Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer

Background: Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. Objective: To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Methods: Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). Results:KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Conclusion: Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available.     

高分辨率熔解曲线技术检测结直肠癌患者KRAS基因突变

目的探讨高分辨率熔解曲线技术(HRM)检测结直肠癌患者KRAS基因突变的可行性及其临床意义。方法采用HRM技术检测60例结直肠癌患者石蜡包埋组织标本KRAS基因2号外显子12和13位密码子突变,并与Sanger测序法检测结果进行对比分析。结果 HRM法检测结直肠癌患者KRAS基因2号外显子12和13位密码子突变检出率为36.67%(22/60),Sanger测序法检测突变检出率为33.33%(20/60)。HRM法检测突变检出率高于Sanger测序法,HRM法检测敏感性为100%,特异性为95.24%。结论 HRM法检测KRAS基因突变,灵敏度高,敏感性和特异性好,具有操作简便、节约时间、成本低的特点,方法可行。     

The prognostic value of KRAS mutations in patients with colorectal cancer

Our aim was to evaluate the KRAS genotypes of Japanese colorectal cancer (CRC) patients and to assess the effect of these genotypes on clinical outcome. A total of 99 patients with stage I-IV CRC who underwent resection were prospectively studied for KRAS mutations by direct sequencing. KRAS mutations were found in 37 (37.4%) of 99?patients. Of these, 11.1% were the KRAS p.G13D mutation and the remaining 26.2% were other KRAS mutations. The cumulative 5-year survival rates for patients with wild-type KRAS, KRAS 12 and KRAS p.G13D mutations were 81.4, 61.4 and 42.0%, respectively (P=0.0397). The KRAS genotype had no effect on stage IV patient prognosis without anti-epithelial growth factor receptor (EGFR) antibody therapy. However, in stage I-III patients significant or trends in prognostic factors for disease-free survival (DFS) were pathological T stage, lymphatic vessel involvement and KRAS p.G13D. Multivariate analysis identified T4 pathological stage (P=0.0076) and the KRAS p.G13D mutation (P=0.0499) as the most significant independent prognostic factors associated with DFS. In Japanese CRC patients KRAS p.G13D had prognostic impact on DFS in stage I-III disease, while the prognosis of stage IV patients without anti-EGFR antibody therapy was unaffected by KRAS status.     

Recurrent KRAS codon 146 mutations in human colorectal cancer

An activating point mutation in codon 12 of the HRAS gene was the first somatic point mutation identified in a human cancer and established the role of somatic mutations as the common driver of oncogenesis. Since then, there have been over 11,000 mutations in the three RAS (HRAS, KRAS and NRAS) genes in codons 12, 13 and 61 reported in the literature. We report here the identification of recurrent somatic missense mutations at alanine 146, a highly conserved residue in the guanine nucleotide binding domain. In two independent series of colorectal cancers from Hong Kong and the United States we detected KRAS A146 mutations in 7/126 and 2/94 cases, respectively, giving a combined frequency of 4%. We also detected KRAS A146 mutations in 2/40 (5%) colorectal cell lines, including the NCI-60 colorectal cancer line HCC2998. Codon 146 mutations thus are likely to make an equal or greater contribution to colorectal cancer than codon 61 mutations (4.2% in our combined series, 1% in the literature). Lung adenocarcinomas and large cell carcinomas did not show codon 146 mutations. We did, however, identify a KRAS A146 mutation in the ML-2 acute myeloid leukemia cell line and an NRAS A146 mutation in the NALM-6 B-cell acute lymphoblastic leukemia line, suggesting that the contribution of codon 146 mutations is not entirely restricted to colorectal cancers or to KRAS.     

Detection of KRAS mutations in circulating tumour DNA from plasma and urine of patients with colorectal cancer

BackgroundCirculating tumour DNA (ctDNA) is very useful for purposes of cancer genetics; however, it has some limitations. Recently, ctDNA in body fluids, such as urine, sputum, and pleural effusion, has been investigated. The aim of this study was to evaluate the quantity of ctDNA derived from urine (trans-renal ctDNA) and the accuracy of KRAS mutation detection in relation to disease stage in colorectal cancer.MethodsUrine, plasma, and tissue samples were collected from consecutively resected colorectal cancer patients. DNA was extracted from each sample and the quantity was determined. From each DNA sample, KRAS mutations were detected using droplet digital PCR.Results200 patients participated and KRAS mutations were detected in 84 patients (42.0%) from tumour tissue. The concentration of trans-renal ctDNA (trtDNA) was significantly lower than that of plasma; however, there was no significant difference between the sensitivity using ctDNA and that using trtDNA (29.8% VS 33.3%, p = 0.62). Concordance between these two tests was only 17.5%. Combination analysis (ctDNA + trtDNA) improved the sensitivity to 53.6%, and sensitivity was significantly higher than that of corresponding single assays (p = 0.003). In early cancer stages, trtDNA had greater sensitivity for detecting KRAS mutations than ctDNA (37.7% vs. 21.3%, p = 0.047). Conversely, it was less useful for advanced cancer stages (21.7% vs. 52.2%, p = 0.07). Notably, KRAS mutations were detected using ctDNA or trtDNA in 12 of 116 (10.3%) patients who had no KRAS mutations in their tissue samples.Conclusions: trtDNA and ctDNA have equal potential and combination analysis significantly improved the sensitivity.     

Characterization of rare transforming KRAS mutations in sporadic colorectal cancer

KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P < 0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10–16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.     

Prognostic relevance of KRAS and BRAF mutations in Japanese patients with colorectal cancer

Background

Mutations of the KRAS or BRAF genes are now recognized as prognostic markers for colorectal cancer (CRC). They are also important predictive markers for resistance to the monoclonal antibodies that target the epidermal growth factor receptor.

Methods

In this retrospective study, KRAS and BRAF mutations were analyzed using a direct sequence method in 254 Japanese CRC patients, and the associations between KRAS or BRAF mutations and clinicopathological characteristics or outcome were evaluated.

Results

KRAS and BRAF mutations were detected in 33.5 and 6.7 % of all patients, respectively. Consistent with previous reports, BRAF mutations were significantly correlated with the anatomical site of the tumor (P < 0.001), tumor grade (P = 0.001) and high frequency of microsatellite instability (P < 0.001). BRAF mutations were correlated with poor overall survival in the full patient cohort (P = 0.009). KRAS mutations were significantly correlated with poor recurrence-free survival (P = 0.03), particularly in patients with stage II CRC (P = 0.007). Cox regression analysis showed that KRAS mutations were a negative predictor of recurrence-free survival in patients with stage II CRC.

Conclusion

KRAS mutation status could be a novel biomarker for predicting disease recurrence in Japanese patients with stage II CRC.     

KRAS, BRAF and PIK3CA mutations in human colorectal cancer: relationship with metastatic colorectal cancer

Many abnormal gene expressions and dysregulated signaling pathways have been found in human colorectal cancer. Activating mutations of the KRAS, BRAF or PIK3CA oncogenes are frequently found in colorectal cancer. The aim of the study was to investigate the molecular occurrence of KRAS, BRAF and PIK3CA mutations in the colorectal tumorigenesis and to study the association of these events with clinicopathological parameters. In our study, DNA was extracted from 200 cases of human colorectal cancer tissue samples. KRAS, BRAF and PIK3CA mutation analysis was performed by PCR and pyrosequencing. Using statistical methods, we analyzed the relationships between the gene mutations and clinicopathological parameters. KRAS point mutations were detected in 63/200 patients (31.5%), with codon 12 mutations in 52/200 patients (26%), codon 13 mutations in 10/200 patients (5%) and codon 12.13 bi-mutations in 1/200 patients (0.5%). The V600E mutations of BRAF were detected in 14/200 patients (7%). PIK3CA point mutations (exon 9, exon 20) were detected in 25/200 (12.5%) patients, exon 9 mutatons in 12/200 patients (6%) and exon 20 mutations in 13/200 (6.5%). Our study suggested that both KRAS and BRAF mutations are exclusive, but KRAS and PIK3CA mutations are coexistent. The mutational status of BRAF did not correlate with Dukes' staging, histological type, age and gender. However, strong associations were found between KRAS, PIK3CA mutations and Dukes' staging (staging D, 12/25, 48%). Notably, our data indicated that colorectal cancers with KRAS and PIK3CA bi-mutations are more likely to develop into liver metastasis.     

KRAS,NRAS and BRAF mutations in colorectal cancer and melanoma

Cancers are the group of diseases, which arise because of the uncontrolled behavior of some of the genes in our cells. There are possibilities of gene amplifications, overexpressions, deletions and other anomalies which might lead to the development and spread of cancer. One of the most dangerous ways to the cancers is the mutations of the genes. The mutated genes can start unstoppable proliferation of cells, their uncontrolled motility, protection from apoptosis, the DNA mutation enhancement as well as other anomalies, leading to the cancer. This review focuses on the genes, which are frequently mutated in various cancers and are known to be important in the advance and progression of colorectal cancer and melanoma, namely KRAS, NRAS and BRAF.     

Both BRAF and KRAS mutations are rare in colorectal carcinomas from patients with hereditary nonpolyposis colorectal cancer

The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal carcinomas. To clarify the extent of BRAF mutations in HNPCC colorectal carcinomas, which are typical mismatch repair deficient carcinomas, we compared the frequency of BRAF mutations between HNPCC, familial adenomatous polyposis (FAP) and sporadic cases. The frequency of KRAS mutations was also compared between these three syndromes. No BRAF mutations were detected in 33 HNPCC colorectal carcinomas, while they were detected in 3 of 26 (12%) FAP carcinomas and 2 of 53 (4%) microsatellite stable sporadic carcinomas. KRAS mutations were detected in 2 of 33 (6%) HNPCC, 9 of 26 (35%) FAP and 18 of 53 (34%) sporadic carcinomas. Such extremely low frequencies of BRAF and KRAS mutations in HNPCC colorectal carcinomas suggest that the participation of RAS-RAF signaling is minor in HNPCC, and that the previously suggested high frequency of BRAF mutations in mismatch repair deficient colorectal carcinomas is not due to mutations of mismatch repair genes.     

KRAS mutations in non-small-cell lung cancer and colorectal cancer: Implications for EGFR-targeted therapies

Background

KRAS mutations are associated with diverse biologic functions as well as prognostic and predictive impact in non-small cell-lung cancer (NSCLC) and colorectal cancer (CRC). In CRC, benefit from monoclonal antibody therapies targeting EGFR is generally limited to patients whose tumors have wild-type (WT) KRAS, whereas data suggest that this association is not present for NSCLC. We hypothesized that the unique tobacco-related carcinogenesis of NSCLC results in a divergence of KRAS MT genotype compared with CRC, contributing to differences in outcomes from EGFR-targeted therapies.

Material and methods

Tumor from 2603 patients (838 CRC and 1765 NSCLC) was analyzed for KRAS mutations. DNA was extracted from microdissected formalin-fixed-paraffin-embedded specimens (FFPE) and 7 different base substitutions in codons 12 and 13 of KRAS were determined.

Results

KRAS mutation genotype differed significantly between NSCLC and CRC in frequency (25% vs. 39%; p < 0.001), smoking-associated G>T transversions (73% versus 27%; p < 0.001), and ratio of transversions to transitions (3.5 vs. 0.79; p < 0.001). In NSCLC GLY12Cys mutations, resulting from a codon 12 GGT>TGT substitution, were observed in 44% compared to 10% for CRC. In contrast, codon 12 or 13 GLY>ASP substitutions (resulting in a G>A transition) were more frequent in CRC (42%) compared with NSCLC (21%).

Conclusion

In this large dataset, KRAS mutation patterns are quantitatively and qualitatively distinct between NSCLC and CRC, reflecting in part differences in tobacco-related carcinogenesis. In light of differences in predictive value for EGFR-directed monoclonal antibody therapy and prognosis for specific KRAS mutations between NSCLC and CRC, these data provide an underlying biologic rationale.     

KRAS mutations in primary tumours and post-FOLFOX metastatic lesions in cases of colorectal cancer

Background:

KRAS mutations are predictive markers for the efficacy of anti-EGFR antibody therapies in patients with metastatic colorectal cancer. Although the mutational status of KRAS is reportedly highly concordant between primary and metastatic lesions, it is not yet clear whether genotoxic chemotherapies might induce additional mutations.

Methods:

A total of 63 lesions (23 baseline primary, 18 metastatic and 24 post-treatment metastatic) from 21 patients who were treated with FOLFOX as adjuvant therapy for stage III/IV colorectal cancer following curative resection were examined. The DNA samples were obtained from formalin-fixed paraffin-embedded specimens, and KRAS, NRAS, BRAF and PIK3CA mutations were evaluated.

Results:

The numbers of primary lesions with wild-type and mutant KRAS codons 12 and 13 were 8 and 13, respectively. The mutational status of KRAS remained concordant between the primary tumours and the post-FOLFOX metastatic lesions, irrespective of patient background, treatment duration and disease-free survival. Furthermore, the mutational statuses of the other genes evaluated were also concordant between the primary and metastatic lesions.

Conclusion:

Because the mutational statuses of predictive biomarker genes were not altered by FOLFOX therapy, specimens from both primary tumours and post-FOLFOX tumour metastases might serve as valid sources of DNA for known genomic biomarker testing.     

CMS-dependent prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer

BackgroundThe prognostic impact of KRAS and BRAF^V600E mutations in primary colorectal cancer (CRC) varies with microsatellite instability (MSI) status. The gene expression–based consensus molecular subtypes (CMSs) of CRC define molecularly and clinically distinct subgroups, and represent a novel stratification framework in biomarker analysis. We investigated the prognostic value of these mutations within the CMS groups.Patients and methodsTotally 1197 primary tumors from a Norwegian series of CRC stage I–IV were analyzed for MSI and mutation status in hotspots in KRAS (codons 12, 13 and 61) and BRAF (codon 600). A subset was analyzed for gene expression and confident CMS classification was obtained for 317 samples. This cohort was expanded with clinical and molecular data, including CMS classification, from 514 patients in the publically available dataset GSE39582. Gene expression signatures associated with KRAS and BRAF^V600E mutations were used to evaluate differential impact of mutations on gene expression among the CMS groups.ResultsBRAF^V600E and KRAS mutations were both associated with inferior 5-year overall survival (OS) exclusively in MSS tumors (BRAF^V600E mutation versus KRAS/BRAF wild-type: Hazard ratio (HR) 2.85, P < 0.001; KRAS mutation versus KRAS/BRAF wild-type: HR 1.30, P = 0.013). BRAF^V600E-mutated MSS tumors were strongly enriched and associated with metastatic disease in CMS1, leading to negative prognostic impact in this subtype (OS: BRAF^V600E mutation versus wild-type: HR 7.73, P = 0.001). In contrast, the poor prognosis of KRAS mutations was limited to MSS tumors with CMS2/CMS3 epithelial-like gene expression profiles (OS: KRAS mutation versus wild-type: HR 1.51, P = 0.011). The subtype-specific prognostic associations were substantiated by differential effects of BRAF^V600E and KRAS mutations on gene expression signatures according to the MSI status and CMS group.ConclusionsBRAF^V600E mutations are enriched and associated with metastatic disease in CMS1 MSS tumors, leading to poor prognosis in this subtype. KRAS mutations are associated with adverse outcome in epithelial (CMS2/CMS3) MSS tumors.     

A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens

Background:

KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain.

Methods:

We conducted a two-site comparison of two commercial KRAS mutation kits – the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit – and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles.

Results:

Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger.

Conclusion:

The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.     

Common KRAS and NRAS gene mutations in sporadic colorectal cancer in Northeastern Iranian patients

ObjectivesMutation analysis of the Epidermal Growth Factor Receptor downstream has been a main part of colorectal carcinoma evaluation. Large prospective clinical trials have shown only colorectal cancer (CRC) with wild-type KRAS and NRAS responds to anti-Epidermal Growth Factor Receptor treatment. Hence, mutation analysis is necessary prior to treatment. It is essential to conduct studies to learn about the mutation signature of such tumors. The aim of this study was to evaluate the frequency of hotspot mutations in KRAS and NRAS genes in Iranian CRC patients and to explore their correlations with clinicopathologic parameters.MethodsWe detected mutations in exon 2 (codons 12 and 13) of the KRAS and NRAS genes using high resolution melting analysis, Intplex design and Sanger sequencing in 87 Iranian CRC patients. Genomic DNA was isolated from fresh tissue samples of CRC patients.ResultsFrom 87 eligible cases, 51 were male and 36 were females. KRAS mutations in codons 12 and 13 were present in 28.7% of all analyzed CRCs. Our findings suggested that the tumors with KRAS mutations are not with well- and moderately differentiated tumors compared to poorly differentiated tumors (P value = 0.32). The most frequent types of mutations were glycine to aspartate on codon 12 (p.G12D), and glycine to aspartate on codon 13 (p.G13D). No mutation was found in the NRAS gene in our patients.ConclusionsBased on this study, the frequency of KRAS mutations seems to be in the spectrum of frequencies of other countries such as China, Japan, India, USA, France, and Germany and NRAS was similar to the West of Iran.     

COLD-PCR-HRM 检测结直肠癌患者外周血KRAS基因突变

目的  评价COLD-PCR-HRM 检测结直肠癌患者外周血KRAS 基因突变的临床应用价值。方法  将已知突变型 KRAS 组织DNA 与野生型DNA 做系列稀释,突变DNA 所占比例分别为50%、25%、10%、5%、3%、2% 和 1%。分别应 用常规PCR-HRM 法和COLD-PCR-HRM 法对不同比例KRAS 突变DNA 样本进行检测,验证两种检测方法的灵敏度;同 时应用 COLD-PCR-HRM 法对 62 例外周血和肿瘤组织配对样本进行一致性验证。结果  PCR-HRM 法最低检测浓度为 3%; COLD-PCR-HRM 法最低检测浓度为1%,两种方法的检测灵敏度均明显高于直接测序法（P ＜ 0.05）。应用COLD-PCRHRM 法检测两种样本中KRAS 基因突变状态,其中血清样本中检测出13 例突变（突变率21.0%,13/62）,组织中检测出 12 例突变（突变率 19.4%,12/62）,两种标本同时存在突变的有 9 例,以组织中检测的 KRAS 状态为准,两者突变一致性 为 75.0%（9/12）。KRAS 基因突变在组织与外周血中存在一致性（κ=0.649;P ＜ 0.001）。结论  COLD-PCR 结合 HRM 明 显提高了 KRAS 突变检测灵敏度,为其应用于低丰度突变样本检测提供了有利条件。