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Molecular biomarker profile of EGFR copy number, KRAS and BRAF mutations in colorectal carcinoma  [cached]
Rong Rong,Jamie Tull,Shengle Zhang
Journal of Solid Tumors , 2012, DOI: 10.5430/jst.v2n4p27
Abstract: Anti-EGFR therapy is approved by US Food and Drug Administration (FDA) as mono-therapy or as part of combination chemotherapy for metastatic colorectal carcinoma. Monoclonal anti-EGFR antibodies cetuximab and panitumumab have shown benefits for those patients with wild-type KRAS gene. Patients with KRAS mutation in codon 12 or 13 are unresponsive to targeted anti-EGFR therapy. Mutations of BRAF, the main downstream target gene of KRAS, also negatively impact the patients’ response to the therapy. On the contrary, EGFR gene amplification in colon cancer predicts a better response to anti-EGFR therapy. The correlation of EGFR amplification with KRAS and BRAF mutation status is not only of academic interests but also of clinical importance. Previous studies have shown that EGFR mutation is mutually exclusive with KRAS and BRAF mutations in lung cancer. However, there is limited data on colorectal cancer so far. In this study, 28 colorectal cancer samples are tested for KRAS and BRAF mutations by PCR and for EGFR gene copy number by fluorescence in situ hybridization (FISH). EGFR high-copies, KRAS mutation and BRAF mutation are found in 15 (54%), 13 (46%) and 3 (11%) samples, respectively. A mutually-exclusive pattern is seen between KRAS mutations (13 positive samples) and BRAF mutations (3 positive samples). However, high EGFR high copy number is not "mutually-exclusive" with KRAS or BRAF mutations. Six samples with KRAS mutations and 2 with BRAF mutation show "co-existing" high EGFR copy number. These account for 29% of total cases tested. Five cases are triple negative for EGFR, KRAS and BRAF alterations. The results from our study indicate that high EGFR copy number with concurrent KRAS or BRAF mutations is quite common in colon cancer and the therapeutic response with anti-EGFR agents in this patient population requires further investigation.
KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment  [PDF]
Nicola Normanno, Carmine Pinto, Francesca Castiglione, Alberto Bardelli, Marcello Gambacorta, Gerardo Botti, Oscar Nappi, Salvatore Siena, Fortunato Ciardiello, GianLuigi Taddei, Antonio Marchetti
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029146
Abstract: 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.
COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma
Colin C Pritchard, Laura Akagi, Poluru L Reddy, Loren Joseph, Jonathan F Tait
BMC Clinical Pathology , 2010, DOI: 10.1186/1472-6890-10-6
Abstract: We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler? instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations.We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.KRAS (Kirsten rat sarcoma virus homolog 2) is a membrane-anchored G-protein that acts downstream of the epidermal growth factor receptor (EGFR) to activate pro-growth and anti-apoptotic pathways, including the MAP kinase and PI3 kinase pathways [1]. Mutations in codons 12 and 13 of the KRAS gene confer resistance to drugs targeted at EGFR by impairing GTPase activity, which results in constitutive EGFR-independent signaling. KRAS is one of the most f
Prevalence of KRAS, BRAF, and PIK3CA somatic mutations in patients with colorectal carcinoma may vary in the same population: clues from Sardinia  [cached]
Palomba Grazia,Colombino Maria,Contu Antonio,Massidda Bruno
Journal of Translational Medicine , 2012, DOI: 10.1186/1479-5876-10-178
Abstract: Background Role of KRAS, BRAF and PIK3CA mutations in pathogenesis of colorectal cancer (CRC) has been recently investigated worldwide. In this population-based study, we evaluated the incidence rates and distribution of such somatic mutations in genetically isolated population from Sardinia. Methods From April 2009 to July 2011, formalin-fixed paraffin-embedded tissues (N = 478) were prospectively collected from Sardinian CRC patients at clinics across the entire island. Genomic DNA was isolated from tissue sections and screened for mutations in KRAS, BRAF, and PIK3CA genes by automated DNA sequencing. Results Overall, KRAS tumour mutation rate was 30% (145/478 positive cases). Distribution of mutation carriers was surprisingly different within the island: 87/204 (43%) in North Sardinia vs. 58/274 (21%) in Middle-South Sardinia (p<0.001). Among 384 CRC cases whose DNA was available, only one (0.3%) patient carried a mutation in BRAF gene; PIK3CA was found mutated in 67 (17%) patients. A significant inverse distribution of PIK3CA mutation rates was observed within Sardinian population: 19/183 (10%) cases from northern vs. 48/201 (24%) cases from central-southern island (p<0.001). This heterogeneity in frequencies of KRAS/PIK3CA somatic mutations is consistent with already-reported discrepancies in distribution of germline mutations for other malignancies within Sardinian population. Preliminary clinical evaluation of 118 KRAS wild-type patients undergoing anti-EGFR-based treatment indicated lack of role for PIK3CA in predicting response to therapy. Conclusions Our findings support the hypothesis that differences in patients’ origins and related genetic backgrounds may contribute to even determine the incidence rate of somatic mutations in candidate cancer genes.
KRAS, BRAF and PIK3CA Mutations and the Loss of PTEN Expression in Chinese Patients with Colorectal Cancer  [PDF]
Chen Mao, Junhua Zhou, Zuyao Yang, Yafang Huang, Xinyin Wu, Hong Shen, Jinling Tang, Qing Chen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036653
Abstract: Background To investigate the frequency and relationship of the KRAS, BRAF and PIK3CA mutations and the loss of PTEN expression in Chinese patients with colorectal cancer (CRC). Methodology/Principal Findings Genomic DNA was extracted from the formalin-fixed paraffin-embedded (FFPE) tissues of 69 patients with histologically confirmed CRC. Automated sequencing analysis was conducted to detect mutations in the KRAS (codons 12, 13, and 14), BRAF (codon 600) and PIK3CA (codons 542, 545 and 1047). PTEN protein expression was evaluated by immunohistochemistry on 3 mm FFPE tissue sections. Statistical analysis was carried out using SPSS 16.0 software. The frequency of KRAS, BRAF and PIK3CA mutations and loss of PTEN expression was 43.9% (25/57), 25.4% (15/59), 8.2% (5/61) and 47.8% (33/69), respectively. The most frequent mutation in KRAS, BRAF and PIK3CA was V14G (26.7% of all mutations), V600E (40.0% of all mutations) and V600L (40.0% of all mutations), and H1047L (80.0% of all mutations), respectivly. Six KRAS mutatant patients (24.0%) harbored BRAF mutations. BRAF and PIK3CA mutations were mutually exclusive. No significant correlation was observed between the four biomarkers and patients' characteristics. Conclusions/Significance BRAF mutation rate is much higher in this study than in other studies, and overlap a lot with KRAS mutations. Besides, the specific types of KRAS and PIK3CA mutations in Chinese patients could be quite different from that of patients in other countries. Further studies are warranted to examine their impact on prognosis and response to targeted treatment.
Impact of KRAS, BRAF, PIK3CA Mutations, PTEN, AREG, EREG Expression and Skin Rash in ≥2nd Line Cetuximab-Based Therapy of Colorectal Cancer Patients  [PDF]
Zacharenia Saridaki,Maria Tzardi,Chara Papadaki,Maria Sfakianaki,Fraga Pega,Aristea Kalikaki,Eleftheria Tsakalaki,Maria Trypaki,Ippokratis Messaritakis,Efstathios Stathopoulos,Dimitris Mavroudis,Vassilis Georgoulias,John Souglakos
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015980
Abstract: To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC) patients treated with cetuximab containing salvage chemotherapy.
Mutations of the KRAS oncogene in endometrial hyperplasia and carcinoma.  [cached]
Bozena Dobrzycka,S?awomir J Terlikowski,Andrzej Mazurek,Oksana Kowalczuk
Folia Histochemica et Cytobiologica , 2009, DOI: 10.5603/4382
Abstract: The aim of this study was to examine the prevalence and clinicopathological significance of KRAS point mutation in endometrial hyperplasia and carcinoma. We analysed KRAS in 11 cases of complex atypical hyperplasia and in 49 endometrial carcinomas using polymerase chain reaction associated with restriction fragment length polymorphism (PCR-RFPL). Point mutations at codon 12 of KRAS oncogene were identified in 7 of 49 (14,3%) tumor specimens and in 2 of 11 (18,2%) hyperplasias. No correlation was found between KRAS gene mutation and age at onset, histology, grade of differentiation and clinical stage. We conclude that KRAS mutation is a relatively common event in endometrial carcinogenesis, but with no prognostic value.
KRAS and TP53 mutations in colorectal carcinoma  [cached]
Al-Kuraya Khawla
Saudi Journal of Gastroenterology , 2009,
结直肠癌KRAS 、NRAS 和BRAF 基因突变与临床病理特征的回顾性分析
Retrospective analysis of KRAS, NRAS and BRAF gene mutations and clinicopathological features in patients with colorectal cancer

- , 2018, DOI: 10.3969/j.issn.1674-8115.2018.01.002
Abstract: 目的· 分析结直肠癌患者KRAS、NRAS 和BRAF 基因突变状态与临床病理特征的相关性。方法· 收集461 例结直肠癌石蜡标 本,利用突变扩增阻滞系统检测标本KRAS、NRAS 和BRAF 基因的热点突变情况,分析突变率与临床病理特征的相关性。通过查阅 PubMed 文献数据库及美国模式培养物集存库相关细胞株资料,在结直肠癌组织和细胞株水平分析KRAS 和BRAF 共突变情况及对下 游靶基因启动子甲基化的影响。结果· KRAS、NRAS 和BRAF 基因突变率分别为44.0%、6.1% 和5.2%;右半结肠KRAS 的突变率显著 高于左半结肠(P=0.000);男性患者NRAS 基因突变率显著高于女性患者(P=0.002)。BRAF 基因突变与患者的年龄、肿瘤的分化程 度、肿瘤位置及神经浸润特征密切相关。203 例KRAS 基因突变样本中,无BRAF 突变。结论· KRAS、NRAS 和BRAF 突变率与结直 肠癌临床病理特征存在关联;KRAS 与BRAF 基因突变存在互斥性。
:Objective · To study the correlation between KRAS, NRAS and BRAF mutations and the clinicopathological features in patients with colorectal cancer (CRC). Methods · The 461 paraffin-embedded CRC tissues were collected. The mutations in hotspot region of KRAS, NRAS and BRAF genes were investigated using amplification refractory mutation system. The relationship between the mutation rates of each gene and the clinicopathological characteristics in CRC patients were analyzed. The KRAS and BRAF mutation profile and the impact on promoter methylation of the downstream genes were further investigated in both CRC tissues and cell lines through literatures and the American Type Culture Collection. Results · KRAS, NRAS and BRAF mutation rates in CRC tissues were 44.0%, 6.1% and 5.2%, respectively. KRAS mutations in the right colon were remarkably higher than the left colon (P=0.000). NRAS mutations were more likely to occur in male patients than female patients (P=0.002). BRAF mutation was closely correlated with age, tumor differentiation, tumor location and nerve invasion, but was exclusive of 203 KRAS mutated samples. Conclusion · KRAS, NRAS and BRAF mutations were significantly correlated with the clinicopathological features of CRC, and the mutation incompatibility was observed between KRAS and BRAF genes
KRAS Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in Prediction of Lung Metastasis  [PDF]
Paloma Cejas,Miriam López-Gómez,Cristina Aguayo,Rosario Madero,Javier de Castro Carpe?o,Cristóbal Belda-Iniesta,Jorge Barriuso,Víctor Moreno García,Javier Larrauri,Rocío López,Enrique Casado,Manuel Gonzalez-Barón,Jaime Feliu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008199
Abstract: KRAS mutations in colorectal cancer primary tumors predict resistance to anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody therapy in patients with metastatic colorectal cancer, and thus represent a true indicator of EGFR pathway activation status.
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