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Published on: July 6, 2017
KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment
Florence Boissière-Michot1, Evelyne Lopez-Crapez, Hélène Frugier
1Department of Pathology, Val d'Aurelle Cancer Institute, Montpellier, France.
Abstract:
KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with anti-epidermal 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 allele-specific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.
Insights
For metastatic colorectal cancer patients, pre-treatment biopsies are the most cost-effective method for KRAS genotyping, especially after radiochemotherapy reduces tumor cellularity in surgical specimens.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- KRAS mutation testing is essential for metastatic colorectal cancer (mCRC) patients undergoing anti-EGFR therapy.
- KRAS mutations confer resistance to anti-EGFR treatments, mandating accurate assessment.
- Assessing KRAS status can be difficult in post-radiochemotherapy rectal cancer specimens due to low tumor cellularity.
Purpose of the Study:
- To determine the most reliable and cost-effective strategy for KRAS mutation detection in challenging colorectal cancer samples.
- To compare the efficacy of different molecular techniques in detecting KRAS mutations in low-cellularity specimens.
Main Methods:
- DNA extraction from 31 post-radiochemotherapy surgical specimens and paired pre-treatment biopsies.
- High-resolution melting (HRM) analysis followed by sequencing for initial mutation screening.
- Further analysis using allele-specific PCR (TheraScreen) and laser microdissection (LMD) with HRM/sequencing for low-yield samples.
Main Results:
- KRAS mutations were identified in 9/31 (29%) post-radiochemotherapy surgical specimens.
- Pre-treatment biopsies revealed 12 mutations (39%) using HRM and sequencing.
- No additional mutations were found with TheraScreen or LMD in the pre-treatment biopsies.
Conclusions:
- Pre-treatment biopsies are the most cost-effective approach for reliable KRAS genotyping in post-radiochemotherapy colorectal cancer specimens with low tumor cellularity.
- While sensitive assays like allele-specific PCR and LMD can detect additional mutations, they incur higher costs and longer turnaround times.
