The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: A comparison between direct sequencing and real-time PCR

Kobunai, Takashi; Watanabe, Toshiaki; Yamamoto, Yasutake; Eshima, Kiyoshi

doi:10.1016/j.bbrc.2010.03.167

Cited by 46 publications

(36 citation statements)

References 19 publications

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“…That the frequency of detected KRAS mutations in clinical samples is influenced by the sensitivity of the method has previously been noted (Kobunai, et al, 2010). However, clinical decisions based upon very low prevalence mutations are controversial.…”

Section: Discussionmentioning

confidence: 94%

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

et al. 2010

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Section: Discussionmentioning

confidence: 94%

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

et al. 2010

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“…However, they are characterized by different sensitivity, turnaround time, and cost. [12][13][14][15] Besides the use of robust KRAS testing methods, the selection of the appropriate tissue to be tested is also critical. Particularly, evaluation of the tumor cellularity is essential to guide the interpretation of the molecular data.…”

mentioning

confidence: 99%

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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“…The mutant could reliably be detected up to a sample containing 5% of mutated alleles in the presence of 95% wildtype alleles (data not shown). This sensitivity is outperforming standard dideoxy sequencing where the minimum content of mutated alleles is reported at the 20-30% level and comparable to other mutation detection techniques, which usually reach down to the same 5% sensitivity level [30,31].…”

Section: Chipce Egfr Exon 21 Mutation Detection By Denaturing Cementioning

confidence: 92%

A novel high‐resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA

Minárik¹,

Gassman

Belšánová³

et al. 2010

Electrophoresis

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There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.

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The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: A comparison between direct sequencing and real-time PCR

Cited by 46 publications

References 19 publications

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

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

A novel high‐resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA

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