Presented by AMP & AACC May 7-8, 2012, Chicago, IL

Molecular Pathology: Principles in Clinical Practice

KT Jerry Yeo, Ph.D.

University of Chicago

Email: jyeo@bsd.uchicago.edu

Validations & Implementation of

Pharmacogenomics: Laboratory

Issues

Objectives

By the end of this presentation participant

should be able to:

1. Identify existing analytical platforms

available for PGx

2. Delineate the various steps needed for

validation PGx assays for clinical use.

3. Articulate the issues in PGx

interpretations and limitations.

Laboratory Issues

Selection and validation of Analytical Platforms

Platforms & Turnaround time (TAT)

LDT vs FDA cleared?

Complex vs Simpler systems (Microarray vs NGS)

PGx panel composition (e.g. 2C19 *1,*2,*3 vs *1-*10,

*17?)

Ranges from 2hrs to >8hrs depending on the number

of specimens

Is TAT going to adequate for what’s needed?

Analytical Systems

Babic et al. CCA 2009;406:143-147

Current US FDA cleared PGx tests

Wu, Babic & Yeo Person Med 2009;6:315

Updates

Test Manufacturer Platform Approval

Date/Status

Drug

CYP2C19

*2/*3/*17

Autogenomics Infiniti 2010 Clopidogrel

*cannot mention

Cyp2C19

*2-*8

Luminex xTAG RUO

CYP2C19

*2/*3

Nanosphere Verigene IUO

CYP2C19

*2-*10, *17

GenMark eSensor RUO

PGx Reference Materials

Pratt et al. J Mol Diagn 2010; 12:835-846

GeT-RM AMP Collaborative Project

Coriell Cell

Line #

CYP2C19 CYP2C9 VKORC1

c.-1639G>A

CYP2D6

GM17289 *2/*2 *1/*1 AA *2/*4

GM17203 (*1/*2) *2/*17 *1/*1 AA *4/*35

GM17272 (*1/*1) *17/*17 *1/*1 AA *4/*10

GM17246 (*1/*8) *8/*17 *1/*2 GA *4/*35

GM17115 *1/*1 (*1/*1) *9/*9 GG *1/*2

GM10005 (1/*1) *1/*17 (*1/*1) *1/*9 GG *17/*29

GM07439 (*2/*2) *2/*10 (*1/*1) *1/*9 GG *4xN/*41

GM 12244 *1/*1 *2/*3 GG *35/*41

GM17052 *1/*3 *1/*1 AA *1/*1

GM02016 *1/*2 *1/*1 GG *2xN/*17

GM17296 (*1/*1) *17/*17 *1/*1 GA *1/*9

Pratt et al. J Mol Diagn 2010; 12:835-846

Genotyping Discrepancies between

Nanochip & Pyrosequencing As illustrated by the Nanogen Validation Studies of 2D6

On 200 anonymized samples that were genotyped by alternative

method

Discrepancies were confirmed using either sequencing or Jurilab

method

Overall concordance between methods is 99.3%, but some genotypic

discrepancies had phenotypic consequences

Lee et al. Clin Chem 2007;53:823

Sample PyroSeq Nanochip Phenotypic Change

DMS 4 *1/*1 *2/*5 EMIM

DMS 7 *1/*1 Dup (*1xN) EMUM

DMS 25 *1/*4 *1xN/*4 IMEM

DMS 109 *4/*4 *4/*6 PMPM

DMS 167 *1/*1 *1/*5 EMIM

Analytical Comparisons

Babic et al. CCA 2009;406:143-147

Validations of Cyp 2C19

Lee et al. CCA 2011;412:1133-1137

Comparison Studies

Lee et al. CCA 2011;412:1133-1137

Variability in Defining SNPs

CDC GeT-RM Collaborative Project 2009-Courtesy of Dr V. Pratt

2D6-

Allele Luminex Roche Amplichip

Autogenomics

INFINITI

*1 assumed assumed assumed

*2

(-1584G, 1661G>C, 2850C>T,

4180G>C)

(-1584G, 1039C>T, 1661G>C,

2850C>T, 4180G>C) 2850C>T

*3 2549delA 2549delA 2549delA

*4

100C>T, 1661G>C, 1846G>A,

4180G>C

100C>T, 1039C>T, 1661G>C,

1846G>A, 2850C>T, 4180G>C 1846G>A

*5 deletion deletion deletion

*6 1707delT 1707delT, 1976G>A, 4180G>C 1707delT

*7 2935A>C 2935A>C 2935A>C

*8

1661G>C, 1758G>T,

2850C>T, 4180G>C

1661G>C, 1758G>T, 2850C>T,

4180G>C 1758G>T

*9 2613-2615 delAGA 2613-2615delAGA 2615_7delAAG

*10 100C>T, 1661G>C, 4180G>C

100C>T, 1039C>T, 1661G>C,

4180G>C 100C>T

Commercial Platforms

Analytical Characteristics

Osmetech eSensor

Autogenomics INFINITI

ParagonDx RT-PCR

Hologic Invader

Luminex Tag-IT

DNA required (ng)

0.1 50 2.5 250 15

No. of Samples per run

8 (can be expanded to 24)

24 96 29 24

Total Assay Time (hr)

4 7 2.5-4.0* 3 8

Dimensions (cm)

40 x 41 x 46 110 x 60 x 66 33 x 46 x 43 38 x 41 x 17 43 x 51 x 25

Wu, Babic & Yeo. Person Med 2009;6:315

POCT PGx (Cyp2C19)-pushing

the envelope?

http://www.spartanbio.com/wp-content/themes/spartan/assets/manuals/Spartan_RX.pdf

• Interrogates only Cyp 2C19 *2 allele

Results of Rapid Gene Study

Roberts et al. The Lancet 2012 DOI:10.1016/S0140-6736(12)60161-5

• POCT PGx testing after PCI can be done effectively at bedside

• Can identify Cyp2C19*2 carriers quickly for treatment with Prasugrel to

reduce HTPR

Summary

Discrepancies can arise due to: Methodology differences & defining SNPs chosen

PCR conditions

Subjective operator interpretations

Contaminations (e.g. PCR, buffers)

Some of these genotypic differences can lead to significant phenotypic differences

Ambiguous genotype results need to be confirmed by an alternative reference method (e.g. bi-directional sequencing)

Size and speed may matter in selection

Each clinical lab needs to validate these PGx assays before implementing these assays for clinical use

Laboratory Issues

Reimbursements?

Regulatory (CLIA) Personnel

Where should it be done

QC materials and frequency of run Coriell human DNA source (cell lines)

Rare variants?

Synthetic oligos?

Laboratory Issues (cont)

Training Issues: especially with non-

molecular lab section

Meeting CAP Molecular checklist

Providing actionable, but yet informative

PGx report

Be aware some state regulations (e.g. IL)

require Clinical interpretive reports by PhD

be co-signed by MD

PGx Interpretations

Genotype-Phenotype

Prediction Issue

Lack of uniform method for translating genotype to useful phenotype information E.g. Non-standardization of genotype-phenotype

prediction for CYP 2D6

CYP 2C19

If panel only detects *1,*2, *3, will call *17/*17 as *1/*1

What is the phenotype of *2/*4/*17 Het?

CYP 2D6 (>70 variants)

Allelic variants mostly uncharacterized for many clinically important drug substrates

E.g *1x N/*4, or *2xN/*17 = phenotype?

Range of 2D6 phenotype activities

Gaedigk et al. Clin Pharm Ther 2008;83:234

Dextromethorphan (DM) Dextrophan (DX) Cyp 2D6

Activity Score Model

Gaedigk et al. Clin Pharm Ther 2008;83:234

Case

79 yo male with EKG and cardiac troponin elevations suggestive of AMI

Three drug eluting stents: First proximal obtuse marginal: 2 stents

Left circumflex: 1 stent

Antiplatelet therapy Plavix (clopidogrel) and aspirin

Patient was high risk clinically, with a lot of underlying coronary artery disease and extensive stenting.

There was a great deal of concern for ensuing thrombosis on re-admission and a platelet aggregation study was requested.

Yeo et al. Manuscript in preparation

Result Name Result Units Reference

P2Y12 Reactivity Unit 306 PRU 210-325

Platelet Reactivity BASE 235 Base PRU 230-370

P2Y12% Inhibition 0 %

Platelet Aggregation Studies

1. Accumetrics VerifyNow Whole Blood Assay

AA 1.2 mM

A23187 25 uM

ADP 8 uM

ADP 2 uM

2. Platelet Lumi-Aggregation Assay

Patient’s Genotyping Results

CYP 450 Genotype Result SNPs

2C19 *1/*17 C/T -806 C>T

2C9 *1/*2 C/T 430 C>T

•So what’s the predicted phenotype???

•Predicted 2C19 phenotype = Ultrametabolizer

•Predicted 2C9 phenotype = Intermediate

metabolizer

•There is an apparent large genotype-observed

phenotype discordance!

Complex Workup & Final

Interpretations Drug Measurement

TOF MS of serum showed presence of parent and metabolite of Clopidogrel

Genotypes CYP 2C19 *1/*17: Extensive-to-Ultrapid metabolizer; sufficient

activation to 2-oxoclopidogrel

CYP 3A4 G>A Mutant: Carriers of A allele has better response to clopidogrel due to less activation of GP IIb/IIIa platelet receptors.

ABCB1 C/C: No increased risk

ITGA2, C/T Het: Carriers of T allele show increased thrombotic risk

P2Y12, C/T Het: Carriers of T allele show increased adverse events with clopidogrel

Summary: Transport and metabolism are sufficient. However clopidogrel resistance is predicted due to variant platelet receptors.

Sample Report

Sample Lab Report

Acknowledgements

Collaborators: Alan Wu, Ph.D. UCSF, Gwen McMillin, Ph.D. ARUP,

Dennis O’Kane, Ph.D. Mayo

U of C Nikolina Babic, Ph.D.

Soma Das, Ph.D.

Christine Lee, AB

Summary

Validations of PGx platform/assays are

required before implementation for clinical

use.

Need to understand the utility and limitations

of PGx panels.

Other non-genetic factors can have a

profound effect and needs to be factored in

decision-making.

Need to generate reports in a timely manner

that are simple to understand and actionable.

Self-Study Questions

List the drugs that currently have FDA

labels for pharmacogenomics testing.

How should analytical validations of PGx

assays be performed?

What are the common concerns regarding

the implementation of PGx for clinical use?

Regulatory

Interpretations