Activities of multiple cancer-related pathways are associated with BRAF mutation and predict the resistance to BRAF/MEK inhibitors in melanoma cells

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.     

BRAF and RKIP are significantly decreased in cutaneous squamous cell carcinoma

Background. Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). However, little is known about the implication of BRAF and RKIP expression, or about the incidence of BRAF mutations in the formation of these cutaneous diseases. The RAS oncogene has been proposed to significantly contribute to skin cancer development. Moreover, numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines.

Objectives. This study aimed to measure the mRNA levels of the genes BRAF and RKIP in AK, as well as their possible implication in the progress of AK to SCC. All biopsy specimens were also screened for BRAF mutations within exons 11 and 15.

Patients and methods. Expression levels of the genes BRAF and RKIP were examined in 16 AKs and 12 SCCs by RT-qPCR. A novel allele-specific qPCR method, in combination with direct DNA sequencing, was performed in order to inspect the frequency of the V600E mutation in exon 15, as well as to examine the mutation status of the gene within exon 11.

Results. Significant down-regulation was noted for both genes in SCC, compared to normal tissue (for BRAF, P=0.002; for RKIP, P     

A simple but highly effective approach to evaluate the prognostic performance of gene expression signatures

Background

Highly parallel analysis of gene expression has recently been used to identify gene sets or ‘signatures’ to improve patient diagnosis and risk stratification. Once a signature is generated, traditional statistical testing is used to evaluate its prognostic performance. However, due to the dimensionality of microarrays, this can lead to false interpretation of these signatures.

Principal Findings

A method was developed to test batches of a user-specified number of randomly chosen signatures in patient microarray datasets. The percentage of random generated signatures yielding prognostic value was assessed using ROC analysis by calculating the area under the curve (AUC) in six public available cancer patient microarray datasets. We found that a signature consisting of randomly selected genes has an average 10% chance of reaching significance when assessed in a single dataset, but can range from 1% to ∼40% depending on the dataset in question. Increasing the number of validation datasets markedly reduces this number.

Conclusions

We have shown that the use of an arbitrary cut-off value for evaluation of signature significance is not suitable for this type of research, but should be defined for each dataset separately. Our method can be used to establish and evaluate signature performance of any derived gene signature in a dataset by comparing its performance to thousands of randomly generated signatures. It will be of most interest for cases where few data are available and testing in multiple datasets is limited.     

Concordance of somatic mutational profile in multiple primary melanomas

This study aimed to determine the frequency and concordance of BRAF and NRAS mutation in tumours arising in patients with multiple primary melanoma (MPM ). Patients with MPM managed at one of three tertiary referral centres in Melbourne, Australia, from 2010 to 2015 were included. Incident and subsequent melanomas underwent mutation testing. Cohen's kappa (κ) coefficient assessed agreement between incident and subsequent primary melanomas for both BRAF and NRAS mutation status (mutant versus wild‐type). Mutation testing of at least two primary tumours from 64 patients was conducted. There was poor agreement for both BRAF and NRAS mutation status between incident and subsequent melanomas (κ = 0.10, 95% CI ?0.10 to 0.42; κ = 0.06, 95% CI ?0.10 to 0.57, respectively). In view of the low concordance in BRAF mutation status between incident and subsequent melanomas, mutational analysis of metastatic tissue, rather than of a primary melanoma, in patients with MPM should be used to guide targeted therapy.     

BRAF initiating mutations in the papillary thyroid carcinoma

Among genetic alterations most important for the initiation of papillary thyroid carcinoma (PTC) is mutation T1799A in the BRAF gene which is the most frequent event (54.5%) in this type of thyroid cancer. It is seen in all stages, from microcarcinoma through clinically overt disease to anaplastic cancer. It has been shown that BRAF mutation is correlated with PTC histotype. It is identified most frequently in classical PTC and in tall cell variant. Moreover, BRAF mutation is described more often in older patients, whereas in young patients RET/PTC rearrangements dominate. In PTC cases with BRAF mutation V600E the prognosis is poorer, with more cancer invasiveness, metastasis and recurrence. The presence of BRAF mutation is related to the specific gene expression signature, different than in cancer cases showing RET/PTC rearrangement or no known initiating mutation.     

BRAF mutation and RASSF1A expression in thyroid carcinoma of southern Italy

Aim of this work is to provide a detailed comparison of clinical‐pathologic features between well‐differentiated and poorly differentiated tumors according to their BRAF and RASSF1A status. We analyzed RASSF1A methylation by MSP and BRAF mutation by LCRT‐PCR with LightMix® kit BRAF V600E in neoplastic thyroid tissues. Immunohistochemical evaluation of RASSF1A expression was also performed by standard automated LSAB‐HRP technique. An overall higher degree of RASSF1A over‐expression than normal thyroid parenchyma surrounding tumors (P < 0.05) has been found in all malignant well‐differentiated lesions. Moreover, statistically significant higher levels of RASSF1A expression were observed in differentiated cancers associated to an inflammatory autoimmune background (P = 0.01). Amplifiable DNA for LC PCR with LightMix® kit BRAF V600E was obtained in nine PTCs, four FVPTCs, five ATCs, and one control. The V600E mutation was found in 13 of 18 (72%) tumors. BRAF was mutated in 6 of 9 (66%) classical PTC, in 2 of 4 (50%) follicular variant PTC and in all ACs (100%). The overall frequency of RASSF1A promoter methylation observed was 20.5% (9 cases out 44). Hypermethylation of RASSF1A in primary tumors was variable according to histotypes ranging from100% (5/5) in ACs to only 12.5% (4/32) in PTCs. We show a correlation between RASSF1A methylation status and RASSF1A protein expression. Finally, we conclude that BRAF V600E mutation and RASSF1A methylation were pathogenetic event restricted to a subgroup of PTC/FVPTCs in early stage and to clinically aggressive ATCs. J. Cell. Biochem. 114: 1174–1182, 2013. © 2012 Wiley Periodicals, Inc.     

Mutation load in melanoma is affected by MC1R genotype

Whole‐genome sequencing of matched germline and tumour pairs in a well‐characterized cohort of melanoma patients allowed investigation of associations between melanoma body site, age at melanoma onset and MC1R variant status with overall mutation burden and specific base pair changes observed in the corresponding melanoma. We observed statistically significant associations between mutation burden in melanoma and body site, age at onset and MC1R genotype, for both ultraviolet radiation (UVR) signature changes (C>T and CC>TT) and non‐UVR base pair substitutions, as well as with overall variant load.