Negative reactions of BRAF mutation‐specific immunohistochemistry to non‐V600E mutations of BRAF

BRAF mutations are rare driver mutations in non‐small cell lung cancer (NSCLC), accounting for 1%–2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non‐V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non‐V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation‐specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non‐V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non‐V600E mutations, using BRAF mutation‐specific IHC. None of the tumors with non‐V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation‐specific IHC is specific without any cross‐reactions to non‐V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.     

Immunohistochemical analysis using a BRAF V600E mutation specific antibody is highly sensitive and specific for the diagnosis of hairy cell leukemia

Hairy cell leukemia (HCL) is usually diagnosed by morphology and flow cytometry studies. However, it is challenging sometimes to distinguish HCL from its mimics. Recently, the BRAF V600E mutation has been described as a disease-defining molecular marker for HCL which is present in nearly all cases of HCL but virtually absent in mimics of HCL. In this study, we investigated the possibility of using immunohistochemical detection of the BRAF V600E mutant protein to differentiate HCL from its mimics. A total of twenty-eight FFPE tissue specimens were studied, including HCL (n=12), HCL variant (HCL-v, n=3), splenic marginal zone lymphoma (SMZL, n=6), and other marginal zone lymphomas (MZL, n=7). Immunohistochemical studies were performed using a mouse monoclonal antibody (clone VE1, Spring Bioscience, CA) specific for BRAF V600E mutation. Molecularly confirmed BRAF V600E mutation positive and negative cases were used as the positive and negative controls respectively. All 12 cases of HCL showed cytoplasmic BRAF V600E protein expression in leukemia cells by immunohistochemical study regardless of tumor burden, whereas all cases of HCL mimics including HCL-v, SMZL, and MZL were negative for BRAF V600E protein. Using this BRAF V600E mutation specific antibody, this immunohistochemical study has 100% sensitivity and 100% specificity for the diagnosis of HCL in our cohort. In conclusion, immunohistochemical detection of the BRAF V600E mutant protein is highly sensitive and specific for the diagnosis of HCL. Compared to PCR or sequencing-based methodologies, immunohistochemistry is a relatively rapid and inexpensive alternative for the differential diagnosis between HCL and its mimics.     

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome

Champion KJ, Bunag C, Estep AL, Jones JR, Bolt CH, Rogers RC, Rauen KA, Everman DB. Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome. BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio‐facio‐cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild‐type BRAF but is less strongly activating than the cancer‐associated p.V600E mutation.     

Identification of the V600D mutation in Exon 15 of the BRAF oncogene in congenital,benign langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a well‐known but rare disease that may occur at any age with markedly variable clinical features: self‐regressive, localized, multiorgan, aggressive, or fatal outcome. Congenital LCH is rare and often clinically benign. While LCH is characterized by a clonal proliferation of Langerhans cells, its etiology is unknown. Although BRAF V600E mutations were recently identified as a recurrent genetic alteration in LCH cases, the clinical significance of this mutation within the heterogeneous spectrum of LCH is also currently unknown. We studied a cutaneous, benign form of congenital LCH that occurred in a newborn male, without recurrence for 8 years. Histopathologically, the skin lesion excised after birth showed the typical cytologic and immunophenotypic features of LCH. Sequencing analysis of Exon 15 of the BRAF gene revealed the V600D mutation, with an allelic abundance of 25–30%, corresponding to the LCH cells being hemizygous for the mutant allele. BRAF V600E‐specific polymerase chain reaction was negative. Our report is the first to identify the rare, variant BRAF V600D mutation in LCH, and provides support for constitutively activated BRAF oncogene‐induced cell senescence as a mechanism of regression in congenital, benign LCH. Further, our clinicopathologic findings provide proof for the first time that the V600D mutation can also occur in the absence of ultraviolet light, and can occur in a clinically benign proliferation, similar to the V600E mutation. Additional clinicopathologic studies in larger numbers of LCH patients may be valuable to ascertain the pathophysiologic role of BRAF mutations in LCH. © 2012 Wiley Periodicals, Inc.     

An immunohistochemical and genetic study of BRAFV600E mutation in Japanese patients with ameloblastoma

Ameloblastoma is an odontogenic tumor of the jaw. It most frequently occurs in the mandible, and less often in the maxilla. Mandibular ameloblastoma harbors a BRAF mutation that causes a valine (V) to glutamic acid (E) substitution at codon 600 (BRAF^V600E). We examined specimens from 32 Japanese patients to detect the prevalence of the BRAF^V600E mutation, and to evaluate the relationship between immunohistochemical (IHC) expression and genetic results, of BRAF^V600E+ ameloblastoma. Among the 32 cases, 22 (69%) were IHC positive for BRAF^V600E protein, and 10 (31%) were IHC negative; and polymerase chain reaction showed 16 of 21 tested cases (76%) carried the BRAF^V600E mutation. Our findings indicate that that samples that stain IHC positive for BRAF^V600E protein are more likely to carry the BRAF^V600E mutation. These results support assessments for BRAF mutations, and the use of BRAF inhibitors as targeted therapy for ameloblastoma in Japanese patients.     

Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes

Aims: The study compares detection rates of oncogenic BRAF mutations in a homogenous group of 236 FFPE cutaneous melanoma lymph node metastases, collected in one cancer center. BRAF mutational status was verified by two independent in-house PCR/Sanger sequencing tests, and the Cobas® 4800 BRAF V600 Mutation Test. Results: The best of two sequencing approaches returned results for 230/236 samples. In 140 (60.9%), the mutation in codon 600 of BRAF was found. 91.4% of all mutated cases (128 samples) represented p.V600E. Both Sanger-based tests gave reproducible results although they differed significantly in the percentage of amplifiable samples: 230/236 to 109/143. Cobas generated results in all 236 cases, mutations changing codon V600 were detected in 144 of them (61.0%), including 5 not amplifiable and 5 negative in the standard sequencing. However, 6 cases positive in sequencing turned out to be negative in Cobas. Both tests provided us with the same BRAF V600 mutational status in 219 out of 230 cases with valid results (95.2%). Conclusions: The total BRAF V600 mutation detection rate didn’t differ significantly between the two methodological approaches (60.9% vs. 61.0%). Sequencing was a reproducible method of V600 mutation detection and more powerful to detect mutations other than p.V600E, while Cobas test proved to be less susceptible to the poor DNA quality or investigator’s bias. The study underlined an important role of pathologists in quality assurance of molecular diagnostics.     

Association between BRAF V600E mutation and regional lymph node metastasis in papillary thyroid carcinoma

Background: BRAF V600E is the most frequent genetic alteration in papillary thyroid carcinoma (PTC); there are ongoing conflicts on its association with regional lymph node metastasis. And we aimed to test this association in a referred sample in a single institute in China. Methods: We analyzed BRAF V600E mutational status in the primary lesion of 150 PTC cases in Peking Union Medical College Hospital (PUMCH) and their corresponding lymph node metastasis (if present and available) using a validated Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) method. Results: Among 150 PTC cases, 121 (80.6%) primary tumors harbored BRAF V600E mutation, 66.9% (81/121) and 79.3% (23/29) had regional lymph node metastasis (LNM) in cases detected with and without BRAF V600E mutation, respectively (P = 0.195). The BRAF V600E mutational status of most of the metastatic lesions was not different to that of their primary foci (73 out of 76 cases, 96.1%, Kappa value = 0.893). The 3 inconsistent cases were all mutation positive for primary tumors and mutation negative for LNM. Conclusion: No association was established between BRAF V600E mutation and regional lymph node metastasis in PTC in Chinese patients.     

The extended spectrum of RAS‐MAPK pathway mutations in colorectal cancer

Current clinical guidelines recommend mutation analysis for select codons in KRAS and NRAS exons 2, 3, and 4 and BRAF V600E to guide therapy selection and prognostic stratification in advanced colorectal cancer. This study evaluates the impact of extended molecular testing on the detection of RAS‐MAPK pathway mutations. Panel next‐generation sequencing results of colorectal cancer specimens from 5795 individuals from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR Project GENIE) were included. Mutations in RAS‐MAPK pathway genes were analyzed and functionally annotated. Colorectal cancers had recurrent pathogenic pathway activating mutations in KRAS (44%), NRAS (4%), HRAS (<1%), BRAF (10%), MAP2K1 (1%), RAF1 (<1%), and PTPN11 (<1%). The proportion of colorectal cancers with pathogenic RAS pathway mutations was 37% when only KRAS codon 12 and 13 mutations were considered, 46% when also including select KRAS and NRAS exons 2, 3, and 4 mutations, 53% when including BRAF V600E mutations, and 56% when including all pathogenic mutations. Panel next‐generation sequencing testing identifies additional RAS‐MAPK pathway driver mutations beyond current guideline recommendations. These mutations have potential implications in treatment selection for patients with advanced colorectal cancer.     

Papillary thyroid carcinomas with and without BRAF V600E mutations are morphologically distinct

Finkelstein A, Levy G H, Hui P, Prasad A, Virk R, Chhieng D C, Carling T, Roman S A, Sosa J A, Udelsman R, Theoharis C G & Prasad M L
(2012) Histopathology  60, 1052–1059 Papillary thyroid carcinomas with and without BRAF V600E mutations are morphologically distinct Aims: The BRAF V600E mutation resulting in the production of an abnormal BRAF protein has emerged as the most frequent genetic alteration in papillary thyroid carcinomas (PTCs). This study was aimed at identifying distinctive features in tumours with and without the mutation. Methods and results: Thirty‐four mutation‐positive and 22 mutation‐negative tumours were identified by single‐strand conformation polymorphism of the amplified BRAF V600E region in the tumour DNA. Mutation‐positive tumours were more common in patients older than 45 years (24/33, P = 0.05), in classic (23/30, P = 0.01), tall cell (4/5) and oncocytic/Warthin‐like (2/2) variants of PTC, and in subcapsular sclerosing microcarcinomas (4/4). In contrast, all 12 follicular variants (P < 0.0001) and two diffuse sclerosing variants were negative for the mutation. Mutation‐positive tumours displayed infiltrative growth (32/34, P = 0.02), stromal fibrosis (33/34, P < 0.001), psammoma bodies (17/34, P = 0.05), plump eosinophilic tumour cells (22/34, P = 0.01), and classic fully developed nuclear features of PTC (33/34, P = 0.0001). Encapsulation was significantly associated with mutation‐negative tumours (15/22, P = 0.02). Conclusions: BRAF V600E mutation‐positive and negative PTCs are morphologically different. Recognition of their morphology may help in the selection of appropriate tumours for genetic testing.     

Distribution of KRAS and BRAF mutations in Moroccan patients with advanced colorectal cancer

Targeted therapies have an increasing importance in digestive oncology. To our knowledge, we are the first to report the distribution of KRAS and BRAF mutations in Moroccan patients with advanced colorectal cancer (CRC) in order to introduce targeted therapy in the arsenal of therapeutic modalities for management of this cancer in Morocco. In this study, 92 samples obtained from patients with CRC were tested for the presence of the nine most common mutations in the KRAS gene and BRAF gene. Among the tested patients, 76.09% of patients had wt-KRAS genotype and 23.91% were KRAS mutants and the majority of mutations would result in an amino acid substitution of glycine by aspartic acid (68.2%) The predominant mutations are G>A transitions and G>T transversions. Around 5% (5.43%) of the tested patients bore the V600E mutation in BRAF gene. Only one patient showing to have the V600E mutation in BRAF was also mutated-KRAS. Summing up the results about the KRAS and the BRAF mutation carriers from our study, the portion of potentially non responsive patients for the anti-EGFR treatment is 28.26%.     

Aberrant BRAF splicing as an alternative mechanism for oncogenic B‐Raf activation in thyroid carcinoma

Activating BRAF mutations have recently been reported in 28–83% of papillary thyroid carcinomas (PTCs). However, it is not known whether aberrant BRAF splicing occurs in thyroid carcinoma. To investigate aberrant BRAF splicing and its association with BRAF mutation in thyroid tumours, we studied aberrant BRAF splicing and BRAF mutation from 68 thyroid tumours. BRAF^V600E mutation was detected in 20 of 43 PTCs and all three anaplastic thyroid carcinomas (ATCs). There is a higher frequency of BRAF mutation in PTC patients with stage III and IV tumours compared with stage I and II. Novel BRAF splicing variants were detected in 12 PTCs, three follicular variants of PTC (FVPTCs), and one ATC, as well as in two thyroid carcinoma cell lines, ARO and NPA. These variants did not have the N‐terminal auto‐inhibitory domain of wild‐type B‐Raf, resulting in an in‐frame truncated protein that contained only the C‐terminal kinase domain and caused constitutive activation of B‐Raf. These variants were significantly associated with advanced disease stage and BRAF^V600E mutation (p < 0.001, Fisher exact test). Furthermore, expression of these variants in NIH3T3 and CHO cells could activate the MAP kinase signalling pathway, transform them in vitro, and induce tumours in nude mice. These data suggest that BRAF splicing variants may function as an alternative mechanism for oncogenic B‐Raf activation. Combination of the BRAF^V600E mutation and its splicing variants may contribute towards disease progression to poorly differentiated thyroid carcinoma. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Co‐occurrence of histone H3 K27M and BRAF V600E mutations in paediatric midline grade I ganglioglioma

Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co‐occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty‐seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co‐occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double‐immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow‐up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow‐up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M‐mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors.     

BRAF mutations in KIT/PDGFRA positive gastrointestinal stromal tumours (GISTs): Is their frequency underestimated?

BRAF V600E mutations in GISTs are considered to be one of the mutational events in KIT/PDGFRA negative or positive GISTs, respectively. BRAF mutated GISTs usually do not respond to imatinib treatment, even more GISTs with imatinib sensitive KIT mutation. However, they are almost phenotypically and morphologically identical with KIT/PDGFRA positive GISTs. In general, due to the small number of BRAF mutations in GIST and because of the rarity of concomitant BRAF/KIT or BRAF/PDGFRA mutations, their frequency may be depreciated. The aim of this study was BRAF mutation detection in KIT/PDGFRA positive GISTs and their verification by other molecular methods. We applied the sensitive droplet digital PCR on 35 randomly selected KIT/PDGFRA positive GISTs to detect V600E mutations. We have established two criteria for the evaluation of samples: false positive rate (FPR) based on the negative controls; Limit of Detection (LoD) based on the serial dilution of positive control from RKO cell line harboring heterozygous V600E mutation in constant wild-type DNA background. Results from ddPCR were verified by other molecular methods: allele-specific PCR, dideoxysequencing, competitive allele-specific TaqMan PCR (castPCR). FPR was determined as 5 (∼4.4) positive droplets, and LoD was assessed to 3.4293 copies/μL what is the method sensitivity of 0.0162 %.We identified eight KIT/PDGFRA positive patients with concomitant V600E mutation. The five of them were in coexistence with KIT mutation and three with PDGFRA mutation. We also included the liver metastasis, but data from primary tumour were not available. We achieved the very high sensitivity of the ddPCR method for detecting BRAF mutation in GISTs to have importance from the point of view of therapy.     

BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma

The serine/threonine‐protein kinase B‐raf (BRAF) is an oncogene mutated in various neoplasms, including 5–15% of colorectal carcinomas. The T1799A point mutation, responsible for a large majority of these alterations, results in an amino acid substitution (V600E) causing the constitutive activation of a protein kinase cascade. BRAF V600E in MLH1 deficient tumors implicates somatic tumor‐only methylation of the MLH1 promoter region instead of a germline MLH1 mutation. BRAF V600E also predicts poor prognosis in microsatellite stable colorectal cancers and may be a marker of resistance to anti‐EGFR therapy in metastatic disease. Currently, only molecular methods are available for assessing BRAF mutational status. An immunohistochemical approach is evaluated here. Colon cancers from 2008 to 2012 tested by pyrosequencing for BRAF V600E mutation were selected. A total of 31 tumors with (n = 14) and without (n = 17) the BRAF V600E mutation were analyzed by immunohistochemistry using a commercially available antibody specific to the V600E‐mutated protein. All 14 colorectal carcinomas with the BRAF V600E mutation demonstrated cytoplasmic positivity in tumor cells with the anti‐BRAF antibody. In a minority of cases, staining intensity for the mutated tumor samples was weak (n = 2) or heterogeneous (n = 4); however, the majority of cases showed diffuse, strong cytoplasmic positivity (8 of 14 cases). None of the 17 BRAF wild‐type colorectal cancers showed immunoreactivity to the antibody. The overall sensitivity and specificity of the immunohistochemical BRAF V600E assay was 100%. Detection of the BRAF V600E mutation in colorectal cancer by immunohistochemistry is a viable alternative to molecular methods. © 2013 Wiley Periodicals, Inc.     

Tumor Heterogeneity Revealed by KRAS,BRAF, and PIK3CA Pyrosequencing: KRAS and PIK3CA Intratumor Mutation Profile Differences and Their Therapeutic Implications

Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.     

Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients’ age but not with tumour aggressiveness

A high prevalence of the BRAF^V600E somatic mutation was recently reported in several series of papillary thyroid carcinomas (PTC). This mutation appears to be particularly prevalent in PTC with a predominantly papillary architecture. Another BRAF mutation (K601E) was detected in a follicular adenoma and in some cases of the follicular variant of PTC. The few studies on record provided controversial data on the relationship between the occurrence of BRAF mutations and clinicopathologic parameters such as gender, age and tumour staging. In an attempt to clarify such controversies we decided to enlarge our previous series to 315 tumours or tumour-like lesions diagnosed in 280 patients, including a thorough analysis of several clinicopathologic features. The BRAF^V600E mutation was exclusively detected in PTC with a papillary or mixed follicular/papillary architecture both of the conventional type (46%) and of other histotypes, such as microcarcinoma (43%), Warthin-like PTC (75%) and oncocytic variant of PTC (55%). The BRAF^K601E mutation was detected in four of the 54 cases of the follicular variant of PTC (7%). The mean age of patients with conventional PTC harbouring BRAF^V600E (46.7 years) was significantly higher (P<0.0001) than that of patients with conventional PTC without BRAF^V600E (29.5 years). The BRAF (BRAF^V600E) mutated PTC did not exhibit signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) and were in fact less often multicentric than PTC without the mutation.V. Trovisco and P. Soares contributed equally to this workFundação para a Ciência e Tecnologia POCTI/FEDER (POCTI/NSE/48171/2002)     

Detection of BRAFV600E mutation on papillary thyroid carcinoma and metastatic malignant melanoma by fine‐needle aspiration cytology

A genetic link between cutaneous melanoma and thyroid cancer (TC) has been identified. A high percentage of both melanomas and papillary carcinomas of the thyroid harbors a recurrent mutation (i.e., BRAF^V600E) in the BRAF oncogene. Herein, we report the case of a 65‐year‐old man with papillary TC and cutaneous malignant melanoma metastatic to masseter muscle, both characterized by BRAF mutation. This is one of the rare reports in which a complete molecular characterization has been performed. As the patients with papillary thyroid carcinoma have a higher risk of malignant melanoma and vice versa, continuous monitoring of such patients, with either of these tumors is necessary. Fine‐needle aspiration cytology is useful as shown in the present case. Diagn. Cytopathol. 2014;42:877–879. © Wiley Periodicals, Inc.     

Clinicopathological characteristics and molecular analysis of lung cancer associated with ciliated muconodular papillary tumor/bronchiolar adenoma

Ciliated muconodular papillary tumor/bronchiolar adenoma (CMPT/BA) is a recently introduced benign lung tumor. It remains unclear whether CMPT/BA is associated with a specific type of lung cancer (LC). We studied the clinicopathological characteristics and genetic profiles of the coexisting primary LC and CMPT/BA (LCCM) cases. We identified eight LCCM (0.4%) from the resected Stage 0–III primary LC (n = 1945). The LCCM cohort was male-dominant (n = 8), elderly (median 72 years old), and most were smokers (n = 6). In addition to the adenocarcinoma (n = 8), we detected two squamous cell carcinomas and one small cell carcinoma—in some cases, multiple cancer. The target sequence/whole exome sequence (WES) revealed no shared mutations between CMPT/BA and LC. One exceptional case was invasive mucinous adenocarcinoma harboring an HRAS mutation (I46N, c.137T>A), but it was likely to be a single nucleotide polymorphism based on variant allele frequency (VAF). Other driver mutations in LC included EGFR (InDel, n = 2), BRAF(V600E) (n = 1), KRAS (n = 2), GNAS (n = 1), and TP53 (n = 2). BRAF(V600E) was the most frequent mutation in CMPT/BA (60%). In contrast, LC showed no specific trend in driver gene mutations. In conclusion, our study revealed differences in the gene mutation profiles of CMPT/BA and LC in coexisting cases, suggesting mostly independent clonal tumorigenesis of CMPT/BA from LC.     

BRAF mutation in sporadic colorectal cancer and Lynch syndrome

The aim of the study was to detect mutations of BRAF oncogene in colorectal cancer and to use this information to identify Lynch syndrome patients. Consecutive cases of primary colorectal cancer (n?=?137) were analyzed for MLH1 protein expression using immunohistochemistry (IHC). BRAF V600E mutation was detected by IHC using a specific monoclonal antibody (VE1) and by qPCR. All MLH1 protein-negative cases were subjected to microsatellite instability analysis and MLH1 promoter methylation assay. MLH1 protein expression deficiency and high microsatellite instability (MSI-H) were detected in 18 of the 137 (13.1 %) consecutive colorectal cancer specimens. Detection of the BRAF V600E mutation by IHC was 100 % sensitive and specific as compared to qPCR, and this mutation was frequently present in the MSI-H group (77.8 %; 14/18) and less frequently in the microsatellite-stable group (7.6 %; 9/118). All BRAF V600E mutated cases of the MSI-H group presented with a MLH1 promoter methylation (14/14) as detected by methylation-specific multiplex ligation-dependent probe amplification. When BRAF was wild type in the MSI-H group, only one MLH1 promoter methylation was detected (1/4), and of the remaining three cases without MLH1 methylation, two were identified to harbor an MLH1 mutation consistent with Lynch syndrome. Finally, 11 previously confirmed Lynch syndrome cases were analyzed for BRAF V600E mutation, and all of them were wild type. In conclusion, detection of BRAF V600E in colorectal cancer specimens by IHC is sensitive and specific and may help to identify Lynch syndrome patients.