Validation of the Ion Torrent PGM sequencing for the prospective routine molecular diagnostic of colorectal cancer

Objectives Treatment individualization based on specific molecular biomarkers is becoming increasingly important in oncology. In colorectal cancer (CRC), the molecular characterization of RAS and BRAF mutation status for prognostic and predictive purposes is commonly performed by different validated methods. However, as the number of clinically relevant mutations to be analyzed increases, the definition of new approaches for more sensitive, rapid and economic patient selection urges. To this aim, we evaluated the Ion Semiconductor sequencing using the Ion Torrent Personal Genome Machine (IT-PGM) in our routine molecular diagnostics for CRC in comparison with the gold standard direct Sanger sequencing. Design and methods Formalin-fixed and paraffin-embedded tumor tissues obtained by surgery or biopsy of 66 CRCs were collected. DNA was extracted and sequenced by IT-PGM and Sanger method. Results The proposed IT-PGM sequencing strategy exceeded the 500 reads of coverage for all clinically relevant RAS/BRAF amplicons in most samples and thus guaranteed optimal determination. Indeed, the frequencies and the mutational spectrum of RAS and BRAF mutations were in agreement with literature data and revealed 100% concordance between the IT-PGM and routine Sanger sequencing approaches. Turnaround time and cost evaluation indicate that the IT-PGM sequencing permits the characterization of the clinically relevant mutational spots at lower cost and turnaround time compared to Sanger sequencing and allows inclusion of additional amplicons whose characterization may acquire significance in the very next future. Conclusion The IT-PGM is a valid, flexible, sensitive and economical method alternative to the Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor therapy for metastatic CRC.