Abstract 937: Molecular analysis of KRAS exon 2 wild type colorectal cancer patients enrolled in the CAPRI clinical trial reveals high degree of tumor heterogeneity
Authors: Rachiglio Anna Maria , Lambiase Matilde , Fenizia Francesca , Esposito Claudia , Roma Cristin , Tonini Giuseppe , Cinieri Saverio , Colucci Giuseppe , Ciardiello Fortunato , Normanno Nicola 
Published: May 2015

Abstract
The CAPRI clinical trial (Optimal strategy in KRAS wild type metastatic colorectal cancer patients: cetuximab plus FOLFIRI followed by FOLFOX +/- cetuximab) enrolls metastatic colorectal carcinoma (mCRC) patients with no codon 12/13 (exon 2) KRAS mutations. Recent data showed that exon 3 and 4 KRAS mutations as well as NRAS exon 2, 3 and 4 mutations might confer resistance to EGFR monoclonal antibodies. In addition, somatic mutations in other genes such as BRAF, PIK3CA, PTEN, have been hypothesized to be prognostic and/or predictive in CRC. Therefore, molecular sub-classification of KRAS exon 2 wild type CRC might allow to identify subgroups of patients highly sensitive or resistant to anti-EGFR agents within the CAPRI trial. We analyzed 182 FFPE samples from mCRC patients enrolled in the CAPRI clinical trial by using a next generation sequencing (NGS) approach based on the Ion AmpliSeq™ technology. In particular, 10 ng of genomic DNA were analyzed with the Ion Ampliseq Colon and Lung cancer panel that targets over 500 hotspot mutations in 22 genes implicated in colon and lung cancers. Data analysis was performed using Ion Reporter™ Software. The analysis with the IonAmpliseq panel was successful in all the tested samples. Among the 182 analyzed cases, 58 (31.9%) showed no mutations in the investigated genes, whereas a total of 206 mutations in 14 genes were detected in 124 cases (68.1%). Surprisingly, 30 exon 2 KRAS mutations were identified in 29 patients (15.9%). Mutations at a frequency &gt;4% were also found in: TP53 (39.5% of the cases mutant), KRAS exon 3 and 4 (7.5%), PIK3CA (13.2), BRAF (8.2%), NRAS (7.1%), FBXW7 (4.9%). A significant degree of tumor heterogeneity was observed: several tumors had multiple mutations in different genes, with some cases showing up to 5 mutations. In particular, 30/45 KRAS, 5/13 NRAS, 12/15 BRAF and 19/24 PIK3CA mutant cases showed variants in additional genes. The frequency of mutant alleles also differed significantly among tumors. For example, among the KRAS exon 2 mutant cases, the frequency of KRAS mutant alleles, after normalization for the neoplastic cell content, ranged between 6% and 130%, suggesting that some KRAS mutant cases were polyclonal and that in some other cases gain of mutant or loss of wild type KRAS alleles might have occurred. These data suggest that molecular classification of KRAS exon 2 wild type CRC with the Ion Ampliseq Colon and Lung cancer panel is feasible. This approach reveals mutations in several key genes involved in CRC tumor progression. The relatively high incidence of codon 12/13 KRAS mutations also suggests that an high rate of false negative results in KRAS testing occurs in clinical practice. Finally, the huge degree of tumor heterogeneity might have important implications for prognosis and response to therapy.
Citation Format: Anna Maria Rachiglio, Matilde Lambiase, Francesca Fenizia, Claudia Esposito, Cristin Roma, Giuseppe Tonini, Saverio Cinieri, Giuseppe Colucci, Fortunato Ciardiello, Nicola Normanno. Molecular analysis of KRAS exon 2 wild type colorectal cancer patients enrolled in the CAPRI clinical trial reveals high degree of tumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 937. doi:10.1158/1538-7445.AM2014-937