Pre-Trial Molecular Validation Across Two Laboratories Ensures Reliable Patient Stratification in FOCUS4 Colorectal Cancer Trial
A rigorous inter-laboratory validation between Leeds and Cardiff tested pyrosequencing for KRAS, NRAS, BRAF, and PIK3CA mutations alongside pTEN and mismatch repair protein immunohistochemistry on 97 metastatic colorectal cancer samples for the FOCUS4 stratified therapy trial. Mutation concordance was near-perfect with only two PIK3CA discrepancies attributable to tumour heterogeneity, while IHC disagreements were resolved on joint review. The study demonstrates that pre-trial analytical validation is both feasible and essential for molecularly stratified cancer trials.
The original study
Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial.
- Authors
- Richman SD, Adams R, Quirke P, Butler R, Hemmings G, Chambers P, et al.
- Journal
- Journal of clinical pathology
- Type
- Journal Article, Multicenter Study, Randomized Controlled Trial, Research Support, Non-U.S. Gov't, Validation Study
- PMID
- 26350752
Original abstract
INTRODUCTION: Molecular characterisation of tumours is increasing personalisation of cancer therapy, tailored to an individual and their cancer. FOCUS4 is a molecularly stratified clinical trial for patients with advanced colorectal cancer. During an initial 16-week period of standard first-line chemotherapy, tumour tissue will undergo several molecular assays, with the results used for cohort allocation, then randomisation. Laboratories in Leeds and Cardiff will perform the molecular testing. The results of a rigorous pre-trial inter-laboratory analytical validation are presented and discussed. METHODS: Wales Cancer Bank supplied FFPE tumour blocks from 97 mCRC patients with consent for use in further research. Both laboratories processed each sample according to an agreed definitive FOCUS4 laboratory protocol, reporting results directly to the MRC Trial Management Group for independent cross-referencing. RESULTS: Pyrosequencing analysis of mutation status at KRAS codons12/13/61/146, NRAS codons12/13/61, BRAF codon600 and PIK3CA codons542/545/546/1047, generated highly concordant results. Two samples gave discrepant results; in one a PIK3CA mutation was detected only in Leeds, and in the other, a PIK3CA mutation was only detected in Cardiff. pTEN and mismatch repair (MMR) protein expression was assessed by immunohistochemistry (IHC) resulting in 6/97 discordant results for pTEN and 5/388 for MMR, resolved upon joint review. Tumour heterogeneity was likely responsible for pyrosequencing discrepancies. The presence of signet-ring cells, necrosis, mucin, edge-effects and over-counterstaining influenced IHC discrepancies. CONCLUSIONS: Pre-trial assay analytical validation is essential to ensure appropriate selection of patients for targeted therapies. This is feasible for both mutation testing and immunohistochemical assays and must be built into the workup of such trials. TRIAL REGISTRATION NUMBER: ISRCTN90061564.