A Proof-of-concept Study Investigating the Comparison of the RAS Mutational Status Between Primary Tumor and Metastasis in Patients Affected by Metastatic Colorectal Cancer on the Basis of Different Chemotherapeutic Regimens

the study implies the subdivision into three groups with a 1:1:1 ratio.

• The first group includes patients treated with surgery of the primary tumor, neoadjuvant chemotherapy plus bevacizumab and, finally, the surgical resection of liver metastasis.
• The second group is similar to group one, with the exception that patients were not treated with a bevacizumab-based regimen.
• The third group, the control group, includes patients presenting with synchronous primary tumour and metastasis resected without any preoperative systemic therapy

Genomic DNA from formalin-fixed paraffin-embedded (FFPE) tissues from the primary tumor and metastatic lesions will be extracted. The genomic DNA will be assessed for the RAS mutational status in a quantitative and qualitative manner using two different approaches: a real-time PCR approach using the SensiScreen® kit (PentaBase Aps) and a next-generation sequencing approach using the Ion Torrent platform by applying the Ion AmpliSeq™ Cancer Hotspot Panel v2 (ThermoFisher Scientific). The real-time PCR is able to provide the relative quantification of the RAS mutant allele by comparing the Ct value of the mutation with respect to the Ct of the reference gene. This ratio will be calculated for both the primary tumor and for the metastasis and then compared. Ion Torrent gives directly the percentage of the mutant allele in each sample. Furthermore, the Cancer Hotspot Panel v2 provides data (both quantitative and qualitative) about the mutational status of additional 49 genes, including the most relevant and frequently mutated genes in CRC (i.e.: APC, TP53, PIK3CA, BRAF and PTEN) and the relative mutational pattern of the primary tumor and the one of the distant metastasis will be compared.