratify progression-free survival in stage II/III cases. TIL was the most significant predictor of progression-free survival among stage II/III CAWMC. It is interesting to note that signet ring cell carcinoma does not portend a worse prognosis for patients with high TIL levels. Combining use the grade of TIL status with the WHO grade of the entire tumor can help identify patients with a high risk of recurrence more accurately., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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.

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment. We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort. We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival. This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin-mediated failure to recruit CD141+ DCs. A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles. The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation. The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants. In summary, we report evidence for a β-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome. We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.

The identification of B-Raf proto-oncongene (BRAF) mutation and the emergence of targeted therapy marked a turning point in the treatment of melanoma. The study of mutation status concordance between primary tumors and metastases in this cancer has major treatment implications as it facilitates the selection of candidates for targeted therapy. This review analyzes the evidence on the level of mutation status concordance between primary tumors and different types of metastases in cutaneous melanoma and provides an overview of the advantages and disadvantages of the various methods used to detect BRAF mutations., (Copyright © 2017 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.)

Aim: VE1 is a monoclonal antibody detecting mutant BRAFV(600E) protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients., Methods: Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers (n = 34), melanomas (n = 23) and thyroid cancers (n = 8). Two prognostic cohorts were evaluated (n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies (n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks (n = 100 blocks)., Results: Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age (p = 0.0175) and MLH1 deficiency (p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender (p = 0.0016), right-sided tumor location (p < 0.0001), higher tumor grade (p < 0.0001) and mismatch repair (MMR)-deficiency (p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks., Conclusion: VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

Background: The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer. The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make-up of individual cancers is not known yet. BRAF and NRAS mutations are commonly acquired during melanoma progression. Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group., Methods: One-hundred-thirteen melanoma metastases underwent microarray analysis and BRAF and NRAS genotyping. Allele-specific PCR was also performed in order to exclude low-frequency mutations., Results: Comparison between BRAF and NRAS mutant versus wild type samples identified mostly constituents or regulators of MAPK and related pathways. When testing gene lists discriminative of BRAF, NRAS and MAPK alterations, we found that 112 BRAF-specific transcripts were able to distinguish the two immune-related phenotypes already described in melanoma, with the poor phenotype associated mostly with BRAF mutation. Noteworthy, such association was stronger in samples displaying low BRAF mRNA expression. However, when testing NRAS mutations, we were not able to find the same association., Conclusion: This study suggests that BRAF mutation-related specific transcripts associate with a poor phenotype in melanoma and provide a nest for further investigation., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The BRAF(V600E) mutation is the most common diagnostic/prognostic marker in papillary thyroid carcinoma (PTC). Its evaluation is typically performed with DNA-based techniques; nonetheless, a few articles have recently proposed the morphological prediction of BRAF(V600E) in histological PTCs. We investigated this morphological parameter in our cytological series., Methods: We re-analyzed all 72 cytohistological samples diagnosed as positive for malignancy (favoring PTC) on fine-needle aspiration cytology from January 2012 to December 2013. We included 22 male patients and 50 female patients. The cytological cases were processed with liquid-based cytology. We performed molecular analysis and immunocytochemistry for the VE1 BRAF(V600E) antibody., Results: We reported 47 mutated cases and 25 wild-type (WT) cases with 100% cytohistological concordance. The cytological evaluations revealed plump cells (abundant eosinophilic cytoplasm and PTC nuclei) in all 47 mutated cases, with only 6 having a focal plump cell component (≤20% cells). Furthermore, 5 of the 25 WT cases showed focal plump cells. A distinctive sickle nuclear shape was found only in the mutated cases. VE1 yielded 100% positivity for all 24 mutated cases that were tested, including 3 cases with focal plump cells., Conclusions: We demonstrated that the BRAF(V600E) mutation might be predicted in cytological samples on the basis of some specific morphological features. Although the detection of plump cells (mainly focal) was also observed in WT cases, the detection of sickle-shaped nuclei provided the highest specificity and sensitivity as a predictive mutational parameter. These morphological features might be a valid tool for selecting cases for molecular analysis., (© 2014 American Cancer Society.)

Malignant melanoma is associated with poor clinical prognosis; however, novel molecular and immune therapies are now improving patient outcomes. Almost 50% of melanomas harbor targetable activating mutations of BRAF that promote RAS-RAF-MEK-ERK pathway activation and melanoma proliferation. Recent evidence also indicates that melanomas bearing mutant BRAF may also have altered immune responses, suggesting additional avenues for treatment of this patient group. The small molecule inhibitors selective for mutant BRAF induce significant but short-lived clinical responses in a proportion of patients, but also lead to immune stimulatory bystander events, which then subside with the emergence of resistance to inhibition. Simultaneous BRAF and MEK inhibition, and especially combination of BRAF inhibitors with new immunotherapies such as checkpoint blockade antibodies, may further enhance immune activation, or counteract immunosuppressive signals. Preclinical evaluation and ongoing clinical trials should provide novel insights into the role of immunity in the therapy of BRAF-mutant melanoma., (©2014 American Association for Cancer Research.)

Treatment of BRAFV600E-mutant melanoma by small molecule inhibitors that target BRAFV600E or MEK kinases is increasingly used in clinical practice and significantly improve patient outcome. However, patients eventually become resistant and therapeutic improvement is required. Molecular diversity within individual tumors (intratumor heterogeneity) and between tumors within a single patient (intrapatient heterogeneity) poses a significant challenge to precision medicine. Using immunohistochemistry, we determined the extent of BRAFV600E intratumor and intrapatient heterogeneity and the influence of morphological heterogeneity in a large series of 171 melanomas of 81 patients. The BRAFV600E mutation rate found in our melanoma series is 44%, with none of 22 (0%) melanoma in situ, 23 of 56 (41%) primary tumors, 28 of 59 (48%) regional metastases, and 24 of 34 (71%) distant metastases harboring the mutation. In general, a diffuse homogeneous immunostaining was seen, even in tumors consisting of more than one cell type, that is, epithelioid, spindle, and/or small cell types. Nevertheless, BRAFV600E-mutant melanomas more often had a purely epithelioid cell population (P=0.063), that is more evident among distant metastases (P=0.014). Only two of 75 (3%) mutated specimens (one primary and one metastasis) displayed heterogeneous BRAFV600E expression. The primary tumor was also morphologically heterogeneous and exclusively displayed BRAFV600E in the epithelioid component, confirming an association between BRAFV600E and epithelioid cells. Twenty-eight of 30 patients (93%) had concordant BRAFV600E mutation status between their tumors. Taken together, BRAFV600E intratumor and intrapatient heterogeneity in melanoma is diminutive, nevertheless, the identified exceptions will have important implications for the clinical management of this disease.

Background: BRAF V600E mutation is the most common genetic alteration in papillary thyroid cancer (PTC). We used a mutation-specific antibody for immunohistochemical (IHC) detection of the BRAF V600E mutation and correlated expression with clinicopathologic features. The study was designed to validate the accuracy and determine the clinical importance of IHC detection of the BRAF V600E mutation in PTC., Methods: Direct sequencing and IHC for BRAF V600E mutation was performed in 37 consecutive patients with PTCs. IHC was scored on an intensity proportion scale. IHC positive tumors were stratified into intensity categories. The categories were assessed for clinicopathologic variables, including age, extrathyroidal extension, lymphovascular invasion, and lymph node metastases., Results: A total of 25 PTCs were BRAF V600E-positive and 12 were BRAF mutation-negative on IHC. The BRAF V600E mutation-specific antibody had a sensitivity of 89% and specificity of 100% for detecting the mutation. Tumors with high-intensity staining were more likely to have extrathyroidal extension., Conclusion: IHC is an accurate method for the detection of the BRAF V600E mutation in PTC, and its ability to quantify the mutation expression may serve as a better predictor of tumor behavior than molecular sequencing. It provides a potentially rapid, easily applicable, and economic alternative to current techniques., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Aims: The presence of a BRAF mutation is a strong marker for poor prognosis of colorectal carcinoma (CRC), and can be used as evidence of a sporadic mechanism of mismatch repair deficiency. BRAF mutation may also predict resistance to EGFR-targeted therapy. A BRAF V600E-specific antibody has recently become commercially available. The aim of this study was to determine whether immunohistochemistry can predict BRAF mutations in CRC., Methods and Results: Immunohistochemistry was performed on 52 genotyped CRC cases (17 BRAF mutant, 18 KRAS mutant, 17 BRAF/KRAS wild-type) with monoclonal antibody VE1. Cytoplasmic staining was observed in 71% of BRAF V600E mutant tumours (moderate or strong staining in 50% of these cases). Weak cytoplasmic staining was observed in 17% of KRAS mutant tumours and 35% of wild-type tumours. Non-specific nuclear staining was common. The sensitivity and specificity of immunohistochemistry with VE1 for BRAF mutation were 71% and 74%, respectively; when only moderate or strong staining was considered to be positive, the specificity was 100%, but the sensitivity only 35%., Conclusions: Immunohistochemistry with VE1 is not a useful surrogate for genotyping in CRC. Although moderate or strong cytoplasmic staining is specific for BRAF V600E mutations, this antibody is insufficiently sensitive to serve as an effective screening tool., (© 2013 John Wiley & Sons Ltd.)

The V600E mutation of BRAF has emerged as both an effective biomarker and therapeutic target for select benign and malignant cutaneous and non-cutaneous human tumors and is typically determined using DNA-based techniques that include allele-specific PCR and direct DNA sequencing. Recently however, the development of new antibodies directed against the V600E protein has opened the door for an easier and more efficient strategy for identifying this mutation. Our present aim was to determine the efficacy of one such antibody, anti-B-Raf (V600E), a mouse monoclonal antibody in which the immunogen is a synthetic peptide derived from the internal region of BRAFV600E. A total of 35 cases of primary cutaneous melanoma were evaluated using a combination of DNA-based techniques that included allele-specific PCR and/or direct DNA sequencing and immunohistochemistry. Cases of papillary thyroid carcinomas (n=5) and colorectal carcinomas (n=5), known to harbor the BRAFV600E mutation, served as positive controls for the study. DNA analyses revealed that 6 of 35 (17%) cases of the primary cutaneous malignant melanoma possessed the BRAFV600E mutation. For immunohistochemical analyses, cytoplasmic positivity with anti-B-Raf was noted in 7 of 35 (20%) cases of primary melanoma and in all 10 positive controls. Statistical analyses of the data demonstrated that the sensitivity of the immunohistochemistry was 100% and specificity was 97%. Findings from the current study support the potential use of immunohistochemistry as an ancillary screening tool to assess the BRAFV600E mutation status in primary cutaneous melanoma.