Ultra-selection of metastatic colorectal cancer patients using Next Generation Sequencing to improve clinical efficacy of anti-EGFR therapy.

Background: Extended RAS analysis is mandatory in metastatic colorectal cancer (mCRC) patients. The optimal threshold of RAS mutated subclones to identify patients most likely to benefit from anti-EGFR therapy is controversial. Our aim was to assess the clinical impact of detecting mutations in RAS, BRAF, PIK3CA and EGFRS492R in basal tissue tumour samples by using a highly sensitive Next Generation Sequencing (NGS) technology in mCRC patients treated with chemotherapy plus anti-EGFR or anti-VEGF.

Patients and Methods: 581 tumour samples from untreated mCRC patients from 7 clinical studies were collected. Mutational analysis was performed by standard-of-care (therascreen pyro) with a sensitivity detection of 5% mutant allele fraction (MAF), and compared to NGS technology using 454GS Junior platform (Roche Applied Science, Germany) with a sensitivity of 1%. Molecular results were correlated with clinical outcomes.

Results: After quality assessment, 380 samples were evaluable for molecular analysis. Standard-of-care mutational analysis detected RAS, BRAFV600E or PIK3CA mutations in 56.05% of samples compared with 69.21% by NGS (p=0.00018). NGS identified coexistence of multiple low-frequency mutant alleles in 96 of the 263 mutated cases (36.5%; range 2 to 7). Response rate (RR), progression-free survival (PFS), and overall survival (OS) were increasingly improved in patients with RAS wild-type, RAS/BRAF wild-type or quadruple (KRAS/NRAS/BRAF/PIK3CA) wild-type tumours treated with anti-EGFR, assessed by standard-of-care. No additional benefit in RR, PFS or OS was observed by increasing the detection threshold to 1% by NGS. An inverse correlation between the MAF of the most prevalent mutation detected by NGS and anti-EGFR response was observed (p=0.039). EGFRS492Rmutation was not detected in untreated samples.

Conclusions: No improvement in the selection of patients for anti-EGFR therapy was obtained by adjusting the mutation detection threshold in tissue samples from 5% to 1% MAF. Response to anti-EGFR was significantly better in patients with quadruple wild-type tumours.