BRAF Immunohistochemistry Research Articles

Effect of Extent of Resection and Adjuvant Radiation on Recurrence of BRAF versus β-Catenin–Mutated Craniopharyngioma: A Single Institutional Case Series

Abstract Objectives The two histologic subtypes of craniopharyngiomas (CPs), papillary and adamantinomatous, harbor mutually exclusive mutations of BRAF V600E and CTNNB1, respectively. Studies suggest that subtotal resection (STR) plus adjuvant radiation therapy (XRT) may result in similar progression-free survival (PFS) as gross total resection (GTR). We hypothesized that STR ± XRT and GTR result in similar PFS for both BRAF and β-catenin–mutated CPs. Design Patients who were surgically treated for a primary CP between 2001 and 2023 at a single institution were included. Immunohistochemical studies were performed retrospectively using BRAF and β-catenin antibodies. Patients with missing immunohistochemistry (IHC) diagnosis were excluded. Differences in PFS for STR ± XRT and GTR groups were assessed with a log-rank test, stratified by BRAF and β-catenin IHC status. Results A total of 77 patients with CP were screened. IHC data were available for 50 patients; 20 had a BRAF mutation, and 30 had a β-catenin mutation. Among BRAF patients, 11 underwent GTR; 9 had STR, and 5 had adjuvant XRT. Among β-catenin patients, 14 underwent GTR; 16 had STR, and 6 had adjuvant XRT. For BRAF patients with GTR, the median PFS was not reached; for BRAF patients with STR ± XRT, the median PFS was 150 days (p < 0.01, log-rank test). For β-catenin patients with GTR, the median PFS was 1,813 days; for β-catenin patients with STR ± XRT, the median PFS was not reached (p = 0.80, log-tank test). Conclusions Both GTR and STR ± XRT seemed to offer similar PFS outcomes only for patients with β-catenin–mutated CP. For patients with BRAF-mutated CP, a greater extent of resection was significantly associated with prolonged PFS.

Multifocal Papillary Thyroid Carcinomas With Discordant Molecular Drivers: Emphasizing the Morphology and Collision Tumors.

Multifocal papillary thyroid carcinomas (PTCs) are common and the majority of the tumors harbor mutual BRAF p.V600E mutation. This study aimed to investigate a contemporary series of multifocal PTCs with discordant molecular drivers. Consecutive thyroidectomies diagnosed with multifocal PTCs ≥0.5cm between 2019 and 2023 were reviewed. Immunohistochemistry (IHC) for BRAF VE1 was performed for all tumors. Cases with discordant BRAF IHC results or morphologic discrepancy were identified, and BRAF IHC-negative tumors were subjected to RAS Q61R IHC and/or targeted RNA next-generation sequencing. A total of 770 patients with a main PTC ≥0.5cm were identified; 255 (33.1%) had multifocal disease, and 142 (18.4%) had at least another PTC ≥0.5cm. Among them, 13 cases (9.2%, 13/142) had discordant molecular drivers. Twelve cases had one or more BRAF -positive PTCs accompanied by a BRAF -negative PTC (3 with CCDC6::RET fusion, 1 with NCOA4::RET fusion, 1 with ACBD5::RET fusion, 2 with ETV6::NTRK3 fusion, 1 with TG::FGFR1 fusion, 1 with LMTK2::BRAF fusion, 1 with AGK::BRAF fusion and RAS p.Q61R mutation, 1 with RAS p.Q61R mutation, and 1 without detectable molecular drivers). The last case had tumors with discordant fusion drivers ( VIM::NTRK3 and TNS1::BRAF ). Most cases showed tumors that were morphologically distinct (92.3%, 12/13) and occurred in the contralateral lobes (76.9%, 10/13). Notably, we identified 4 cases (30.8%) that presented as collision tumors and 6 cases (46.2%) that showed lymph node metastases, including 2 with simultaneous involvement by tumors with discordant molecular drivers, as novel findings. In summary, a subset (9.2%) of multifocal PTCs had discordant molecular drivers and 84.6% of them were a combination of BRAF -positive and kinase gene fusion-associated PTCs, most with distinct morphologies. Almost half of the cases had nodal metastasis and a third of them showed simultaneous involvement by tumors with discordant molecular drivers. The results highlight the clinical importance of identifying such cases, given the potentially different treatments.

Utility of BRAF V600E immunohistochemistry in the diagnosis of mandibular ameloblastomas

Ameloblastoma, odontogenic keratocyst (OKC), and dentigerous cyst (DC) can have similar radiographic and histological appearances. The purpose of this study was to determine the utility of BRAF immunohistochemistry in discerning mandibular ameloblastomas from OKCs and DCs. This retrospective cohort study included patients treated between 1998 and 2018. Inclusion criteria include incisional biopsy-proven mandibular ameloblastoma, OKC, or DC, and sufficient tissue for immunohistochemistry. The primary predictor variable was the type of lesion. The primary outcome variable was the presence/absence of BRAF V600E immunoreactivity. The cohort consisted of 43 patients (19 female, 24 male; mean age 48 ± 17 years). There were 22 ameloblastomas, 11 OKCs, and 10 DCs. Among ameloblastomas, 68.2% (15/22) stained positive for BRAF V600E; no OKC or DC was positive (P < 0.001). By subtype, the majority of the follicular (83.3%), unicystic (83.3%), desmoplastic (66.7%), and acanthomatous (100%) subtypes were positive, but only 33.3% of the plexiform subtype were positive. BRAF immunohistochemistry may be a useful adjunct in the differentiation of ameloblastoma from OKCs and DCs on incisional biopsies. It may be particularly useful for small samples with a prominent cystic component or equivocal histopathology. Mandibular lesions that are BRAF immunohistochemistry positive are unlikely to be DCs or OKCs.

Comparison of Two Rapid Assays for the Detection of BRAF V600 Mutations in Metastatic Melanoma including Positive Sentinel Lymph Nodes

Testing for the BRAF mutation is mandatory for the management of patients with locally advanced or metastatic melanoma. Molecular analysis based on DNA sequencing remains the gold-standard method for the screening of the different BRAF mutations. These methods must be rapid, sensitive, and specific enough to allow optimal therapeutic management in daily practice and also to include patients in clinical trials. Here, we compared the Idylla BRAF Mutation Test and the anti-BRAF V600E (clone VE1) immunohistochemistry (IHC) in 90 melanoma samples, with a focus on a challenging cohort of 32 positive sentinel lymph nodes. The BRAF status was assessed with both methods independently of the percentage of tumor cells. The concordance rate was calculated excluding both non-contributory analyses and BRAF V600K/R/M mutants due to the specific V600E-IHC test design. The incidence of the BRAF V600E mutation was 33% with both BRAF Idylla and BRAF IHC. The agreement rate was 91% (72/79). Although the agreement rate was high, we suggest that the use of IHC is more suitable for rapid BRAF testing on sentinel lymph node biopsies when associated with a low percentage and scattered tumor cells, which gave a high risk of non-contributory analysis and/or false negative results with the IdyllaTM BRAF Mutation Test.

Pathologist initiated reflex BRAF mutation testing in metastatic melanoma: experience at a specialist melanoma treatment centre

Testing for BRAF mutations in metastatic melanoma is pivotal to identifying patients suitable for targeted therapy and influences treatment decisions regarding single agent versus combination immunotherapy. Knowledge of BRAF V600E immunohistochemistry (IHC) results can streamline decisions during initial oncology consultations, prior to DNA-based test results. In the absence of formal guidelines that require pathologist initiated ('reflex') BRAF mutation testing, our institution developed a local protocol to perform BRAF V600E IHC on specimens from all stage III/IV melanoma patients when the status is otherwise unknown. This study was designed to evaluate the application of this protocol in a tertiary referral pathology department. A total of 408 stage III/IV melanoma patients had tissue specimens accessioned between 1 January and 31 March in three consecutive years (from 2019 to 2021), reported by 32 individual pathologists. The BRAF mutation status was established by pathologists in 87% (352/408) of cases. When a prior BRAF mutation status was previously known, as confirmed in linked electronic records (202/408), this status had been communicated by the clinician on the pathology request form in 1% of cases (3/202). Pathologists performed BRAF V600E IHC in 153 cases (74% of cases where the status was unknown, 153/206) and testing was duplicated in 5% of cases (20/408). Reflex BRAF IHC testing was omitted in 26% of cases (53/206), often on specimens with small volume disease (cytology specimens or sentinel node biopsies) despite adequate tissue for testing. Incorporating BRAF IHC testing within routine diagnostic protocols of stage III/IV melanoma was both feasible and successful in most cases. Communication of a patient's BRAF mutation status via the pathology request form will likely improve implementation of pathologist initiated BRAF mutation testing and may result in a reduction of duplicate tests. To improve pathologist reflex testing rates, we advocate for the use of an algorithmic approach to pathologist initiated BRAF mutation testing utilising both IHC and DNA-based methodologies for stage III/IV melanoma patients.

BRAF Immunoexpression Can Be Intralesionally Heterogeneous but BRAF V600E Mutation Status Is Intralesionally Homogeneous and Interlesionally Concordant in Melanoma: A Study of 140 Lesions From 98 Patients.

This study sought to confirm the homogeneity of BRAF V600E mutation status in melanoma. BRAF immunohistochemistry was performed on 102 lesions from 60 patients of melanoma with BRAF V600E mutation and 38 negative-control melanoma lesions from 38 patients, both of which were confirmed by real-time PCR or the MassARRAY System. In the positive-control lesions, 9 lesions from 7 patients with preceding BRAF-inhibitor therapy were included. Of the 102 BRAF-mutant lesions, 101 (99.0%) showed diffuse BRAF immunoexpression, but 39 (38.2%) of them showed various heterogeneous intensities. The heterogeneous intensity of immunostaining was due to necrosis (n = 10), minimal or clear cytoplasm (n = 5), tissue crush (n = 8), insufficient fixation (n = 24), or technical error (n = 4). Only 1 lesion (1.0%) with nondiffuse immunoexpression harbored 80% weakly BRAF-positive tumor area and 20% BRAF-negative area with tissue damage. Sanger sequencing performed on the weak or negative regions in 7 lesions revealed BRAF V600E mutation in all the tested lesions. By contrast, all 38 negative-control lesions demonstrated no BRAF immunoexpression. This study demonstrated intralesional homogeneity and interlesional concordance for BRAF V600E mutation status and intralesional frequent heterogeneity for BRAF immunoexpression. The abovementioned 5 phenomena caused substantial reduction in BRAF immunostaining intensity. In 9 lesions within this study, BRAF immunoexpression and BRAF V600E point mutation status were not affected by preceding BRAF inhibitor therapy. Our data would also support the position that it does not matter whether we select primary or metastatic samples for BRAF mutation analysis.

High Prevalence of Gene Fusions and Copy Number Alterations in Pediatric Radiation Therapy-Induced Papillary and Follicular Thyroid Carcinomas.

Background: Childhood cancer survivors and bone marrow transplant recipients treated with radiation therapy (RT) are at increased risk for subsequent thyroid cancer. However, the genetic landscape of pediatric thyroid cancer, both primary and RT-induced, remains poorly defined, as pediatric papillary thyroid carcinoma (PTC) has been understudied compared with adults and data on pediatric follicular thyroid carcinoma (FTC) are virtually nonexistent. The objective of this study was to characterize and compare the molecular profiles of pediatric RT-induced PTC and FTC cases with primary pediatric thyroid cancers. Methods: A total of 41 differentiated thyroid carcinomas (11 RT cases and 30 primary cases) from 37 patients seen at Phoenix Children's Hospital between January 1, 2010 and December 31, 2019 were evaluated by targeted next-generation sequencing and/or BRAF immunohistochemistry. Results: Eighty-six percent (6/7) of RT-PTC harbored a gene fusion (GF) compared with 56% (14/25) of primary PTC; 14% (1/7) of RT-PTC had a single-nucleotide variant (SNV; specifically, a point mutation in the DICER1 gene) compared with 44% (11/25) of primary PTC (all of the latter had the BRAFV600E mutation). An exceedingly rare ROS1 fusion was identified in a child with RT-PTC. With respect to FTC, copy number alterations (CNAs) were seen in 75% (3/4) of RT cases compared with 40% (2/5) of primary cases. None of the RT-FTC had SNVs compared with 100% (5/5) of primary FTC. Conclusions: In children, the molecular profile of subsequent RT-induced thyroid cancers appears to differ from primary (sporadic and syndromic) cases, with a high prevalence of GFs in RT-PTC (similar to PTC occurring after the Chernobyl nuclear reactor accident) and CNAs in RT-FTC. A better understanding of the molecular mechanisms underlying these cancers may lead to more accurate diagnosis, prognosis, and treatment, as some of the genomic alterations are potentially targetable.

Use of BRAF immunohistochemistry as a screening test in detecting BRAFV600E mutation in melanomas.

BRAF mutation is detected in 50-70% of melanomas. The molecular methods used to detect BRAF mutations are 80-90% sensitive, specific, and expensive methods. Immunohistochemistry is a relatively common, rapid, relatively inexpensive method in pathology practice compared to molecular techniques. We aimed to compare immunohistochemical and molecular methods in our case of malign melanoma in which we investigated BRAF mutation with "real time PCR" method and to investigate the compatibility of molecular test results of BRAF immunohistochemistry results as a preliminary test. Selected blocks of 30 patients with metastatic melanoma who came to our department for BRAF mutation detection were subjected to real time PCR molecular method and immunohistochemical study was performed with BRAF primer antibody. BRAF mutation was detected by molecular method in 7 of 30 cases (23.33%). In all of these 7 cases, positive immunohistochemical staining was identified (100%). In conclusion, the use of BRAF immunohistochemistry as a screening test in the detection of mutant disease will allow the cost-effective use of molecular testing.

Immunohistochemistry as a Surrogate Marker of Underlying Molecular Derangements in Sporadic Colorectal Carcinoma in Children – A Series of Three Cases

Background: Colorectal carcinomas (CRCs) are uncommon tumors in children. Here, we elucidate three cases of childhood CRCs with their underlying molecular derangements using immunohistochemistry (IHC) with emphasis on BRAF mutation. Case summary: All three CRCs were sporadic tumors involving the left colon with two of them having a mucinous phenotype. We performed IHC for BRAF, p53 and β-catenin along with markers of microsatellite instability (MSI) in all three tumors. All the tumors had diffuse strong cytoplasmic BRAF positivity, with focal p53 positivity in two cases and cytoplasmic β-catenin staining in one case. One case showed CpG island hypermethylation with isolated loss of PMS2 staining. None of the cases had any family history of CRC. Conclusions: IHC can be used as a surrogate marker for determining the underlying molecular derangements in CRC. Sporadic CRCs in children are a cumulative effect of multiple mutations, of which BRAF mutation is significant and critical for planning targeted therapy.

Real-world assessment of the BRAF status in non-squamous cell lung carcinoma using VE1 immunohistochemistry: A single laboratory experience (LPCE, Nice, France)

IntroductionInternational guidelines recommend BRAF mutational status assessment in treatment-naive advanced non-squamous non-small cell lung carcinoma (NSCLC) patients since the presence of a BRAFV600 mutation enables specific BRAF inhibitor treatment. For this purpose, the mutational status needs to be obtained in 10 working days. Herein, we prospectively evaluated the feasibility of systematic assessment of the BRAF status using immunohistochemistry (IHC) in a single institution (LPCE, Nice) at baseline for NSCLC diagnosed. Methods1317 NSCLC were evaluated using BRAF IHC from 2011 to 2019. Initially the BRAF status was prospectively assessed using NGS and/or pyrosequencing in 618 consecutively diagnosed NSCLC patients from 2012 to 2016; BRAFV600E and BRAF nonV600E mutated tumors detected in this cohort were retrospectively evaluated using BRAF IHC. Secondarily, 699 biopsies of NSCLC were prospectively analyzed between 2017 and 2019 using BRAF IHC. BRAF IHC positive tumors were tested using a rapid BRAF specific PCR based assay. ResultsInitially, 21/618 (3%) of tumors (15 early and 6 late stage tumors) were BRAFV600E mutated according to the results of NGS and/or pyrosequencing. BRAF IHC was positive in 21/21 of these cases and negative in 51/51 (100 %) BRAF non V600E mutated cases. In the prospective BRAF IHC tested cohort of patients, 24/699 (3%) tumors (13 early and 11 late stage tumors) were positive with VE1 IHC. The BRAF PCR assay was positive in 20/24 (83 %) of these cases. ConclusionBRAFV600E IHC screening of treatment-naïve NSCLC patients is a rapid, specific and very sensitive method which can lead in advanced stage positive NSCLC tumors to a BRAF inhibitor treatment. This test can be routinely integrated into mandatory predictive biomarker ‘testing of NSCLC. According to the organization of patient care and the physician’s request, this practice can be proposed as an alternative to NGS-based tissue biopsy made at baseline.

A Systematic Review and Meta-Analysis of the Diagnostic Performance of BRAF V600E Immunohistochemistry in Thyroid Histopathology.

Immunohistochemistry (IHC) in evaluating thyroid surgical specimens may facilitate diagnostic and prognostic evaluation, with potential therapeutic implications. We performed a systematic review and meta-analysis examining the analytic validity of IHC in detecting BRAFV600E mutations in thyroid cancer (primary or metastatic). We screened citations from three electronic databases (until December 20, 2018), supplemented by a hand search of authors' files and cross-references of reviews. Citations and full-text papers were independently reviewed in duplicate, and consensus was achieved on inclusion of papers. Two reviewers independently critically appraised and abstracted data from included papers. Random-effect meta-analyses were conducted for sensitivity and specificity estimates. We reviewed 1499 unique citations and 93 full-text articles. We included 1 systematic review and 30 original articles. The published review (from 2015) needed to be updated as there were multiple subsequent original studies. The pooled sensitivity of IHC in detecting a BRAFV600E mutation was 96.8% (95% confidence interval [CI] at 94.1%, 98.3%) (29 studies, including 2659 BRAFV600E mutant tumors). The IHC pooled specificity was 86.3% (95% CI 80.7%, 90.4%) (28 studies, including 1107 BRAFV600E wild-type specimens). These meta-analyses were subject to statistically significant heterogeneity, partly explained by antibody type (sensitivity and specificity) and tissue/tumor type (specificity). In conclusion, BRAF IHC is highly sensitive and reasonably specific in detecting the BRAFV600E mutation; however, there is some variability in analytic performance.

BRAF immunohistochemistry predicts sentinel lymph node involvement in intermediate thickness melanomas.

BackgroundSentinel node biopsy (SNB) is an important step in melanoma staging and prognostication. It is commonly performed for patients with intermediate thickness melanomas, based on clinicopathological features. However, only 20–25% of patients eventually demonstrate nodal involvement. The aim of this study was to evaluate whether tissue biomarkers with links to melanoma biology, together with clinicopathological parameters, could aid in the prediction of sentinel node involvement and improve selection of patients for SNB. In addition, we examined the role of these clinical or biological markers in disease outcome.MethodsWe collected a case-control cohort of 140 intermediate thickness (Breslow 0,9–4,0mm) melanoma patients with or without SNB involvement matched for age, gender, Breslow thickness and location. From this cohort, we tested the predictive value of common clinicopathological parameters (ulceration, mitotic count and tumor regression) and FMNL-2, ezrin and BRAF V600E immunoreactivity, for sentinel node involvement and survival. We further analyzed the correlations in the superficial spreading melanoma subtype.ResultsBased on our case control analysis, of the markers, BRAF V600E status (p = 0.010) and mitotic count (p = 0.036) correlated with SNB involvement. SNB status was a strong independent prognosticator for recurrence free survival (RFS p<0.001), melanoma specific survival (MSS p = 0.000) and overall survival (OS p = 0.029). In the superficially spreading melanoma subgroup, BRAF V600E positivity indicated poorer RFS (p = 0.039) and OS (p = 0.012). By combining the Breslow thickness, mitotic count and BRAF immunohistochemistry, we identified a group of superficially spreading melanomas with an excellent survival probability independent of SNB status.ConclusionsThese results demonstrate that BRAF immunohistochemistry could serve as a useful addition to a marker panel for selecting intermediate thickness melanoma patients for SNB.

Innate immune cell infiltration in melanoma metastases affects survival and is associated with BRAFV600E mutation status.

Little is known about the infiltrative pattern of innate immune cells in primary melanoma compared with their paired metastases and in BRAF-mutated tumors. Therefore, our aim was to characterize the inflammatory microenvironment in primary ulcerated and nonulcerated melanomas and paired metastases, to investigate the relation between inflammation and BRAF mutation in primary melanoma and paired metastases, and to evaluate the effect of the analyzed biomarkers on melanoma-specific survival. A total of 385 primary tumors and 96 paired metastases were stained with immunohistochemistry for BRAF, CD163+ macrophages, CD123+ plasmacytoid dendritic cells, CD66b+ neutrophils, and E-cadherin and estimated using objective computer-assisted image analysis. BRAF was semiquantitatively scored as either present or absent. In metastases of nonulcerated melanomas, we observed higher neutrophil (P=0.02) and macrophage (P=0.01) numbers. In the metastases of ulcerated melanomas, we found a higher number of macrophages (P<0.0001). Increase in the neutrophil numbers in the metastases was associated with poor patient survival after first relapse (hazard ratio=1.19, 95% confidence interval: 1.03-1.38, P=0.02). BRAF-positive primary tumors (P=0.02) and metastases (P=0.01) exhibited increased plasmacytoid dendritic cell numbers compared with BRAF-negative tumors. Lastly, primary melanomas in men had higher neutrophil numbers than women (P≤0.0001), and men had worse melanoma-specific survival (hazard ratio=1.52, 95% confidence interval: 1.04-2.21, P=0.03). Our data show that melanoma metastases are densely infiltrated with neutrophils, which affects survival. Our results also highlight the importance of recognizing the presence of inflammatory cells in the metastases as a prognostic marker, and that they may potentially be used to improve the precision of immunotherapy and BRAF targeted therapy.

Ectopic Thyroid Tissue: Immunohistochemistry and Molecular Analysis.

Ectopic thyroid tissue is rare and controversial. Some experts consider it to always be metastatic thyroid carcinoma, whereas others consider it benign as long as it is restricted to few follicles without cytoarchitectural features of papillary thyroid carcinoma. Immunohistochemistry (IHC) and molecular studies have not yet been performed to further characterize this entity. We retrospectively searched our pathology files for all ectopic thyroid inclusions and reviewed clinicopathologic characteristics and concurrent thyroid pathologic findings. We identified 8 cases from 7 patients. Ectopic thyroid tissue was present in the following locations: neck soft tissue: 3, thymus: 2, neck lymph nodes: 2, perihilar soft tissue: 1. All patients had histologically benign thyroid specimens. BRAFV600E (VE1) IHC, HBME-1 IHC, galectin-3 IHC, BRAFV600E allele-specific polymerase chain reaction (PCR) and NRAS/KRAS pyrosequencing were performed. To assess the sensitivity and specificity of BRAFV600E IHC compared with PCR; we tested 13 cases of primary and metastatic papillary and follicular thyroid carcinomas. All the ectopic cases were HBME-1, galectin-3, BRAFV600E (IHC, PCR), and NRAS/KRAS mutation negative (specificity=100%). Compared with PCR, BRAF IHC had 89% sensitivity and 100% specificity. Lack of common carcinoma-associated mutations supports benign nature of this entity. BRAF, HBME-1, and galectin-3 IHC are accurate and helpful when not enough tissue is available for molecular studies. IHC and molecular studies are more helpful than morphology alone in identifying benign thyroid rests.

Association of malignant potential of BRAF mutant colorectal cancer with coexpression of PD-L1.

643 Background: PD-L1 is a member of the B7/CD28 family that regulates immune response, and also well known as targets for immune checkpoint inhibitor. On the other hand BRAF mutation (MT) is recognized as a strongly poor prognostic factor in colorectal cancer (CRC). Recently in addition to the RAS gene, a lot of new knowledge about some molecules playing an important role in the process of carcinogenesis or drug resistance, and emerging molecules as targets for new treatments have been reported. In this research, we aim to address the clinical significance of PD-L1 expression in colorectal carcinoma correlation between emerging molecules and differences through clinical stages in CRC using tissue array. Methods: Consecutive patients who underwent surgery in our hospital from June 2003 to March 2011 were enrolled in this study. Tissue array based profiling of emerging molecules was performed on archival samples using immunohistochemistry for BRAF, MLH1/MSH2/MSH6/PMS2, CDX2, HER2 and PD-L1. We analyzed PD-L1 expression and correlation with molecular profile, survival, clinicopathological findings and location of primary site. Results: A total of 1030 CRC from stage 0 to IV were analyzed. The expression rates of PD-L1 were 3.6% and increased significantly in BRAF MT, dMMR, lack of CDX-2, tumor grade3 (TG3), right sided colon of primary site (25%, 37.8%, 21.6%, 22.1%, 6.9%, respectively; P &lt; 0.001). In the univariate analysis, lack of CDX2, BRAF MT and TG3 were identified as prognostic factors in whole population of this cohort. Furthermore the survival of BRAF MT and lack of CDX2 with co-expression of PD-L1 were extremely poor (median OS in stage IV; 2.6 months in both). In Particular the co-expression rates of PD-L1 in BRAF MT were increasing as the clinical stage progress regardless of dMMR. Conclusions: Our study comprehensively summarized the significance of PD-L1 expression associated with the recent emerging molecules and suggested that malignant potential of BRAF mutant CRC may be explained in part due to escape from immunity by co-expression of PD-L1. Immune checkpoint inhibitor could be a candidate of target of BRAF mutant CRC with PD-L1expression irrespective of MSI status.

Pulmonary metastasectomy for colorectal cancer: analysis of prognostic factors affecting survival.

Pulmonary metastasectomy is considered a standard procedure in the treatment of metastatic colorectal cancer (CRC). Different prognostic factors including multiple metastatic nodules, the presence of extra-pulmonary metastases and BRAF mutation status have been associated with poor survival. The aim of this study was to evaluate which factors influenced survival in CRC patients undergoing pulmonary metastasectomy by studying primary tumors and pulmonary metastases. All patients treated for primary CRC who presented pulmonary metastases in a 10-year period were considered (group A). A control group treated for primary CRC who did not develop any pulmonary or extra-pulmonary metastases was taken for comparison (group B). Different prognostic factors including gender, age, tumor location, histological type, inflammatory infiltrate, BRAF, CDX2 and extra-pulmonary metastases were analyzed. Overall survival (OS) and patients' survival after pulmonary metastasectomy were also considered. Fifty-four patients were evaluated in group A and twenty-three in group B. In group A, BRAF immunohistochemistry did not significantly differ between primary tumors and pulmonary metastases; no difference of BRAF expression was found between group A and B. Even the expression of CDX2 was not significantly different in primary tumors and metastases. Similarly, in group B CDX2 did not significantly differ from primary CRC of group A. The most significant prognostic factor was the presence of extra-pulmonary metastases. Patients with extra-pulmonary metastases experienced a significant shorter survival compared to patients with pulmonary metastases alone (P=0.001 with log-rank test vs. P=0.003 with univariate Cox regression). Interestingly, patients with right pulmonary metastases presented a significant longer survival than those with left pulmonary metastases (P=0.027 with log-rank test vs. 0.04 with univariate Cox regression). The main prognostic factor associated with poor survival after lung resection of CRC metastases is a history of extra-pulmonary metastases. BRAF and CDX2 did not have a significant role in this small series of patients.

Local validation of B-Raf immunohistochemistry in hairy cell leukaemia

Local validation of B-Raf immunohistochemistry in hairy cell leukaemia

P2.01-035 Protein and Molecular Alterations in EMT Pathways of Lung Cancer: A Comparative Analysis between NSCLCs

The adoption of next-generation sequencing (NGS) may help to identify single nucleotide variants (SNVs), small insertions–deletions (indels), and larger structural variations including chromosomal rearrangements. Many molecular alterations have protein-level associations that can be questioned using immunohistochemistry (IHC). The goal of our work was investigated molecular patterns of predictive biomarkers and new genes involved as potential therapeutic targets with an emphasis on protein IHC and their translational promise. We studied 212 formalin fixed and paraffin embedded tissues: 8 high-grade and 20 low-grade neuroendocrine carcinomas(NEC), 102 adenocarcinomas(ADC), 65 squamous cell carcinomas (SCC) and 17 large cell carcinomas(LCC), placed in tissue microarrays(TMAs). EGFR,P53,KRAS,ALK,ERBB2,PTEN,BRAF,VEGF,CD24 and CD44 were examined using IHC and Aperio system. DNA extracted(QIAmp) from a subset was used to analyze several variants including EGFR, ERBB2, PIK3CA, MMP2, SNAI, VGFA, VIM, ZEB1, AXL, CD44, CD276, and CDH1, using the TruSeq Custom Amplicon assay on the Illumina MiSeq System, resulting data set for 80 LC were analyzed using the Variant-Studio software and correlated them with the clinocopathological data. The median age of the patients was 64 yrs (minimum-maximum, 24-88yrs). The population included 98(44.5%) women; 32(14,5%) never smokers and 100(45,5%) former smokers; only 2(1%) Asians. Our image analysis showed that the median IHC protein expressions were similar between low and high-grade NEC, but those were different compared to others (P<0.05). Indeed, EGFR, EBB2, P53 and BRAF IHC expression were significantly lower in NEC group compared to other subtypes (P<0.05). Overall, LCC have lower protein expression than ADC and SCC, specially P53 and VEFG. We detected several drivers mutation including EGFR 22%(19/80), ERBB2 2%(5/80), immune regulated genes CD276 6.8%(17/80) and CTLA 15.4%(39/80). We observed also EMT gene mutations as CD44 30.7%(78/80), MMP2 2.8%(7/80), VGFA 2%(5/80), CDH1 2.4%(6/80), SNAI 2.8%(7/80), VIM 1.2%(3/80), and ZEB1 4%(12/80). Interestingly, a significant higher AXL and CD44 gene expression was found in ADC and SCC specimens compared to NEC(P=0.001 and P=0.04,respectively) Similar to the protein expression in overall low gene expressions was also observed in LCC compared to others. We detected different patterns of protein and gene alteration in LC with predominant low expression in NEC. Furthermore, the high expression of EMT genes as AXL and CD44 observed among ADC and SCC can be a evidence that those genes might be a distinctive RTK in these tumor than in NEC tumor suggesting that targeting these genes will be benefit as anti-cancer treatment.

A UK feasibility and validation study of the VE1 monoclonal antibody immunohistochemistry stain for BRAF-V600E mutations in metastatic melanoma.

Background:Determining the BRAF mutation status of patients with advanced metastatic melanoma is essential in order to assess patients' eligibility for targeted BRAF inhibitor therapy. The aim of this study was to validate the utility of immunohistochemistry (IHC) to rapidly obtain the BRAF status in the UK cancer centre setting.Methods:All samples sent for molecular testing for detection of the BRAF mutation over a 26-month period were prospectively tested using the VE1 monoclonal antibody IHC stain.Results:Two-hundred and nineteen samples from 214 patients were identified. All patients were AJCC stage III/IV, except one. There was an overall 95.0% (208/219) concordance rate, with a sensitivity of 94.4% (84/89) and a specificity of 95.4% (124/130) when using genomic assays as the gold standard. Discordance resulted from the inability of the molecular technique to detect the V600E2 mutation and an inability of the IHC staining technique to detect non-V600E mutations. Molecular testing on smaller tumour deposits was also unreliable.Conclusions:IHC staining has good sensitivity and excellent specificity for BRAF V600E mutations. BRAF IHC can be incorporated into a BRAF mutation testing algorithm for UK cancer centres to as a feasible first-line assay and identify a subset of cases that require subsequent genomic testing. It has the additional major advantages of reduced cost and rapid turnaround time.

Diagnostic Accuracy of BRAF Immunohistochemistry in Colorectal Cancer: a Meta-Analysis and Diagnostic Test Accuracy Review.

The aim of this study was to evaluate the concordance between the BRAF (V600E) mutation test and immunohistochemistry (IHC) and to evaluate the diagnostic accuracy of BRAF IHC for colorectal cancer (CRC) through a systematic review, meta-analysis, and diagnostic test accuracy review. The current study included 1021 CRCs from eight eligible studies. The concordance rates were investigated between BRAF IHC and the mutation test. In addition, diagnostic test accuracy review was conducted and calculated using the value of area under curve (AUC) on the summary receiver operating characteristic (SROC) curve. The positive rate of BRAF IHC was 30.5% (range; 13.2-66.2%), and the BRAF mutation was found in 30.2% (range; 11.7-66.2%). The overall concordance rate between BRAF IHC and the mutation test was 0.944 (95% confidence interval (CI) 0.873-0.977). In the BRAF IHC-positive and -negative groups, the concordance rates between BRAF IHC and the mutation test were 0.895 (95% CI 0.800-0.945) and 0.956 (95% CI 0.878-0.985), respectively. The pooled sensitivity and specificity were 0.94 (95% CI 0.91-0.96) and 0.96 (95% CI 0.95-0.98), respectively. The diagnostic odds ratio was 272.86 (95% CI 46.11-1614.88), and the value of AUC on SROC curve was 0.9846. Taken together, our results suggest that BRAF IHC is strongly concordant with the BRAF mutation test and has high diagnostic accuracy in BRAF mutation analysis of CRCs. Further cumulative studies on detailed evaluation criteria are needed before application in daily practice.