KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program

Krieken, J.H.J.M. van; Jung, Andreas; Kirchner, Thomas; Carneiro, Fátima; Seruca, Raquel; Ft, Bosman; Quirke, Phil; Fléjou, Jean François; Hansen, Tine Plato; Hertogh, G De; Jares, Pedro; Langner, Christine; Höefler, Gerald; Ligtenberg, M. J. L.; Tiniakos, D.; Tejpar, Sabine; Bevilacqua, Giuseppe; Ensarı, Arzu

doi:10.1007/s00428-008-0712-8

Cited by 52 publications

(76 citation statements)

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“…11,28 Manual dissection of formalin-fixed paraffin-embedded tissues is a widely used and powerful tool to enrich the sample with tumor cells. 17,27 This pre-analytical step is easy to perform, not time consuming and suitable for a routine setting.…”

Section: Discussionmentioning

confidence: 99%

“…[8][9][10][11] Various laboratory-based or commercial KRAS mutation assays are used in the routine practice and the majority of them have been optimized to be compatible with DNA extracted from formalin-fixed paraffin-embedded samples, which represent the most available source of tumor tissue. However, they are characterized by different sensitivity, turnaround time, and cost.…”

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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%

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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“…The recent advice from the American Society of Clinical Oncology and a European expert panel to perform KRAS mutation detection before therapy with cetuximab in metastatic colorectal cancer 5 and in stage II and III colon cancer, 6 respectively, has made the need urgent for a sensitive, flexible, and fast assay that is easy to implement in daily practice. Therefore, we compared three currently available techniques for implementation in routine diagnostics.…”

Section: Discussionmentioning

confidence: 99%

“…5 Moreover, in Europe, KRAS mutation analysis in stage II and III colon cancer has been recommended by an expert panel. 6 Thus, KRAS mutation detection plays an important role in colon cancer therapy decision making and could very well become one of the most frequently performed tests in diagnostic pathology laboratories in the future.…”

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confidence: 99%

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

Sarasqueta

Moerland

Bruyne

et al. 2011

The Journal of Molecular Diagnostics

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Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2-specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives.

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“…Examples are of course mutations and amplifications in oncogenes, translocations resulting in abnormal gene activity or even novel proteins. In all these situations, there are important quality issues and external quality assurance is mandatory [6][7][8]. In these tests the interaction of pathologists and molecular biologists should not be underestimated, but it is not in comparison with clonality testing.…”

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confidence: 99%

Clonality testing: teamwork by pathologist and molecular biologist

Krieken

Cabeçadas²,

Groenen

2012

J Hematopathol

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KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program

Cited by 52 publications

References 0 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

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

Clonality testing: teamwork by pathologist and molecular biologist

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