KRAS and BRAF mutation analysis can be reliably performed on aspirated cytological specimens of metastatic colorectal carcinoma

N. K. B. Pang, M. E. Nga, S. Y. Chin, T.‐M. Ismail, G. L. Lim, R. Soong and M. Salto‐Tellez
KRAS and BRAF mutation analysis can be reliably performed on aspirated cytological specimens of metastatic colorectal carcinoma Background: Sanger sequencing is one of several reliable methods in use to detect KRAS and BRAF mutations to facilitate clinical patient selection for anti‐epidermal growth factor receptor (EGFR) monoclonal antibody therapy in unresectable metastatic colorectal adenocarcinoma (CRC). Most analyses are made on pretreatment biopsy or resection specimens. There is a scarcity of published studies on the suitability of cytological samples for KRAS testing in this setting. Methods: DNA extraction was attempted on 11 search‐retrieved paired cases of histological resections or excisions of CRC and their corresponding cytological samples (representing metastases) and tested for KRAS mutations in exon 2 and 3, as well as BRAF exon 15 mutations by Sanger sequencing. Only KRAS wild‐type cases were subjected to BRAF analysis because this is the setting with true diagnostic value, as these mutations are mutually exclusive. Results: Of the 11 paired cases analysed, only eight histology cases showed satisfactory DNA quality for sequencing. Thus, only eight of the corresponding cytology cases were analysed. Seven of the eight cases tested showed the same KRAS genotype on both the aspirated cytology specimen of metastatic carcinoma and the primary tumour (histological specimen), from which we derive an overall concordance rate of 87.5%. The single discordant case was likely to be a true difference as it was demonstrated again on repeat testing of both samples. No BRAF mutations were detected on the four KRAS wild‐type cases. Conclusion: A range of cytological samples are suitable for KRAS and BRAF mutation testing, be it from previously stained preparations or cell blocks. These samples would be highly valuable in cases where cytological samples are the only material available for mutation testing.     

Biochip detection of KRAS,BRAF, and PIK3CA somatic mutations in colorectal cancer patients

Molecular Biology - Somatic mutations of KRAS, PIK3CA, and BRAF cause insensitivity of colorectal tumors to therapy with anti-EGFR monoclonal antibodies, necessitating a genetic testing prior to...     

Gene expression profile and mutational analysis of DNA mismatch repair genes in carcinoma prostate in Indian population

DNA mismatch repair (MMR) plays a role in promoting genetic stability by repairing DNA replication errors, inhibiting recombination between nonidentical DNA sequences, and participating in responses to DNA damage. Although the role of MMR in prostate carcinogenesis remains unclear, MMR deficiency in Carcinoma Prostate (Pca) could prove to be clinically significant. Thus, the present study investigated the gene expression profile of six major MMR genes, viz. hMLH1, hMSH2, hPMS1, hPMS2, hMSH3, and hMSH6, and polymorphism in hMLH1 and hMSH2 in Pca in Indian population. Further, correlation with clinicopathological parameters was evaluated to establish their role as a potential prognostic marker. A significant downregulation of hMLH1, hMSH2, and hPMS2 expression was observed in Pca compared to benign prostatic hyperplasia (BPH). A greater loss of hPMS2 protein in poorly differentiated tumors was demonstrated, which was in concordance with a significant inverse correlation of hPMS2 gene expression with the Gleason score indicating its significance as a marker for Pca progression. An important association of hMLH1-93G>A polymorphism with the risk of Pca was also identified. The results of the present study suggest that an altered MMR has important biological and clinical significance in Pca in Indian population.     

Mutation analysis of p53, K-ras, and BRAF genes in colorectal cancer progression

Gene mutations in APC, K-ras, and p53 are thought to be essential events for colorectal cancer development. Recent data seem to indicate that K-ras and p53 mutations rarely co-exist in the same tumor, indicating that these alterations do not represent a synergistic evolutionary pathway. Moreover, an inverse relation between K-ras gene activation and BRAF mutations has been demonstrated, suggesting alternative pathways for colorectal cancer transformation. To reconstruct the chronological modulation of these gene mutations during cell transformation and colorectal cancer progression, mutations of p53, K-ras, and BRAF genes were analyzed by Single Strand Conformation Polymorphism (SSCP) or sequencing analysis in 100 colorectal cancer samples, evenly distributed among different Dukes' stages. We found mutations in p53, K-ras, and BRAF genes in 35%, 30%, and 4% of tumors, respectively, and observed a minimal or no co-presence of these gene alterations. Moreover, the frequency of molecular p53 mutations increased as tumor stage increased, suggesting an important role for this gene in the progression of colorectal cancer. Conversely, K-ras or BRAF genes were not related to tumor stage or location. These data seem to indicate the absence of a co-presence of the genes, highlighting the possibility of multiple pathways for colorectal tumor progression. Moreover, mutations in p53, K-ras, and BRAF are not present in about one-third of colorectal cancers and therefore other gene mutations need to be investigated to better understand molecular mechanisms at the basis of cell transformation and the progression of colorectal cancer.     

Low frequency of BRAF and KRAS mutations in Chinese patients with low-grade serous carcinoma of the ovary

Background

Mounting evidence has shown that KRAS and BRAF are somatic mutations associated with low grade serous carcinoma (LGSC) of the ovary. However, the frequency of KRAS or BRAF mutation was variable in literatures, with a frequency of 16–54% for KRAS mutation and 2–33% for BRAF mutation. Meanwhile, the prognostic significance of KRAS or BRAF mutation remains controversial.

Methods

Codons 12 and 13 of exon 2 of KRAS gene and exon 15 of BRAF gene were analyzed using direct Sanger sequencing in 32 cases of LGSC of the ovary. The associations between KRAS or BRAF mutation and clinicopathological characteristics, overall survival (OS) and disease-free survival (DFS) were statistically analyzed.

Results

KRAS mutation was observed in nine cases (9/32, 28%) and BRAF mutation in two cases (2/32, 6%). KRAS and BRAF mutations were mutually exclusive. Neither KRAS nor BRAF mutation was statistically associated with OS or DFS in our cohort, although there was a favorable prognostic trend in patients with KRAS G12D mutation than those with KRAS G12 V mutation or wild-type KRAS for OS.

Conclusions

The present study indicated a low frequency of BRAF or KRAS mutation in Chinese patients with LGSC of the ovary, and neither KRAS nor BRAF mutation is a prognostic factor.

     

Intra-tumoral heterogeneity of KRAS and BRAF mutation status in patients with advanced colorectal cancer (aCRC) and cost-effectiveness of multiple sample testing

KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE) tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor). We also investigated the utility and efficiency of genotyping a 'DNA cocktail' prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1%) showed intratumoral heterogeneity; 5 (7.2%) at KRAS codons 12, 13 and 2 (2.9%) at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in 'DNA cocktail' samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a 'DNA cocktail' from two or more tumor blocks, improves mutation detection at minimal extra cost.     

NRAS and BRAF Mutations in Melanoma-Associated Nevi and Uninvolved Nevi

According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF^V600E-mutation specific antibody VE1. The BRAF^V600E mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF^V600E-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.     

Effectors of Epidermal Growth Factor Receptor Pathway: The Genetic Profiling of KRAS,BRAF, PIK3CA,NRAS Mutations in Colorectal Cancer Characteristics and Personalized Medicine

Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.     

Association between methylation in mismatch repair genes,V600E BRAF mutation and microsatellite instability in colorectal cancer patients

Colorectal cancer (CRC) corresponds to the third most prevalent type of cancer. Its origins can either be sporadic or inherited, being Lynch syndrome the most common form of hereditary CRC. The activation of BRAF oncogene, inactivation of mismatch repair genes by methylation of CpG islands, and microsatellite instability (MSI) have been reported to be involved in CRC development. The goal of the study was to characterize CRC tumors using clinical and molecular criteria through association and cluster analysis. Amsterdam II and Bethesda guidelines and molecular variables were analyzed in 77 patients from Brazil. The replication error (RER) status, based in microsatellite instability, showed association with metachronous tumor, MLH1 gene methylation and inverse association with left-sided and synchronous tumors. The PMS2 gene was considered the best predictor for differentiating levels of methylation and the mononucleotide were considered the best markers to evaluate RER status. The cluster 1 was characterized of individuals over 60 years of age, female, right-sided tumor, high microsatellite instability, and metachronous or synchronous tumors. The individuals in cluster 2 were younger than 45 years of age, male and showed left sided or rectum tumors, and microsatellite stability. Even though it was not observed a significant association, a higher number of individuals with family history of cancer and tumors without promoter methylation were found in cluster 2. The V600E mutation did not show association with clinical or molecular characteristics. Evaluation of MSI and methylation of MLH1 and PMS2 genes should be considered in order to assist with clinical diagnosis.     

KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment

Background

Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients.

Methodology/Principal Findings

In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass.

Conclusions

The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers.     

Sex differences in the prognostic significance of KRAS codons 12 and 13, and BRAF mutations in colorectal cancer: a cohort study

Background

Activating KRAS and BRAF mutations predict unresponsiveness to EGFR-targeting therapies in colorectal cancer (CRC), but their prognostic value needs further validation. In this study, we investigated the impact of KRAS codons 12 and 13, and BRAF mutations on survival from CRC, overall and stratified by sex, in a large prospective cohort study.

Methods

KRAS codons 12 and 13, and BRAF mutations were analysed by pyrosequencing of tumours from 525 and 524 incident CRC cases in The Malmö Diet and Cancer Study. Associations with cancer-specific survival (CSS) were explored by Cox proportional hazards regression, unadjusted and adjusted for age, TNM stage, differentiation grade, vascular invasion and microsatellite instability (MSI) status.

Results

KRAS and BRAF mutations were mutually exclusive. KRAS mutations were found in 191/ 525 (36.4%) cases, 82.2% of these mutations were in codon 12, 17.3% were in codon 13, and 0.5% cases had mutations in both codons. BRAF mutations were found in 78/524 (14.9%) cases. Overall, mutation in KRAS codon 13, but not codon 12, was associated with a significantly reduced CSS in unadjusted, but not in adjusted analysis, and BRAF mutation did not significantly affect survival. However, in microsatellite stable (MSS), but not in MSI tumours, an adverse prognostic impact of BRAF mutation was observed in unadjusted, but not in adjusted analysis. While KRAS mutation status was not significantly associated with sex, BRAF mutations were more common in women. BRAF mutation was not prognostic in women; but in men, BRAF mutation was associated with a significantly reduced CSS in overall adjusted analysis (HR = 3.50; 95% CI = 1.41–8.70), but not in unadjusted analysis. In men with MSS tumours, BRAF mutation was an independent factor of poor prognosis (HR = 4.91; 95% CI = 1.99–12.12). KRAS codon 13 mutation was associated with a significantly reduced CSS in women, but not in men in unadjusted, but not in adjusted analysis.

Conclusions

Results from this cohort study demonstrate sex-related differences in the prognostic value of BRAF mutations in colorectal cancer, being particularly evident in men. These findings are novel and merit further validation.

     

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC₅₀ value between 70 and 110 μM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.