Determination of KRAS Status in Highly Challenging Rectal Adenocarcinomas after Neoadjuvant Treatment.
Florence Boissiere-Michot, Helene Frugier, Evelyne Lopez-Crapez, Marie-Laurence Berthe, Frederic Bibeau. CRLC Val d'Aurelle, Montpellier, France; CHU – A. de Villeneuve, Montpellier, France
Background: KRAS status is a pre-requisite in patients with metastatic colorectal carcinoma candidate for treatment with monoclonal antibodies targeting EGFR. Indeed, KRAS mutations are associated with resistance to treatment. However, KRAS status determination may be very challenging when tumor cellularity is poor, notably when major tumor regression (TR) is achieved after radiochemotherapy (RCT).
Design: We aimed to determine the most reliable strategy to detect KRAS mutations in poor cellular samples of rectal adenocarcinomas after RCT. 31 tumors with major TR and paired pre-treatment (PT) biopsies were analyzed. Following manual dissection of tumor from surgical specimens and PT biopsies, extracted DNA were submitted to High Resolution Melting (HRM) analysis. DNA displaying an altered shape of the melting curves were submitted to sequencing, as in our daily practice. DNA with altered melting curve without identification of mutation by sequencing were analyzed by an allele-specific PCR assay (a-sPCR). Wild type (wt) KRAS surgical samples, after HRM/sequencing, were microdissected and submitted to the same molecular processes.
Results: Among the 31 manually dissected surgical samples, 7 mutations were identified by HRM/sequencing. 3 additional mutations were detected with a-sPCR. Laser microdissection allowed the detection of 2 supplementary mutations but 2 mutations previously identified with a-sPCR were no more identified. Altogether, 12 surgical specimens displayed a mutated KRAS status. Manual dissection of PT biopsies followed by HRM/sequencing allowed the detection of 12 mutations, of which two were only detected on PT biopsies. Conversely, 2 wt KRAS PT biopsies were found mutated in their paired surgical samples. Overall, when combining the 3 molecular assays and manual/laser dissection, 14 patients displayed a mutated KRAS status. This mutation rate was twice that obtained after our routine procedure.
Conclusions: The recourse to PT biopsies is the best strategy for the management of poor cellular surgical specimens after RCT, allowing the detection of 12/14 mutations. Thus, we recommend a tumor PT biopsy dedicated to molecular diagnosis before any neoadjuvant treatment. If not available, more sensitive assays such as a-sPCR, or laser microdissection, could be helpful in case of wt KRAS status, but at the expense of higher costs and longer delays. These results may impact patients'management, avoiding the use of inefficient, costly and potentially toxic targeted therapy.
Monday, February 28, 2011 9:00 AM
Platform Session: Section E, Monday Morning