KRAS Mutations In Clinical Samples Research Articles

The mutation rates of EGFR in non-small cell lung cancer and KRAS in colorectal cancer of Chinese patients as detected by pyrosequencing using a novel dispensation order

BackgroundThe purpose of this study was to develop a cost-effective approach for the determination of EGFR and KRAS mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) samples from Chinese patients based on a sensitive pyrosequencing (PS) technique.MethodsThe NSCLC and CRC cell lines were tested to determine the limitation of detection and reproducibility of the PS method. In addition, 494 NSCLC and 1099 CRC patient samples were assayed by PS to evaluate the EGFR or KRAS mutation patterns according to the clinicopathological features.ResultsThe PS assay was able to reproducibly detect as few as 2 % mutant alleles with excellent linearity. EGFR mutations were detected in 35.63 % of the NSCLC samples, and KRAS mutations were detected in 39.76 % of the CRC samples. EGFR mutations were more frequently observed to be significant by multivariate analysis in NSCLC patients who were 65 years old or younger (OR = 2.51), had a nonsmoking history (OR = 3.63), and adenocarcinoma (OR = 3.57), but not in females (OR = 0.64). KRAS mutations were more frequently detected in CRC patients who were female (OR = 1.64) and 50 years old or older (OR = 4.17), and had adenocarcinoma (OR = 2.41).ConclusionsThis is the first extensive validation of PS on FFPE samples using the detection of EGFR exons 18–21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of PS analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and provide important clinical and molecular characteristics of NSCLC and CRC from Chinese patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0179-9) contains supplementary material, which is available to authorized users.

A New Fast and Highly Sensitive Assay for the Detection of Kras Mutations

ABSTRACT Aim: The demonstrated efficacy of anti-EGFR drugs (mAbs) in KRAS wild-type (wt) patients affected by colorectal cancer (CRC) resulted in increasing demand for mutation analyses. Mutation testing techniques have therefore become an urgent concern as a powerful mean to select wt patients. However, it is still unclear which is the most sensitive, specific and efficient protocol to detect KRAS mutations in clinical samples. The aim of this study was to compare the new Pentabase kit with direct sequencing (DS) (gold standard) and mutant-enriched PCR (ME-PCR) (highly sensitive method) with regard to sensitivity, specificity and efficiency of testing. Methods: Eight tumour cell lines, 7 specific for each KRAS mutation and 1 with the KRAS wt sequence, were used to perform a sensitivity assay. Seven different percentages of mutated DNA in fixed amounts of wt DNA were tested by all the methods (10%, 1%, 0,5%, 0,1%, 0,05%, 0,01% and 0%). KRAS mutations in exons 2 (codons 12 and 13) were evaluated by DS, ME-PCR and the Pentabase kit in a sensitivity assay and in 100 consecutive CRC patients. The Pentabase kit is based on real-time PCR using SuPrimers™ (DNA primers with increased specificity), BaseBlockers™ (oligos suppressing amplification of wt genes) and HydrolEasy probes (hydrolysis probe with increased signal-to-noise ratio and sensitivity). Results: In the cell line experiments, DS had a sensitivity of 10%, ME-PCR of up to 0,1% and the Pentabase kit of Conclusions: Overall, through the application of highly sensitive KRAS analysis methods, we detected additional KRAS alterations in up to 13/71 (18%) and 2/60 (3%) patients after DS and ME-PCR had shown them to be KRAS wt, respectively. The new kit allows the KRAS mutational typing in only 2 hours, compared to DS and ME-PCR, which require at least 2 days. Therefore the Pentabase kit represents a new, faster, more sensitive and more reliable method for the analysis of KRAS mutations in CRC and can be proposed for the identification of patients resistant to anti-EGFR mAbs. Disclosure: All authors have declared no conflicts of interest.

Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay

ObjectivesMutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples. Design and methodsWe developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues. ResultsOverall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61. ConclusionThe RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.

Specific and sensitive detection of BRAF and KRAS mutations in clinical samples.

10587 Background: The development of highly sensitive and specific genotyping assays that are suitable for clinical research and molecular diagnostics opens new opportunities for the detection, assessment, and research of cancer. Swift Biosciences has developed myT Primer qPCR technology which has unique structural and thermodynamic properties that enables highly sensitive mismatch discrimination. The myT Primer reagents can detect 1% mutant BRAF or KRAS present in a background of 103 wild-type genomic DNA copies with no background signal from wild-type. A separate ultrasensitive BRAF format has been developed which enables the detection of a single mutant BRAF copy in > 104 wild-type genomic DNA copies, representing 0.01% detection. Methods: To evaluate the performance on clinical samples, DNA was prepared from 26 melanoma, 40 colorectal, and 49 lung tumor fresh frozen and FFPE samples, and their corresponding normal adjacent samples. qPCR was performed using standard cycling conditions in single tube format with allele-specific myT primers to assess two BRAF alleles or seven KRAS alleles. Performance of the assay was evaluated on the ABI 7500, CFX96, and Roche LightCycler. A subset of samples were selected for validation by Sanger sequencing. Results: Mutations of BRAF in melanoma, colorectal, and lung tumors were observed to be 46%, 5%, and 2%, in concordance with published data for the frequency of BRAF mutations. Mutations of KRAS in colorectal cancer were observed to be in 17/40, or 42% of samples, consistent with published reports of KRAS in colorectal cancer. 34% of lung cancer samples had a KRAS mutation, also consistent with available statistics. Mutation status was confirmed with Sanger sequencing in the subset of selected samples. Conclusions: The extreme selectivity of the myT BRAF Primers makes them well suited for genotyping of conventional FFPE and frozen tissue samples, whereas the ultrasensitive format is ideal for use with difficult samples such as needle biopsies, CTCs, and serum. The extreme selectivity of these primers results in a definitive Yes/No answer and eliminates the need to use a delta Ct method.

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10–20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80°C (vs 94°C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

BackgroundEpithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case.MethodsWe developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues.ResultsAll 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive.ConclusionThis is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings.