Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial

Abstract

IntroductionMolecular 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.MethodsWales 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.ResultsPyrosequencing 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.ConclusionsPre-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 numberISRCTN90061564.

Colorectal Cancer Stratification in the Routine Clinical Pathway: A District General Hospital Experience

“…Other potential targeted therapies may be beneficial to CRC patients. For example, HER- (31). Our study complements this trial by demonstrating the feasibility of using these tests for routine stratification in a small DGH.…”

This is a platform alteration: a trial management perspective on the operational aspects of adaptive and platform and umbrella protocols

“…Two academic laboratories (Leeds and Cardiff) process and analyse tumour samples for biomarkers. To ensure concordance of results and molecular cohort stratification between laboratories, a Quality Assurance (QA) process was set up for pre-trial and ongoing checks [13].…”

“…Since the initiation of FOCUS4, the definition of the molecular cohorts has changed, new biomarkers have been added and there have been improvements to the analysis methodology used to detect genetic mutations. Implementation of these changes posed logistical challenges; hence, the following were considered before making the changes:◦ Development time and cost for new biomarker assays in the laboratories;◦ Cross-laboratory QA [13], specifically for new biomarkers and new analysis methodologies;◦ Timing of implementation of changes to the biomarker panel.…”

Additional loss of MSH2 and MSH6 expression in sporadic deficient mismatch repair colorectal cancer due to MLH1 promoter hypermethylation

“…IHC for the MMR proteins was carried out as previously described on a DAKO Autostainer Link 48 using DAKO reagents (DAKO, Ely, UK) 3. Loss of expression was defined as negative staining in tumour cell nuclei in the presence of adjacent positive internal controls (stromal cells, infiltrating lymphocytes or normal colonic crypts).…”

“…For BRAF mutational testing, tumour-rich cell content was macro-dissected and DNA extracted using the QIAGEN QIAamp DNA Extraction Kit (QIAGEN, Manchester, UK). Analysis of mutation hotspots within BRAF codon 600 (exon 15) was carried out by pyrosequencing as previously described 3. Testing for MLH1 promoter methylation was carried out in Leeds Teaching Hospitals NHS Trust.…”