A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations

Angulo, Bárbara; García-García, Elena; Martínez, Rebeca; Suárez-Gauthier, Ana; Conde, Esther; Hidalgo, Manuel; López‐Ríos, Fernando

doi:10.2353/jmoldx.2010.090139

Cited by 100 publications

(78 citation statements)

References 29 publications

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“…We and others have previously demonstrated the inverse relationship between tumor cell content of a specimen and ability to detect mutations by direct sequencing. 16,18,19,27 Thus, the method used to detect somatic mutations must be sensitive enough to avoid false-negative results, particularly in samples contaminated by non-tumor elements.…”

Section: Discussionmentioning

confidence: 99%

“…Due to its exhaustiveness, the multi-step sequencing methodology is the gold standard for the detection of unknown mutations, but it lacks sensitivity. Consequently, more rapid and sensitive methods have been developed and validated, including highresolution melting, 29,31 allele-specific PCR, 16,29 and pyrosequencing. 32 Performed before DNA sequencing, high-resolution melting is very attractive because the analysis of the real-time amplification curves allows (i) the calculation of the Ct value that is directly related to the amount of amplifiable DNA and (ii) the identification of heterozygous genetic changes even in samples containing only 10% of mutated cells.…”

Section: Discussionmentioning

confidence: 99%

“…For instance, false-negative results can occur when the starting sample is contaminated by an important number of non-tumor cells. [16][17][18] In the case of metastatic colorectal cancer, poor tumor cellularity is mainly observed when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy or in liver metastases after chemotherapy. 10,19,20 Manual dissection to remove the non-invasive cell components before nucleic acid extraction is a powerful tool for tumor cell enrichment.…”

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confidence: 99%

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KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Boissière‐Michot¹,

Lopez-Crapez²,

Frugier³

et al. 2012

Modern Pathology

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KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with antiepidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allelespecific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Boissière‐Michot¹,

Lopez-Crapez²,

Frugier³

et al. 2012

Modern Pathology

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“…Sequencing has long been the most widely used method to detect point mutations. The major disadvantage is that sequencing is not very sensitive [18], and in samples with a low tumor content, in particular, analysis might be difficult [19]. Allele-specific PCR is more sensitive but tests for only a subset of the most common mutations, whereas sequencing can detect all possible mutations.…”

Section: Discussionmentioning

confidence: 99%

External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds

et al. 2011

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Learning Objectives After completing this course, the reader will be able to: Identify the most frequent errors made in KRAS testing in this study and the possible consequences for a patient. Describe factors that could increase the chance of an error during KRAS testing. This article is available for continuing medical education credit at http://CME.TheOncologist.com The use of epidermal growth factor receptor–targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild‐type KRAS tumors by the European Medicines Agency since 2008, based on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors. In an effort to ensure optimal, uniform, and reliable community‐based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use their own preferred method for histological evaluation, DNA isolation, and mutation analysis. In this study, we analyze the setup of the KRAS scheme. We analyzed the advantages and disadvantages of the regional scheme organization by analyzing the outcome of genotyping results, analysis of tumor percentage, and written reports. We conclude that only 70% of laboratories correctly identified the KRAS mutational status in all samples. Both the false‐positive and false‐negative results observed negatively affect patient care. Reports of the KRAS test results often lacked essential information. We aim to further expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization.

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“…In this technique, allele-or mutation-specific amplification is achieved by ARMS (amplification refractory mutation system) technology and its detection is conducted using Scorpions (bifunctional molecules containing a PCR primer covalently linked to a probe; ref. 31). The following mutations were evaluated: KRAS G12A, G12D, G12R, G12C, G12S, G12V, G13D and PIK3CA H1047R, E542K, E545D, E545K.…”

Section: Tissue Samples and Molecular Analysesmentioning

confidence: 99%

Molecular Profiling of Patients with Colorectal Cancer and Matched Targeted Therapy in Phase I Clinical Trials

Dienstmann

Serpico

Rodón

et al. 2012

Molecular Cancer Therapeutics

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Clinical experience increasingly suggests that molecular prescreening and biomarker enrichment strategies in phase I trials with targeted therapies will improve the outcomes of patients with cancer. In keeping with the exigencies of a personalized oncology program, tumors from patients with advanced chemorefractory colorectal cancer were analyzed for specific aberrations (KRAS/BRAF/PIK3CA mutations, PTEN and pMET expression). Patients were subsequently offered phase I trials with matched targeted agents (MTA) directed at the identified anomalies. During 2010 and 2011, tumor molecular analysis was conducted in 254 patients: KRAS mutations (80 of 254, 31.5%), BRAF mutations (24 of 196, 12.2%), PIK3CA mutations (15 of 114, 13.2%), KRAS and PIK3CA mutations (9 of 114, 7.9%), low PTEN expression (97 of 183, 53.0%), and high pMET expression (38 of 64, 59.4%). In total, 68 patients received 82 different MTAs: phosphoinositide 3-kinase (PI3K) pathway inhibitor (if PIK3CA mutation, n ¼ 10; or low PTEN, n ¼ 32), PI3K pathway inhibitor plus MEK inhibitor (if KRAS mutation, n ¼ 10; or BRAF mutation, n ¼ 1), second-generation anti-EGF receptor monoclonal antibodies (if wild-type KRAS, n ¼ 11), anti-hepatocyte growth factor monoclonal antibody (if high pMET, n ¼ 10), mTOR inhibitor plus anti-insulin-like growth factor-1 receptor monoclonal antibody (if low PTEN, n ¼ 5), and BRAF inhibitor (if BRAF mutation, n ¼ 3). Median time-to-treatment failure on MTA was 7.9 versus 16.3 weeks for their prior systemic antitumor therapy (P < 0.001). Partial response was seen in 1 patient [1.2%, PI3K inhibitor with PIK3CA mutation] and stable disease >16 weeks in 10 cases (12.2%). These results suggest that matching chemorefractory patients with colorectal cancer with targeted agents in phase I trials based on the current molecular profile does not confer a significant clinical benefit. Mol Cancer Ther; 11(9); 2062-71. Ó2012 AACR.

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A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations

Cited by 100 publications

References 29 publications

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds

Molecular Profiling of Patients with Colorectal Cancer and Matched Targeted Therapy in Phase I Clinical Trials

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