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Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial
  1. Susan D Richman1,
  2. Richard Adams2,
  3. Phil Quirke1,
  4. Rachel Butler3,
  5. Gemma Hemmings1,
  6. Phil Chambers1,
  7. Helen Roberts3,
  8. Michelle D James4,
  9. Sue Wozniak4,
  10. Riya Bathia5,
  11. Cheryl Pugh5,
  12. Timothy Maughan6,
  13. Bharat Jasani7
  14. on behalf of FOCUS4 Trial Management Group
  1. 1Department of Pathology and Tumour Biology, Leeds Institute of Cancer and Pathology, St James Hospital, Leeds, UK
  2. 2Institute of Cancer & Genetics, Cardiff University School of Medicine, Velindre Hospital, Cardiff, UK
  3. 3Cardiff and Vale UHB-Medical Genetics University Hospital of Wales, Heath Park, Cardiff, UK
  4. 4Cardiff and Vale UHB- Histopathology University Hospital of Wales, Heath Park, Cardiff, UK
  5. 5MRC Clinical Trials Unit at UCL, London, UK
  6. 6Gray Laboratories, CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
  7. 7Institute of Cancer and Genetics, Heath Park, Cardiff, UK
  1. Correspondence to Dr Richard Adams, Institute of Cancer & Genetics, Cardiff University School of Medicine, Velindre Hospital, Cardiff CF14 2TL, UK; Richard.Adams{at}wales.nhs.uk

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.

  • COLORECTAL CANCER
  • ANTIBODIES
  • LABORATORY TESTS
  • MOLECULAR PATHOLOGY

This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/

https://doi.org/10.1136/jclinpath-2015-203097

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