Clinical performance evaluation of a rapid, low throughput test for KRAS, BRAF and EGFR mutation analysis using formalin fixed Paraffin Embedded tissue blocks.

BACKGROUND

Genomics plays an important role in cancer diagnostics and treatment .Over 500 genes are known to play a part in cancer development of which EGFR, KRAS and BRAF are becoming increasingly popular in cancer investigations. Different molecular methods exist for testing oncogenes in tissue blocks but testing is limited by sensitivity and speed of analysis. The aim of this study was to establish the clinical performance of the novel fully automated polymerase chain reaction based Idylla mutation tests.

METHOD

This was a retrospective study carried out with known archived formalin fixed paraffin embedded tissue blocks and Horizon EQA samples. Results were compared between the EQA samples and known archived samples previously run on Cobas 4800 to determine correlation. 10 samples for each test I.e. KRAS, BRAF and EGFR were tested on the Idylla instrument. These samples were inclusive of Horizon EQA samples and archived formalin fixed paraffin embedded tissue block. A reduced number of samples was used in this study due to cost implications.

RESULTS

Data from the 30 samples was used to show overall agreement between the two instruments.
 

5 Horizon samples and 5 archived samples were tested for KRAS mutations in exons 2,3 and 4 of the KRAS oncogenes where the tests consists of five allele specific multiplex PCR reactions designed to amplify KRAS gene sequences with mutations in codons 12,13,59,61,117 and 146. The 5 Horizon EQA samples shown 100% correlation with all having mutations detected in KRAS codon 13.The other 5 archived samples; 3 had no mutation detected (wild type) and 2 other had mutations detected in KRAS codon 12.
 

2 Horizon samples and 8 archived samples were used in verification for EGFR mutation assay intended for the qualitative detection of exon 18(G719A/C/S), exon 21(L858R, L861Q), exon 20 (T790M, S768I) mutations, exon 19 deletions and exon 20 insertions in the EGFR oncogene. The 2 Horizon samples shown 100% correlation with no mutations detected and the archived samples; 3 had no mutation detected, 4 had mutations detected on L858R on exon 21 and 1 had mutations on L858R and S7681I on exon 20.

Horizon sample and 9 archived samples were used in verification of BRAF mutation assay intended for qualitative detection of V600E/E2/D and V600K/R/M mutations in codon 600 of the BRAF gene. 1 horizon shown 100% correlation with mutation detected. 5 of the archived samples had no mutation detected in BRAD codon 600 while 4 had mutations detected in BRAF codon 600.

CONCLUSION

It is therefore recommended that Genexpert Proficiency testing Panels should be done by all the sites that have Genexpert machine to check the performance/functionality and quality of the test results of the Laboratory that conducts the genexpert testing.