Delineation of the infrequent mosaicism of KRAS mutational status in metastatic colorectal adenocarcinomas Ce ´line Bossard, 1 Se ´bastien Ku ¨ry, 1 Philippe Jamet, 2 He ´le `ne Senellart, 3 Fabrice Airaud, 1 Jean-Franc ¸ois Rame ´e, 4 Ste ´phane Be ´zieau, 1 Tamara Matysiak-Budnik, 1,2 Christian L Laboisse, 1 Jean-Franc ¸ois Mosnier 1 ABSTRACT This study addresses the extent of the heterogeneity of KRAS status, present in a minority of metastatic colorectal carcinomas (mCRCs), on the basis of a thorough analysis of surgical resection specimens. Eighteen patients with mCRC were included. KRAS mutations (exon 2, codons 12 and 13) were determined using PCR and subsequent direct sequencing. This analysis included primary tumours (n¼21), synchronous (n¼10) and metachronous (n¼18) matched metastases, and pelvic recurrence (n¼1). Heterogeneity of KRAS status consisted in KRAS mutated in (i) the primary tumour but not in its synchronous metastasis, (ii) the metastasis but not in the primary tumour, (iii) the pelvic recurrence but not in the primary tumour, (iiii) some metastases and not in others from the same patient. Finally, the KRAS status varied among different areas of the same metastatic focus. This study defines the concept of KRAS mosaicism that affects a minority of mCRCs. INTRODUCTION The improved outcome of patients with metastatic colorectal cancer (mCRC) is in part due to the introduction of targeted therapies such as the anti- epidermal growth factor receptor (EGFR) anti- bodies. Several studies have shown that KRAS mutation in a primary tumour predicts its resis- tance to anti-EGFR antibodies, 1e3 and thus only patients with a wild-type KRAS primary tumour are eligible for anti-EGFR therapy. In fact, a high degree of concordance in KRAS mutational status has been reported between the primary tumour and its matched metastasis in a given patient. 4e6 In a minority of cases (5e10%), the KRAS mutational status is heterogeneous between primary tumour and metastasis. 7e11 However, the degree of hetero- geneity of the KRAS status remains unknown, especially between and within metastases. Therefore, in this study, based on a cohort of 18 consecutive patients with a mCRC, we analysed the heterogeneity of the KRAS mutational status in each patient (1) between the primary tumour and its resected matched synchronous and/or meta- chronous metastases, (2) between the different synchronous and/or metachronous metastases, and finally (3) inside a given metastasis. PATIENTS AND METHODS Eighteen consecutive patients (median age 61 years) treated for a mCRC between 2001 and 2010, and whose primary tumour tissue and matched metastatic tissues were available for histological review and mutational analysis, were included. Formalin-fixed and paraffin-embedded tissues, including 21 primary tumours, 28 synchronous or metachronous metastases and one pelvic recurrence, were processed according to the guidelines of the French Ethics Committee for Research on Human Tissues. All tumour blocks were reviewed for quality and tumour content. For each sample, tumour tissue was identified on a H&E-stained section and macrodissected under direct visualisation to obtain at least 50% of the tumour cells, and thus to reduce the contamination by non-neoplastic cells. Genomic DNA was extracted using a classical protocol (proteinase K digestion and phenolechloroform purification). The KRAS mutational analysis (exon 2, codon 12 and 13) was performed by means of direct sequencing of PCR products on ABI 3130XL, using Big Dye terminator V1.1 chemistry (Applied Biosystems, Foster City, California, USA). When possible, the mutational analysis was performed in multiple areas of arbitrarily selected metastatic lesions to analyse the intratumoral heterogeneity within a given metastasis. RESULTS The main clinicopathological characteristics of the 18 patients are summarised in the table 1. In 11 patients, metastatic or recurrence tissues were confined to one site, whereas in seven patients, metastasis occurred at two different sites at least (one to four per patient). KRAS mutations in primary tumours and in matched metastases and recurrence are shown in table 2. In 14 out of 18 patients, the KRAS status was concordant between the primary tumour and matched metastases. However, in four of 18 patients, the KRAS status was discordant (table 3). In two patients, a mutation was detected in the primary colorectal cancer but not in the synchro- nous liver metastasis. In one patient, the KRAS mutation was detected only in the pelvic recur- rence. In one patient, the primary tumour and one synchronous liver metastasis were wild-type, while another synchronous metastasis and a further metachronous metastasis were mutated. One patient had two synchronous colonic adenocarci- nomas, one of the right colon and one of the left colon, differing in their KRAS mutational profile: 1 Universite ´ de Nantes, Faculte ´ de Me ´decine de Nantes, EA 4273 Biometadys, Nantes, France 2 Institut des Maladies de l’Appareil Digestif et Service d’He ´pato-Gastroente ´rologie, CHU de Nantes, France 3 Centre de Lutte contre le Cancer Nantes Atlantique, Service d’Oncologie Me ´dicale, Nantes, France 4 Centre Catherine De Sienne, Service d’Oncologie Me ´dicale, Nantes, France Correspondence to Dr Ce ´line Bossard, CHU Nantes, Service d’Anatomie et Cytologie Pathologiques, EA 4273 Biome ´tadys, Universite ´ de Nantes, Faculte ´ de Me ´decine, 1 rue Gaston Veil, Nantes 44035, France; celine.bossard@chu-nantes.fr Accepted 23 December 2011 Bossard C, Ku ¨ry S, Jamet P, et al. J Clin Pathol (2012). doi:10.1136/jclinpath-2011-200608 1 of 4 Short report JCP Online First, published on January 18, 2012 as 10.1136/jclinpath-2011-200608 Copyright Article author (or their employer) 2012. Produced by BMJ Publishing Group Ltd under licence. group.bmj.com on January 20, 2012 - Published by jcp.bmj.com Downloaded from