Presentation Title February 23, 2011 - PowerPoint Presentation

Slide1

Presentation Title

February 23, 2011

Establishing Clinical-grade Assays for Support of Drug Trials

May 3, 2012

Patrick Hurban

Expression AnalysisSlide2

Topics

Validated assays in clinical trialsAnalytical validationMulti-analyte testsAn ADME case study

The impact on Proficiency TestingTransitioning to NGSSlide3

Validated assays in clinical trialsInclusion/Exclusion versus a treatment decision

Cytochrome P450s (CYP)Ultra-rapid, Extensive, Intermediate, Poor metabolizersSelecting dosage based on metabolism profileExclusion of patients from a clinical trial based on risk of an adverse eventSelection of patients for efficacySlide4

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)Slide5

CriteriaCriteria differ for quantitative and qualitative tests

Category

Example

AccuracyThe 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 presentSlide6

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.Slide7

Multi-analyte testsWhen tests are comprised of multiple analytes, is there a requirement to affirm each reportable analyte?

During validationFor each batchHow does this impact quantitative versus qualitative tests?Is a pattern of analytes sufficient?Slide8

Multi-analyte assays in clinical trials

Identify multiple exclusion/inclusion criteria simultaneouslyUse marker combinations to refine criteriaPre-screen populations of potential candidate to create pools of individuals with known marker setsIdentify individuals within pools to create tailored cohorts

ExamplesADMEOncologySlide9

DMET

Affymetrix array

MIP-based amplification of target DNA231 genes

1936 SNP, CNV and IndelsCore ADME genes plus additional contentValidation71 genomic DNA samples fully sequenced (Sanger) across the core setBut only 36% of the known rare alleles were known to be includedReportable range confirmed for some, but not all of the content of the assaySlide10

Impact on proficiency testing

DMET assay utilized for analysis of PGX samples in CAP Proficiency ProgramDMET assay reports CYP2D6 *1A/*65 but CAP expects *1/*10CYP 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 *10

AlleleGenotypes*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 Slide11

Next Generation Sequencing

Each base position can exist in multiple discrete statesWe can set criteria for variant calling sensitivity dependent upon factors such as quality and depth of coverageWhat 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 websiteSlide12

SummaryValidation 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.Slide13

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