Background It has long been recognized that the epidermal growth factor receptor (EGFR) pathway is frequently activated in colorectal cancer. Therefore targeted therapies against EGFR such as cetuximab and panitumumab have been developed and are currently approved for the treatment of metastatic disease. It has been shown that activating mutations of KRAS or NRAS lead to a consecutive activation of the RAS-RAF pathway downstream of EGFR and consequently result in resistance to anti-EGFR therapy. Therefore mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy. Moreover BRAF status in conjunction with microsatellite testing adds prognostic information and is increasingly demanded by clinical collegues. As patients with PIK3CA mutations may benefit from nonsteroidal anti-inflammatory drugs, testing may further be extented for PIK3CA. Thus, reliable, fast, sensitive and cost effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion. Methods We developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test for the presence/absence of KRAS, NRAS BRAF and PIK3CA mutations. We applied this assay to 371 samples from patients with CRC, compared the results to Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). Results MS detected KRAS mutations in 160 (43%), NRAS mutations in 7 (2%), BRAF mutations in 34 (9%) and PIK3CA mutations in 57 (15%) out of 371 cases. Interestingly, Co-mutations of PIK3CA occured in 68% of cases. Conclusions Mutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.
Detection of KRAS, NRAS, BRAF and PIK3CA by Mass Spectrometry: A flexible Technology for Routine Diagnostics
Casadonte R;
2016-01-01
Abstract
Background It has long been recognized that the epidermal growth factor receptor (EGFR) pathway is frequently activated in colorectal cancer. Therefore targeted therapies against EGFR such as cetuximab and panitumumab have been developed and are currently approved for the treatment of metastatic disease. It has been shown that activating mutations of KRAS or NRAS lead to a consecutive activation of the RAS-RAF pathway downstream of EGFR and consequently result in resistance to anti-EGFR therapy. Therefore mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy. Moreover BRAF status in conjunction with microsatellite testing adds prognostic information and is increasingly demanded by clinical collegues. As patients with PIK3CA mutations may benefit from nonsteroidal anti-inflammatory drugs, testing may further be extented for PIK3CA. Thus, reliable, fast, sensitive and cost effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion. Methods We developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test for the presence/absence of KRAS, NRAS BRAF and PIK3CA mutations. We applied this assay to 371 samples from patients with CRC, compared the results to Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). Results MS detected KRAS mutations in 160 (43%), NRAS mutations in 7 (2%), BRAF mutations in 34 (9%) and PIK3CA mutations in 57 (15%) out of 371 cases. Interestingly, Co-mutations of PIK3CA occured in 68% of cases. Conclusions Mutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
