Presentation TitleFebruary 23, 2011

Establishing Clinical-grade Assays for Support of Drug

TrialsMay 3, 2012

Patrick HurbanExpression

Analysis

Topics

• Validated assays in clinical trials• Analytical validation• Multi-analyte tests• An ADME case study• The impact on Proficiency Testing• Transitioning to NGS

Validated assays in clinical trials

• Inclusion/Exclusion versus a treatment decision

• Cytochrome P450s (CYP)– Ultra-rapid, Extensive, Intermediate, Poor

metabolizers• Selecting dosage based on metabolism profile• Exclusion of patients from a clinical trial based

on risk of an adverse event• Selection of patients for efficacy

Analytical validation

• “The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose” (ICH Harmonised Tripartite Guideline).

• “Confirmation, through the provision of objective evidence, that the requirements for a specific intended use or application have been fulfilled” (ISO 9000:2005)

Criteria

• Criteria differ for quantitative and qualitative tests

Category Example

Accuracy The genotype is called correctly

Reproducibility The genotype is called correctly within and between runs

Robustness The genotype is called correctly even when the sample was stored incorrectly

Reportable Range The genotypes called by the assay at a given locus

Reference Range The genotypes that are known at a given locus

Limit of detection Minimal amount of DNA that produces a valid genotype call

Sensitivity A genotype is called when it comprises at least 20% of the starting sample

Specificity A genotype is not called when it is not present

A simple RFLP test

Controls Unknowns

• The reportable range is tested during validation using known reference material.

• Batch controls used during ongoing conduct of the assay are designed to produce a positive result for each value (genotype) in the reportable range.

Multi-analyte tests

• When tests are comprised of multiple analytes, is there a requirement to affirm each reportable analyte?– During validation– For each batch

• How does this impact quantitative versus qualitative tests?

• Is a pattern of analytes sufficient?

Multi-analyte assays in clinical trials

• Identify multiple exclusion/inclusion criteria simultaneously

• Use marker combinations to refine criteria• Pre-screen populations of potential candidate to

create pools of individuals with known marker sets• Identify individuals within pools to create tailored

cohorts

• Examples– ADME– Oncology

DMET

• Affymetrix array– MIP-based amplification of target

DNA• 231 genes• 1936 SNP, CNV and Indels• Core ADME genes plus

additional content

• Validation– 71 genomic DNA samples fully

sequenced (Sanger) across the core set

– But only 36% of the known rare alleles were known to be included

– Reportable range confirmed for some, but not all of the content of the assay

Impact on proficiency testing

• DMET assay utilized for analysis of PGX samples in CAP Proficiency Program

• DMET assay reports CYP2D6 *1A/*65 but CAP expects *1/*10

• CYP Nomenclature database no longer has an entry for *1 – supplanted by *1A, *1B, *1C, *1D, *1E, *1XN

• *65 is a more is an extension of *10Allele Genotypes*10 *10A: 100C>T; 1661G>C; 4180G>C

*10B: -1426C>T; -1237_-1236insAA; -1235A>G; -1000G>A; 100C>T; 1039C>T; 1661G>C; 4180G>C *10D: 100C>T; 1039C>T; 1661G>C; 4180G>C, CYP2D7-like 3'-flanking region

*65 100C>T; 310G>T; 843T>G; 1661G>C; 2850C>T; 3384A>C; 3584G>A; 3790C>T; 4180G>C; 4481G>A

Next Generation Sequencing

• Each base position can exist in multiple discrete states• We can set criteria for variant calling sensitivity dependent

upon factors such as quality and depth of coverage

• What happens when we identify mutations of interest for a subject but have not specifically validated for these mutations?

• How do we deal with potential complications such as allelic drop-out?

Courtesy Promega website

Summary

• Validation is contextual and must produce an assay that is suitable for the intended purpose.

• Multi-analyte assays complicate validations because it may not be feasible to validate the entire reportable range.

• The traditional use of batch control material also becomes complicated with multi-analyte assays.

• Understanding the performance of an assay against reference methods can be difficult when the assay has greater resolution.

Recommended reading

• ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology

• “Recommended Principles and Practices for Validating Clinical Molecular Pathology Tests,” Jennings, et al, Arch Pathol Lab Med, V133, p. 743, 2009

• “A Standardized Framework for the Validation and Verification of Clinical Molecular Genetic Tests,” Mattocks, et al, Eur J Hum Gen V18, p. 1276, 2010.

• “Multiplex Assay for Comprehensive Genotyping of Genes Involved in Drug Metabolism, Excretion, and Transport,” Daly, et al, Clin Chem V53, p. 1222, 2007.

• “Influence of Cytochrome P450 Polymorphisms on Drug Therapies: Pharmacogenetic, Pharmacoepigenetic and Clinical Aspects,” Ingelman-Sundberg, et al, Pharmacology & Therapeutics V116, P. 496, 2007