Molecular aspects of small bowel adenocarcinoma for new therapeutic approaches.

Small Bowel Adenocarcinoma (SBA) is a rare and aggressive neoplastic disease. Several aspects of SBA carcinogenesis still need to be elucidated, but risk factors and histomorphological similarities seem to indicate that SBA can follow a carcinogenetic development similar to that proposed for colorectal cancer. At molecular level, at odds with adenocarcinoma arising in the large intestine, very few, and fragmented, information is available for SBA. In general, it has been suggested that the two models of colorectal carcinogenesis can be valid also for SBA. For that reason, chemotherapies set up for the cancers of the large intestine have been applied also for SBA. Therefore, since recent studies led to the introduction of EGFR-targeted therapies in colorectal cancer, the treatment with anti-EGFR drugs can be proposed also for SBA patients. In particular, in colorectal cancer patients it has been demonstrated that KRAS mutations are correlated with the absence of efficacy of EGFR-targeted therapies, and it has been proposed by few studies to investigate additional markers of the EGFR pathway (EGFR gene copy number, BRAF and PIK3CA mutations, as well as PTEN protein expression), in order to increase the predictive power of the efficacy of anti-EGFR drugs. Information regarding these markers in SBA is quite completely missing. Primary aim of the present work was the evaluation, in the same cohort, of all the alterations involved in colorectal carcinogenesis, in order to shed light more deeply about the molecular similarity between SBA and colorectal cancer. Second aim of the present work was to investigate in the same cohort of SBA the aforementioned markers involved in the EGFR pathway, in order to verify if the pattern of these alterations could justify the possible introduction of these therapies also in patients affected by SBA. To do this, for the first aim we investigated -catenin protein expression by immunohistochemistry (IHC), and KRAS and TP53 mutations by direct sequencing, as well as microsatellite instability (MSI) and allelic imbalance of Chromosome 18q MSI by fragment analysis on genomic DNA extracted from formalin-fixed paraffinembedded tissue sections. For the second aim we investigated EGFR gene status by Fluorescent in situ hybridization (FISH), BRAF and PIK3CA mutational status by direct sequencing, as well as PTEN protein expression by IHC, in the same cohort of SBA. We recruited 40 SBA cases from the Institute of Pathology of Locarno (Canton Tessin, Southern Switzerland) and from three institutions of Northern Italy. First aim. -catenin overexpression was observed in 23.6% at nuclear level and in additional 47.3% of cases only at cytoplasmic level, MSI was found in 23.6% of cases, KRAS mutations in 43.6% of cases, TP53 mutations in 29% of cases and allelic imbalance of Chromosome 18q in 75% of cases. All the percentages of alterations and the types of mutations are in line with those identified in the analysis of colorectal cancer patients. Therefore, by the analysis of all these markers in a same cohort, we can confirm that SBA shares the carcinogenetic development with colorectal cancer also at molecular level. Second aim. We identified a copy number gain of EGFR gene in 57.5% of cases, BRAF and PIK3CA mutations in 2.5% and 10.5% of cases respectively, and PTEN loss of expression in 25.6% of cases. Also for the EGFR pathway analysis, percentages of alterations and types of mutations found in SBA are in line with colorectal cancer, even if we did not detect the classical V600E change in the BRAF gene (where, on the contrary, we found a rare mutation, the G596R change). Taking into account the molecular algorithm proposed for the administration of EGFR-targeted therapies in colorectal cancer patients, if we look only at KRAS mutations, we can propose the administration of EGFR-targeted therapies to about 60% of patients (i.e.: KRAS wildtype cases), and to 23% of cases if we base our evaluation on the whole EGFR pathway (i.e.: cases showing, at the same time, EGFR copy number gain, KRAS, BRAF and PIK3CA wild-type sequences, and PTEN normal expression). The treatment with anti-EGFR therapies of a SBA patient of our cohort who developed a metastatic lesion confirmed the relevance of the molecular characterization of the tumor to predict the response to these therapies. In conclusion, our analyses of SBA confirm the feeling that the mechanisms of carcinogenesis of such disease are superimposable with those proposed for colorectal cancer. Therefore, the hypothesis that therapeutic protocols valid for the large intestine can be applied also to SBA patients is supported. As a consequence, the targeted therapies recently introduced in colorectal cancer can be proposed for SBA patients, pending tumor molecular characterization as demonstrated by our case report