Accumulation of molecular alterations, including EGFR overexpression and mutations in KRAS and BRAF, contribute to colorectal carcinogenesis. Since intestinal-type adenocarcinoma (ITAC) of the nasal cavity and paranasal sinus has morphologic and phenotypic features that are usually indistinguishable from colorectal cancer (CRC), it is likely that both tumor types share equivalent genetic alterations. Data from a series of 43 patients treated surgically for ITAC in Montpellier, France between November 1998 and December 2012 were collected. Tumors were characterized for mutations in KRAS and BRAF as well as EGFR overexpression. Kaplan-Meier survival curves were constructed using overall survival as the primary end points. Patient survival was analyzed using the hazards ratio. Twenty seven tumors (63%) showed EGFR positivity and 30% exhibited a high expression level (+2/+3). KRAS mutations were detected in 43% of cases. BRAF mutations were identified in 3.6% of specimens. Patients with age superior to 60 years, metastatic status, and KRAS mutations had significant overall survival values (p = 0.026, p = 0.001 and p = 0.03, respectively). Our results indicate that KRAS mutations and EGFR expression are frequent in ITAC and that KRAS mutations predict good patient prognosis in ITAC. Finally, EGFR directed molecular treatments could be investigated in a subset of patients affected by ITAC.

BackgroundSince patients diagnosed with BRAF V600E and V600K mutated advanced melanoma show response to treatment with MAP kinase inhibitors, several sensitive methods have been developed to determine the V600 allele status of melanoma patients. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar) are specific BRAF V600 inhibitors recently approved by the US FDA as single agent treatments for unresectable or metastatic melanoma in patients with the BRAF V600 mutation.MethodsWe assessed the new CE THxID™-BRAF diagnostic test, which is also FDA-approved as a companion diagnostic test in the US under a specific reference and compared the results of this assay with both High Resolution Melting (HRM) and Sanger sequencing in 113 melanoma FFPE samples.ResultsInvalid results were observed in 0/113 specimen with HRM, 5/113 (4.4%) with Sanger sequencing, and 1/113 (0.9%) with the THxID™-BRAF test. Positive percentage agreement (PPA) was 93.5% (95% CI 82.5 - 97.8) for V600E and V600K mutations combined for the THxID™-BRAF test and HRM, and negative percentage agreement (NPA) was 100.0% (95% CI 94.5 - 100.0). For the THxID™-BRAF test and Sanger, PPA was 100.0% (95% CI 92.1 - 100.0) and NPA 100.0% (95% CI 94.2 - 100.0). One V600E sample identified by THxID™-BRAF test was detected as wild-type by HRM and uninterpretable by Sanger. All V600K (n = 3) were detected using the 3 different approaches. Finally, percent agreement values were not significantly different when using punches (n = 77) vs. slides (n = 36) or depending on samples characteristics such as pigmentation, necrosis, and tumor content.ConclusionsThis study demonstrated the high agreement between the FDA approved THxID™-BRAF assay, HRM, and Sanger sequencing. It has also highlighted the potential of THxID™-BRAF to be applied to a broader range of sample types than claimed in the current “instructions for use”, an extension that would require the ad hoc validation and approval.

The inhibitors of mutant BRAF that are used to treat metastatic melanoma induce squamoproliferative lesions. We conducted a prospective histopathological and molecular study on 27 skin lesions from 12 patients treated with vemurafenib. Mutation hot spots in HRAS, NRAS, KRAS, BRAF, and Pi3KCA were screened. HPV and HPyV infection status were also determined. The lesions consisted of 19 verrucal papillomas, 1 keratoacanthoma and 7 squamous cell carcinomas. No mutations were found within BRAF and NRAS. KRAS, HRAS, and Pi3KCA oncogenic mutations were found in 10 (83.3%), 7 (58.3%), and 4 (33.3%) patients respectively; however, these mutations were not consistent within all tumors of a given patient. Pi3KCA mutation was always associated with a mutation in HRAS. Finally, no correlation was found between the mutated gene or type of mutation and the type of cutaneous tumor or clinical response to vemurafenib. P16 protein level was not indicative of HPV infection. HPV was detected in only two lesions. Two cases had MCPyV, and one had HPyV7. In conclusion, neither HPV nor HPyV seem to be involved in the development of squamoproliferative lesions induced by verumafenib. By contrast, HRAS and KRAS play a predominant role in the physiopathology of these tumors.

Next-generation sequencing (NGS) has revolutionized the therapeutic care of patients by allowing high-throughput and parallel sequencing of large numbers of genes in a single run. However, most of available commercialized cancer panels target a large number of mutations that do not have direct therapeutic implications and that are not fully adapted to low quality formalin-fixed, paraffin-embedded (FFPE) samples. Here, we designed an amplicon-based NGS panel assay of 16 currently actionable genes according to the most recent recommendations of the French National Cancer Institute (NCI). We developed a panel of short amplicons (<150 bp) using dual-strand library preparation. The clinical validation of this panel was performed on well-characterized controls and 140 routine diagnostic samples, including highly degraded and cross-linked genomic DNA extracted from FFPE tumor samples. All mutations were detected with elevated inter-laboratory and inter-run reproducibility. Importantly, we could detect clinically actionable alterations in FFPE samples with variant allele frequencies as low as 1%. In addition, the overall molecular diagnosis rate was increased from 40.7% with conventional techniques to 59.2% with our NGS panel, including 41 novel actionable alterations normally not explored by conventional techniques. Taken together, we believe that this new actionable target panel represents a relevant, highly scalable and robust tool that is easy to implement and is fully adapted to daily clinical practice in hospital and academic laboratories.

Lynch syndrome (LS) is the most frequent cause of hereditary colorectal cancer. A subset of patients with a history of LS shows no causal germline pathogenic alteration and are identified as having Lynch‐like syndrome (LLS). Alu retrotransposons are the most abundant mobile DNA sequences in the human genome and have been associated with numerous human cancers by either disrupting coding regions or altering epigenetic modifications or splicing signals. We report a family first classified as having LLS by Sanger sequencing analysis. Next‐generation sequencing (NGS) analysis identified an AluY5a insertion in MLH1 exon 6 that led to exon skipping. This splicing alteration inducing a pathogenic frameshift was found in patients who developed colorectal adenocarcinomas. Retroelement insertion might thus be an important but underestimated mechanism of cancer genetics that could be systematically tested in patients with a phenotype suggesting LS to accurately assess family risk and surveillance approaches.