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

Susan D. Richman, Richard Adams, Phil Quirke, Rachel Butler, Gemma Hemmings, Phil Chambers, Helen Roberts, Michelle D. James, Sue Wozniak, Riya Bathia, Cheryl Pugh, Timothy Maughan, Bharat Jasani, FOCUS4 Trial Management Group

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 crossreferencing. 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, edgeeffects 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.

Original languageEnglish
Pages (from-to)35-41
Number of pages7
JournalJournal of Clinical Pathology
Volume69
Issue number1
DOIs
Publication statusPublished - Jan 1 2016
Externally publishedYes

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Neoplasms
Mutation
DNA Mismatch Repair
Therapeutics
Immunohistochemistry
Wales
Mucins
Random Allocation
Patient Selection
Colorectal Neoplasms
Necrosis
Clinical Trials
Drug Therapy
Research
Proteins

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Richman, S. D., Adams, R., Quirke, P., Butler, R., Hemmings, G., Chambers, P., ... FOCUS4 Trial Management Group (2016). Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial. Journal of Clinical Pathology, 69(1), 35-41. https://doi.org/10.1136/jclinpath-2015-203097

Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial. / Richman, Susan D.; Adams, Richard; Quirke, Phil; Butler, Rachel; Hemmings, Gemma; Chambers, Phil; Roberts, Helen; James, Michelle D.; Wozniak, Sue; Bathia, Riya; Pugh, Cheryl; Maughan, Timothy; Jasani, Bharat; FOCUS4 Trial Management Group.

In: Journal of Clinical Pathology, Vol. 69, No. 1, 01.01.2016, p. 35-41.

Research output: Contribution to journalArticle

Richman, SD, Adams, R, Quirke, P, Butler, R, Hemmings, G, Chambers, P, Roberts, H, James, MD, Wozniak, S, Bathia, R, Pugh, C, Maughan, T, Jasani, B & FOCUS4 Trial Management Group 2016, 'Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial', Journal of Clinical Pathology, vol. 69, no. 1, pp. 35-41. https://doi.org/10.1136/jclinpath-2015-203097
Richman, Susan D. ; Adams, Richard ; Quirke, Phil ; Butler, Rachel ; Hemmings, Gemma ; Chambers, Phil ; Roberts, Helen ; James, Michelle D. ; Wozniak, Sue ; Bathia, Riya ; Pugh, Cheryl ; Maughan, Timothy ; Jasani, Bharat ; FOCUS4 Trial Management Group. / Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial. In: Journal of Clinical Pathology. 2016 ; Vol. 69, No. 1. pp. 35-41.
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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 crossreferencing. 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, edgeeffects 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.",
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AU - Hemmings, Gemma

AU - Chambers, Phil

AU - Roberts, Helen

AU - James, Michelle D.

AU - Wozniak, Sue

AU - Bathia, Riya

AU - Pugh, Cheryl

AU - Maughan, Timothy

AU - Jasani, Bharat

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N2 - 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 crossreferencing. 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, edgeeffects 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.

AB - 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 crossreferencing. 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, edgeeffects 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.

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