ArticleBMC clinical pathology2010
COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma.
Article in BMC clinical pathology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 28 citations in OpenAlex.
- A novel PIK3CA hot-spot mutation in breast cancer patients detected by HRM-COLD-PCR analysis.Breast disease · 2024Article
- Mutation enrichment in human DNA samples via UV-mediated cross-linking.Nucleic acids research · 2022Article
- Role of oncogenic KRAS in the diagnosis, prognosis and treatment of pancreatic cancer.Nature reviews. Gastroenterology & hepatology · 2020Review
- Review
- A novel RFLP-ARMS TaqMan PCR-based method for detecting the BRAF V600E mutation in melanoma.Oncology letters · 2018Article
- DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples.Clinical chemistry · 2015Article
- Enhanced ratio of signals enables digital mutation scanning for rare allele detection.The Journal of molecular diagnostics : JMD · 2015Article
- Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.Clinical chemistry · 2015Article
- Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.Molecular diagnosis & therapy · 2014Article
- COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.PloS one · 2014Article
- Colorectal cancer patients with low abundance of KRAS mutation may benefit from EGFR antibody therapy.PloS one · 2013Article
- Colorectal carcinoma: Pathologic aspects.Journal of gastrointestinal oncology · 2012Article
- Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment.Clinical chemistry · 2012Article
- Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan(®) -MGB probe genotyping method.Experimental and therapeutic medicine · 2012Article
- Article
Corrections and comments
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Authors and funding
5 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundKRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.
methodsWe 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.
resultsWe 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.
conclusionsWe 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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.