Detection Of K-ras Gene Mutations Research Articles

K-ras gene mutation analysis to diagnosis pancreatic adenocarcinoma from endoscopic ultrasound-guided tissue acquisition; a systematic review and meta-analysis

BackgroundEndoscopic ultrasound-guided tissue acquisition (EUS-TA) has high sensitivity for the pathological diagnosis of pancreatic masses, but also a high false-negative rate. K-ras gene mutations occur in over 75 % of pancreatic ductal adenocarcinomas (PDAC), and this meta-analysis evaluated the utility of detecting K-ras gene mutations from EUS-TA specimens for the diagnosis of PDAC. MethodsRelevant studies in PubMed, the Cochrane Library, and Web of Science were systematically searched. Meta-analysis was performed on data from the selected studies using a bivariate model to provide pooled values of sensitivity, specificity, and their 95 % confidence intervals (CIs). ResultsThis meta-analysis included 1521 patients (from 10 eligible studies) who underwent EUS-TA with K-ras gene mutation analysis for diagnosis of pancreatic solid masses. The pooled estimates of sensitivity and specificity were 76.6 % (95 % CI, 70.9–81.5 %) and 97.0 % (95 % CI, 94.0–98.5 %), respectively, for pathological diagnosis, 75.9 % (95 % CI 69.5–81.4 %) and 95.3 % (95 % CI, 92.3–97.2 %) for K-ras gene mutation analysis, and 88.7 % (95 % CI 87.1–91.7 %) and 94.9 % (95 % CI, 91.5–97.0 %) for pathological diagnosis in combination with K-ras gene mutation analysis. The sensitivity for diagnosis of PDAC was significantly higher for pathological diagnosis in combination with K-ras gene mutation analysis than for pathological diagnosis or K-ras gene mutation analysis alone (both, p < 0.001). There was no difference in specificity between pathological diagnosis in combination with K-ras gene mutation analysis and both either (p = 0.234, 0.945, respectively). ConclusionsK-ras gene mutation analysis in combination with to pathological diagnosis of EUS-TA increases the accuracy of differential diagnosis of PDAC.

A Chamber-Based Digital PCR Based on a Microfluidic Chip for the Absolute Quantification and Analysis of KRAS Mutation.

The Kirsten rat sarcoma virus gene (KRAS) is the most common tumor in human cancer, and KRAS plays an important role in the growth of tumor cells. Normal KRAS inhibits tumor cell growth. When mutated, it will continuously stimulate cell growth, resulting in tumor development. There are currently few drugs that target the KRAS gene. Here, we developed a microfluidic chip. The chip design uses parallel fluid channels combined with cylindrical chamber arrays to generate 20,000 cylindrical microchambers. The microfluidic chip designed by us can be used for the microsegmentation of KRAS gene samples. The thermal cycling required for the PCR stage is performed on a flat-panel instrument and detected using a four-color fluorescence system. "Glass-PDMS-glass" sandwich structure effectively reduces reagent volatilization; in addition, a valve is installed at the sample inlet and outlet on the upper layer of the chip to facilitate automatic control. The liquid separation performance of the chip was verified by an automated platform. Finally, using the constructed KRAS gene mutation detection system, it is verified that the chip has good application potential for digital polymerase chain reaction (dPCR). The experimental results show that the chip has a stable performance and can achieve a dynamic detection range of four orders of magnitude and a gene mutation detection of 0.2%. In addition, the four-color fluorescence detection system developed based on the chip can distinguish three different KRAS gene mutation types simultaneously on a single chip.

The Application of the RNA Interference Technologies for KRAS: Current Status, Future Perspective and Associated Challenges.

KRAS is a member of the murine sarcoma virus oncogene-RAS gene family. It plays an important role in the prevention, diagnosis and treatment of tumors during tumor cell growth and angiogenesis. KRAS is the most commonly mutated oncogene in human cancers, such as pancreatic cancers, colon cancers, and lung cancers. Detection of KRAS gene mutation is an important indicator for tracking the status of oncogenes, highlighting the developmental prognosis of various cancers, and the efficacy of radiotherapy and chemotherapy. However, the efficacy of different patients in clinical treatment is not the same. Since RNA interference (RNAi) technologies can specifically eliminate the expression of specific genes, these technologies have been widely used in the field of gene therapy for exploring gene function, infectious diseases and malignant tumors. RNAi refers to the phenomenon of highly specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. There are three classical RNAi technologies, including siRNA, shRNA and CRISPR-Cas9 system, and a novel synthetic lethal interaction that selectively targets KRAS mutant cancers. Therefore, the implementation of individualized targeted drug therapy has become the best choice for doctors and patients. Thus, this review focuses on the current status, future perspective and associated challenges in silencing of KRAS with RNAi technology.

KRAS, NRAS, and BRAF Mutation Pattern in Metastatic Colorectal Cancer: A Study from Northwest Iran

Introduction: Colorectal cancer is currently the third most common cancer in terms of incidence and mortality in Iran. Different gene mutations may confer different degrees of biological aggressiveness and reduce the effectiveness of targeted therapeutic strategies in metastatic Colorectal Cancer (mCRC) patients. Aim: To evaluate any mutation pattern in mCRC, and then evaluate clinical and epidemiological correlations with the detected mutations. Materials and Methods: This study was a cross-sectional analytical study, and all mCRC cases referred to two main central hospitals and oncologists’ clinics, in Tabriz, Iran, from January 2016 to November 2018 were enrolled. KRAS, NRAS, and BRAF mutation tests were performed routinely for all mCRC cases before considering any treatment strategies. Idylla Biocartis NV system{Test Type Package (TTP)}, determined the presence of mentioned mutations. Logistic regression models were used to statistically analysis. Results: The present authors included 173 cases with confirmed mCRC. Among 102 patients with KRAS gene mutation detection, the frequency of mutations was 38.23% (n=39) while most were in exon 2, codon 12 (61.54%), followed by patients who had mutations in codon 13 (n=5, 12.82%). NRAS mutations were only observed in one patient (1.33%) from among the 75 cases who were tested. BRAF codon 600 was tested in 39 cases, and only one case (2.56%) had the mutation. Patients with left-sided tumours had about 9.5 times higher likelihood of KRAS mutation than right-sided tumours (OR=9.64; 95% CI=1.24-75.27). Smoking increased the odds of KRAS mutation about 50%, and mCRC who had alcohol consumption had about two times more likelihood of mutation. Conclusion: The overall frequency of KRAS mutations in the present study was high, while the frequency of KRAS mutations in mCRC patients is lower in Asian populations. KRAS mutation results in this study were most similar to European populations.

Performance of probe polymerization-conjunction-agarose gel electrophoresis in the rapid detection of KRAS gene mutation.

This study aimed to develop a simple and rapid method to detect KRAS gene mutations for conventional clinical applications under laboratory conditions. The genotype of mutation sites was determined based on the occurrence of target bands in the corresponding lanes of the reaction tubes through polymerization-conjunction of the probes, probe purification and amplification, and agarose gel electrophoresis. Circulating DNA samples were obtained from the plasma of 72 patients with lung cancer, which were identified based on six mutation sites (G12S, G12R, G12C, G12D, G12A, and G12V) of codon 12 of the KRAS gene. The detection results were compared with direct sequencing data. The proposed detection method is characterized by simple operation, high specificity, and high sensitivity (2%). This method can detect the mutations of three samples at G12S, G12R, and G12A. In the direct sequencing spectra of these samples, the genotype could not be determined due to the lack of evident sequencing peaks that correspond to the basic group of mutations. In conclusion, a simple and rapid method was established based on probe polymerization-conjunction-agarose gel electrophoresis for detecting KRAS gene mutations. This method can be applied to the conventional mutation detection of inhomogeneous samples.

Sensitive detection of cancer gene based on a nicking-mediated RCA of circular DNA nanomachine

We propose a DNA nanomachine (simplified as MB-PP) by inserting molecular beacon into a padlock probe for the sensitive and specific detection of K-ras gene mutation. In the presence of target DNA, MB-PP is able to be cyclized by ligase because its two ends are pulled together by target species. In this case, rolling circle amplification (RCA) occurs via the extension of primer by polymerase on cyclized MB-PP (CMB-PP), and thus two complete binding sites of nicking endonuclease are obtained due to the formation of RCA product/CMB-PP duplex. Naturally, a steady stream of two types of nicked fragments flow out from continuous rolling CMB-PP nanomachine after introduction of nickase, one of which can in turn triggers strand-displacement amplification (SDA), leading to the dramatic accumulation of nicked fragments. As a result, even in the presence of a trace amount of wild target DNA, the fluorescence intensity will substantially increase. Utilizing this DNA machine, the K-ras gene can be detected down to 50pM with the linear response range from 50pM to 10nM. Moreover, the point mutation existing in the codon 12 of K-ras can be easily distinguished from the wild type.

Development and Clinical Application of Liquid chip Luminex Assay in theDetection of Epidermal Growth Factor Receptor and K-Ras Mutation

Purpose: The success of molecular targeted cancer therapy relies on the accurate detection of the mutated gene. We attempted to develop a rapid, accurate, high sensitive and specific liquid chip luminex method for the detection of EGFR and K-ras mutation, both of which are an important biomarker for the personalized treatment of advanced lung cancer patients. Materials and Methods: Using the liquid chip technology, we developed a luminex system by combining PCR-LDR (Polymerase Chain Reaction-Ligase Detection Reaction) with Luminex platform for the detection of EGFR and K-ras mutation. To verify the clinical application of this liquid chip luminex system, we compared its detection results with those from the gold standard sequencing method through analysis of 100 patients. Results: The developed luminex system showed high flux, sensitivity and specificity for EGFR and K-ras gene mutation detection. Compared with sequencing for the EGFR and K-ras gene mutation detection, this luminex system showed no obvious difference in the mutation rates among different ages, histological classification and TNM stages. However, for the exon21 L585R and exon19 (including the E746-A750 deletion mutant), the luminex method showed even more effective and specificity and demonstrated obvious difference to sequencing (p Conclusion: Our liquid chip luminex system has a wide prospect of clinical application, especially for the detection of EGFR exon 21 L585R and 19 and can beused for early screening and individual therapy of patients with lung cancer.

Effectiveness of circulating tumor DNA for detection of KRAS gene mutations in colorectal cancer patients: a meta-analysis.

Circulating tumor DNA (ctDNA) can be identified in the peripheral blood of patients and harbors the genomic alterations found in tumor tissues, which provides a noninvasive approach for detection of gene mutations. We conducted this meta-analysis to investigate whether ctDNA can be used for monitoring KRAS gene mutations in colorectal cancer (CRC) patients. Medline, Embase, Cochrane Library and Web of Science were searched for the included eligible studies in English, and data were extracted for statistical analysis according to the numbers of true-positive (TP), true-negative (TN), false-positive (FP) and false-negative (FN) cases. Sensitivity, specificity and diagnostic odds ratio (DOR) were calculated, and the area under the receiver operating characteristic curve (AUROC) was used to evaluate the diagnostic performance. After independent searching and reviewing, 21 studies involving 1,812 cancer patients were analyzed. The overall sensitivity, specificity and DOR were 0.67 (95% confidence interval [CI] =0.55–0.78), 0.96 (95% CI =0.93–0.98) and 53.95 (95% CI =26.24–110.92), respectively. The AUROC was 0.95 (95% CI =0.92–0.96), which indicated the high diagnostic accuracy of ctDNA. After stratified analysis, we found the higher diagnostic accuracy in subgroup of patients detected in blood sample of plasma. The ctDNA may be an ideal source for detection of KRAS gene mutations in CRC patients with high specificity and diagnostic value.

Development and Clinical Application of Liquidchip Luminex Assay in the Detection of Epidermal Growth Factor Receptor and K-Ras Mutation

Purpose: The success of molecular targeted cancer therapy relies on the accurate detection of the mutated gene. We attempted to develop a rapid, accurate, high sensitive and specific liquidchip luminex method for the detection of EGFR and K-ras mutation, both of which are important biomarkers for the personalized treatment of advanced lung cancer patients.Materials and methods: Using the liquidchip technology, we developed a luminex system by combining PCRLDR (Polymerase Chain Reaction- Ligase Detection Reaction) with luminex platform for the detection of EGFR and K-ras mutation. To verify the clinical application of this liquidchip luminex system, we compared its detection results with those from the gold standard sequencing method through analysis of 100 patients.Results: The developed luminex system showed high flux, sensitivity and specificity for EGFR and K-ras gene mutation detection. Compared with sequencing for the EGFR and K-ras gene mutation detection, this luminex system showed no obvious difference in the mutation rates among different ages, histological classification and TNM stages. However, for the exon 21 L585R and exon 19 (including the E746-A750 deletion mutant), the luminex method showed even more effective and specificity and demonstrated obvious difference to sequencing (p<0.05).Conclusion: Our liquidchip luminex system has a wide prospect of clinical application, especially for the detection of EGFR exon 21 L585R and 19 and can be used for early screening and individual therapy of patients with lung cancer.

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

BackgroundPatients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.MethodsWe developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.ResultsOverall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.ConclusionThe MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

Modified mismatch polymerase chain reaction-restriction fragment length polymorphism detected mutations in codon 12 and 13 of exon 2 of K-ras gene in colorectal cancer patients and its association with liver metastases: Data from a South Asian country.

Mutations in K-ras codon 12 and 13 of exon 2 are known to affect prognosis and impart resistance to anti-epidermal growth factor monoclonal antibody therapy in colorectal carcinoma (CRC). Our aim was to investigate the utility value of modified mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to detect mutation in K-ras codons of CRC patients and to relate the mutational status to liver metastasis. Mismatch PCR-RFLP was developed to detect K-ras mutations in DNA isolated from paraffinized tumor tissue of thirty CRC patients. All patients had 5 year follow-up data to detect liver metastasis. Cross-tabulations were generated between K-ras mutations and the metastatic status. The Chi-square test was used to indicate statistical significance of the association. Of the 30 CRC patients investigated, K-ras mutations of codons 12 and/or 13 of exon 2 were detected in 14 (46.6%). Meanwhile, 13 patients (43.3%) were observed to have developed liver metastases. There was a significant association between the presence of the K-ras mutation in codon 12 and the occurrence of liver metastasis (χ2 = 4.693, P = 0.030) on the contrary to the mutation in codon 13 to which such occurrence of liver metastases was not seen (χ2 = 1.884, P = 0.169). Codon 12 of exon 2 of K--ras gene detected by modified mismatch PCR-RFLP assay is significantly associated with liver metastasis in CRC patients during the first 5 years after surgery. Thus, modified mismatch PCR-RFLP protocol is a suitable method in this setting to detect K-ras gene mutations predicting liver metastasis in CRC patients.

Result of 2014 external quality assessment for KRAS mutation testing

Objective To evaluate the performance of KRAS gene mutation detection in 2014 external quality assessment (EQA) program and discuss the problems in clinical laboratories. Methods The sample panel of 2014 EQA program contained 5 artificial formalin–fixed, paraffin–embedded (FFPE) samples. The participating laboratories were asked to report their results before the deadline. The scores of EQA and the rate of overall coincidence, false positive and false negative were calculated. Results The EQA program for KRAS testing was set twice a year. In 2014, 58 and 57 valid lab results were submitted respectively. About 79.31%(46/58)and 94.73%(54/57) of the laboratories were correct for all samples. The coincidence rate of positive samples were 93.53% (217/232) and 96.49% (165/171). The coincidence rate for negative ones were 100% (58/58) and 98.25% (112/114). The false–negative ratio was 1.29% (3/232) and 0%. The false–positive ratio was 4.14% (12/290) and 3.15% (9/285). Conclusions The results of 2014 EQA for KRAS gene mutation testing suggested that the performance of laboratories had been improved significantly, however, the false–negative and false–positive results had always been the major problems affecting the accuracy of KRAS mutations testing. Laboratories needed to standardize the testing process and manufacturers should optimize the reagents and the way of interpretation, to guarantee the performance of KRAS gene mutation detection.(Chin J Lab Med, 2015, 38: 661–665) Key words: Colorectal neoplasms; Proto–oncogene proteins; ras Proteins; Mutation; Individualized medicine

761 Detection of K-RAS Gene Mutation by Liquid Biopsy in Patients With Pancreatic Cancer

761 Detection of K-RAS Gene Mutation by Liquid Biopsy in Patients With Pancreatic Cancer

Detection of KRAS gene mutations in colorectal carcinoma: a study of 6 364 patients

To investigate the mutation rate and types of KRAS gene in colorectal carcinoma of Chinese patients, and to document the regional distribution of the mutation. A total of 6 364 colorectal carcinoma tumor specimens were obtained from 27 provinces and autonomous regions in China from 2009 to 2013. Pyrosequencing was used to detect mutations in codons 12 and 13 of KRAS gene. The mutation types of KRAS and the difference of regional distribution were statistically analyzed. KRAS mutation rate in Chinese colorectal carcinoma patients was 37.43 % (2 382/6 364). Ten types of single-base mutations of KRAS codons 12 and 13 were detected, including six common types: 12GGT > GAT (14.77%), 13GGC > GAC (8.19%), 12GGT > GTT (7.89%), 12GGT > TGT (2.20%), 12GGT > AGT (2.00%), and 12GGT > GCT (1.48%). Other four less occurring mutation types (<1%) included 12GGT > CGT, 13GGC > TGC, 13GGC > CGC, and 13GGC > AGC. In addition, 8 other mutation types were identified in 13 tumor samples. The rates of KRAS mutation in patients from different regions were between 35.68% and 38.04% and no significant differences were observed (P > 0.05). Abundant mutation types of KRAS gene exist in colorectal cancers among Chinese patients. The six common mutation types occur with a frequency of not less than 1%. There are no significant differences of KRAS mutation rate among Chinese patients from various areas. Pyrosequencing provides a rapid and accurate method of KRAS mutation detection for clinical application.

Establishment of a sequencing method for detecting KRAS gene mutations

Objective To establish a sequencing method for detecting KRAS gene mutations.Methods A sequencing method for KRAS gene mutations detection was established with specific primers that targeted hotspot mutation region (codon12 and codon13),and wild-type and mutation samples were selected as negative sample and positive sample respectively.Then the performance of the method was assessed.Results A sequencing method for KRAS gene mutations detection was established successfully which owned high sensitivity (3.9 ng/μl) and good repeatability.The mutation rate was 23.5％ in 17 colorectal cancer samples.Conclusion The sequencing method can be used in clinical samples to detect KRAS gene mutations. Key words: KRAS; Mutation; Gene sequencing; Colorectal cancer

The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation. Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results. Results The sensitivity of liquid chip technology detection was 10%-20%,higher than the traditional sequencing method by 1%.Average CV value was 4%-15% and showed good repeatability.5 K-ras mutations in 100 patients (5%) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Gly12Val and 2 Gly12 Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing. Conclusions The novel detection method of K-ras mutations based PCR-LDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.(Chin J Lab Med,2013,36:704-707) Key words: Microfluidic analytical techniques; Polymerase chain reaction; Ligase chain reaction; Genes; ras; Genotype; Carcinoma; non-small-cell lung

Using low sensitivity of direct sequencing to detect K-ras mutations? A new method for improving diagnostic accuracy.

e22100 Background: Identifying K-ras mutation has became a standard procedure in cancer treatment. Colorectal cancer patients with K-ras mutation are likely to poorly respond to cetuximab. Therefore, detecting K-ras gene mutations should be suggested before the selection of personalized treatment in colorectal cancer. To date, general molecular biology techniques contain HRM, PCR-RFLP, TaqMan PCR and CE-IVD-validated Cobas 4800 KRAS (Roche Diagnostics) are used for K-ras mutation detection in molecular diagnosis, but the sensitivity limitation of these method is approximately 1%. Methods: Therefore, we used a new approach, a universal genetic detecting method (FemtoPath), which improves sensitivity of K-ras mutation detection and the limitation of sensitivity is closed to 0.1% of mutation type. Results: We compared the sensitivity between FemtoPath/direct sequencing test and Cobas KRAS real-time PCR. Cobas real-time PCR identifies mutations in 21 (40.38%) of the 52 tumors. Surprisingly, the FemtoPath/direct sequencing test identified mutations in 40 (76.92%) of the 52 tumors. Our data showed that the FemtoPath/direct sequencing test can identify 19 additional mutation samples. In addition, the FemtoPath/direct sequencing test can identify more unknown K-ras mutations adjacent to codon 12 and 13. Conclusions: Ensure the most timely and appropriate therapy for cancer patients is the first priority of clinical application. FemtoPath/direct sequencing test is more sensitive, accurate and inexpensive and needs fewer sample amount than Cobas real-time PCR.

Detection of KRAS gene mutation and its clinical significance in colorectal adenocarcinoma

To explore the clinical significance of KRAS mutation detection in colorectal adenocarcinoma. Paraffin-embedded tissue specimens were obtained from 440 patients with colorectal adenocarcinoma. The genomic DNA was extracted. Mutations of exon 2 of KRAS gene were examined by PCR and direct sequencing. Somatic mutations of KRAS gene were identified in 146 cases, with the mutation rate of 33.2% (146/440). Among these 146 patients, KRAS mutation involved codon 12 in 118 patients, including 35G > A (Gly12Asp, 62 cases), 35G > T (Gly12Val, 35 cases), 34G > T (Gly12Cys, 9 cases), 34G > A (Gly12Ser, 6 cases), 35G > C (Gly12Ala, 5 cases), and 34G > C (Gly12Arg, 1 case); in 27 patients the mutation involved codon 13, including 38G > A (Gly13Asp, 25 cases), 38G > C (Gly13 Val, 1 case) and 37G > T (Gly13 Cys, 1 case); and in one patient, the mutation involved codon 14 with 40G > A (Val14Ile). The status of KRAS or codon 12 mutations in colorectal adenocarcinoma was related to patients' gender (P = 0.021 and P = 0.030, respectively), and this significant correlation to females was conserved in clinical stage III (P = 0.007 and P = 0.003, respectively), but not in stages I, II, and IV. The status of KRAS or codon 12 mutations was also related to tumor stage. Between stage II and stage IV, the mutation rate of KRAS and codon 12 showed significant difference (P = 0.028 and 0.034, respectively). Between stage III and stage IV, only the codon 12 mutation rate showed significant difference (P = 0.011). Codon 13 mutation was not related to tumor stage. About one third of patients with colorectal adenocarcinoma have KRAS gene mutation, which might be related to patients' gender; and could be consistently detected by PCR and direct sequencing.

Consensus for KRAS gene mutation detection in colorectal carcinoma

全球结直肠癌每年新发病例数约120万,年病死人数超过60万,发病率居第3位,病死率居第4位[1].近些年,结直肠癌的发病率呈明显上升趋势[1].因此,结直肠癌的治疗一直是国内外学者研究的热点.随着靶向治疗药物表皮生长因子受体(EGFR)抑制剂西妥昔单抗(Cetuximab)和帕尼单抗(Panitumumab)的问世,结直肠癌的治疗进入了靶向治疗时代。

Comparison of real-time PCR-optimized oligonucleotide probe method and Sanger sequencing for detection of KRAS mutations in colorectal and lung carcinomas

To investigate the feasibility of real-time PCR-optimized oligonucleotide probe method for detection of KRAS mutations in lung and colorectal carcinomas, as compared with Sanger sequencing method. Genomic DNA was extracted from formalin fixed paraffin embedded samples of 221 lung carcinomas and 131 colorectal carcinomas after tumor cell content assessment and macrodissection. Real-time PCR-optimized oligonucleotide probe method and Sanger sequencing were performed to detect KRAS gene mutations. The frequency of KRAS mutation, mutation types, and their concordance were analyzed. KRAS mutation was detected in 6.3% (14/221) and 4.5% (10/221) of 221 lung cancer samples by using real-time PCR-optimized oligonucleotide probe method and Sanger sequencing, respectively, while in 41.2% (54/131) and 40.5% (53/131) of 131 colorectal cancer samples, respectively. There was no significant correlation between KRAS gene mutations and patients' gender and age (P > 0.05). The positive rate of KRAS codon 12 mutation was significantly higher than that of KRAS codon 13 mutation (P < 0.05). The overall concordance between real-time PCR-optimized oligonucleotide probe method and Sanger sequencing for KRAS mutation detection was 97.4%. Real-time PCR-optimized oligonucleotide probe method provides an alternative method with high consistency and sensitivity as compared to Sanger sequencing in gene mutation detection.