Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT:

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.

SUBMITTER: Gonzalez de Castro D

PROVIDER: S-EPMC3394984 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

Project description:PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

| S-EPMC3983811 | biostudies-other

Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens.

Project description:We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.

| S-EPMC4651363 | biostudies-literature

Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens.

Project description:The utilization of archived, formalin-fixed paraffin-embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here, we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 µm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C · G > T · A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.

| S-EPMC3413110 | biostudies-literature

Performance of a novel KRAS mutation assay for formalin-fixed paraffin embedded tissues of colorectal cancer.

Project description:We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

| S-EPMC4320212 | biostudies-literature

Detecting BRAF Mutations in Formalin-Fixed Melanoma: Experiences with Two State-of-the-Art Techniques.

Project description:BackgroundMelanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition.Patients and methodsWe used 2 methods to analyze the BRAF mutation status in 52 of 62 melanoma patients. Method 1 (mutation-specific real-time PCR) specifically detects the most frequent BRAF mutations, V600E and V600K. Method 2 (denaturing gel gradient electrophoresis and direct sequencing) identifies any mutations affecting exons 11 and 15.ResultsEighteen BRAF mutations and 15 wild-type mutations were identified with both methods. One tumor had a double mutation (GAA) in codon 600. Results of 3 samples were discrepant. Additional mutations (V600M, K601E) were detected using method 2. Sixteen DNA samples were analyzable with either method 1 or method 2. There was a significant association between BRAF V600E mutation and survival.ConclusionStandardized tissue fixation protocols are needed to optimize BRAF mutation analysis in melanoma. For melanoma treatment decisions, the availability of a fast and reliable BRAF V600E screening method may be sufficient. If other BRAF mutations in exons 11 and 15 are found to be of predictive value, a combination of the 2 methods would be useful.

| S-EPMC3383294 | biostudies-literature

Performance comparison of three BRAF V600E detection methods in malignant melanoma and colorectal cancer specimens.

Project description:Personalized cancer care requires reliable biomarkers. While the BRAF V600E mutation is implemented in the clinic, no method for its detection has so far been established as reference. We aimed to perform a comprehensive comparison of three methods currently being used for V600E detection in clinical samples. We analysed genomic DNA from 127 malignant melanomas (77 patients) and 389 tumours from 141 colorectal cancer patients (383 liver metastases and 6 primary tumours) by Sanger sequencing and a single probe-based high-resolution melting assay (LightMix). Formalin-fixed paraffin-embedded (FFPE) tissue from a subset of these lesions (n = 77 and 304, respectively) was analysed by immunohistochemistry (IHC) using the V600E-specific antibody VE1. In a dilution series of V600E-mutated DNA in wild-type DNA, the detection limit for the LightMix assay was 1:1000 mutated alleles while it was 1:10 for Sanger sequencing. In line with this, we detected 15 additional mutated melanoma samples and two additional mutated metastatic colorectal cancer samples by the LightMix assay compared to Sanger sequencing. For the melanoma samples, we observed high concordance between DNA-based methods and analysis by IHC. However, in colorectal samples, IHC performed poorly with 12 samples being scored as V600E positive exclusively by IHC and nine samples being scored as V600E negative exclusively by IHC. In conclusion, the VE1 antibody is not recommendable for clinical tests of colorectal cancer samples. For melanoma samples, IHC may be useful as a screening tool guiding further analytical approaches.

| S-EPMC4342512 | biostudies-literature

Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens.

Project description:Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.

| S-EPMC4912568 | biostudies-literature

Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens.

Project description:DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species.

| S-EPMC6992170 | biostudies-literature

Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing.

Project description:Purpose:We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis. Methods:This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool. Results:Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge. Conclusions:Next generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

| S-EPMC5770184 | biostudies-literature

Combination of KRAS and SMAD4 mutations in formalin-fixed paraffin-embedded tissues as a biomarker for pancreatic cancer.

Project description:Formalin-fixed paraffin-embedded (FFPE) tissues used for routine pathological diagnosis are valuable for cancer genomic analysis; however, the association between mutation status derived from these specimens and prognosis in pancreatic ductal adenocarcinoma (PDAC) remains unclear. We analyzed 50 cancer-related gene mutations including driver genes in PDAC, using next-generation sequencing (NGS) to clarify the association between gene mutations and prognosis. DNA was extracted from FFPE tissues obtained from 74 patients with untreated resectable PDAC who underwent surgery at our institution between 2013 and 2018. Fifty of the 74 patients with DNA extracts from FFPE samples suitable for NGS were analyzed. The prevalence of driver gene mutations was as follows: 84% for KRAS, 62% for TP53, 32% for SMAD4, and 18% for CDKN2A. There were no cases of single SMAD4 mutations; its rate of coincidence with KRAS or TP53 mutations was 30% and 2%, respectively. The combination of KRAS and SMAD4 mutations resulted in significantly shorter relapse-free survival (RFS; median survival time [MST], 12.3 vs. 28.9 months, P = .014) and overall survival (OS; MST, 22.3 months vs. not reached, P = .048). On multivariate analysis, the combination of KRAS and SMAD4 mutations was an independent prognostic factor for RFS (hazard ratio [HR] 4.218; 95% confidence interval [CI], 1.77-10.08; P = .001) and OS (HR 6.730; 95% CI, 1.93-23.43; P = .003). The combination of KRAS and SMAD4 mutations in DNA obtained from FFPE tissues is an independent poor prognostic factor in PDAC.

| S-EPMC7293095 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data