Pharmacogenomics: Focus on breast cancer Kathy Giacomini Deanna Kroetz Joan Venticinque

Personalizing Medicines Tumor Genomics E.g., Trastuzumab (Herceptin) Germline DNA

Tamoxifen: Used to treat estrogen-positive breast cancer

Tamoxifen: Is a Pro-drug CP Jin Y et al: J Natl Cancer Inst 97:30, 2005 Tamoxifen is a Pro-Drug

CYP2D6 Has Reduced Function Alleles Tamoxifen Inactive Prodrug Endoxifen Active Drug CYP2D6 CYP3A4 CYP2D6 Polymorphisms *4 Allele CYP2D6 Polymorphisms *10 Allele In Japanese

Jin Y et al: J Natl Cancer Inst 97:30, 2005 CYP2D6*4 (most common genetic variant associated with the CYP2D6 poor metabolizer state) P<0.001, r 2 =0.24 Plasma Endoxifen (nM) Individuals with CYP2D6*4 Have Lower Levels of Endoxifen CYP2D6 *4/*4 Polymorphisms

% With Relapse Free Survival % With Relapse Free Survival Years after randomization CYP2D6 WT/WT CYP2D6 *4/WT P=0.020 CYP2D6 *4/*4 Goetz et al J Clin Oncol. 2005;23(36): Individuals with CYP2D6*4 Have Have Poorer Relapse Free Survival CYP2D6 *4/*4 Polymorphisms

Schroth, W. et al. JAMA 2009;302: Women with CYP2D6 Polymorphisms Have Poorer Response to Tamoxifen CYP2D6 Polymorphisms *4, and other N = 1325

Cancer Science, May 2008, page Japanese Women with CYP2D6 Polymorphism, Have Poorer Response to Tamoxifen

Jin Y et al: J Natl Cancer Inst 97:30, 2005 Wt/Wt, no inhibitor VenlafaxineSertralineParoxetine*4/*4, no inhibitor Plasma Endoxifen (nM) Japanese Women with CYP2D6 Polymorphism, Have Poorer Response to Tamoxifen

EUROPE CANADA TAIWAN ALASKA JAPAN Hawaii Research Groups Collaborating Sites Network Resources PARC PPII PAPI PGBD NWAP XGEN PHAT PAAR PEAR PAT PHRAT PMT PNAT PAAR4KIDS PHONT P-STAR PG-POP PGRN-CGM UW-EXOME WU-NGS BCM-HGSC NIH Pharmacogenomics Research Network Cancer Pharmacogenomics GAP-J

Riken- Center for Genomic Medicine: NIH Pharmacogenomics Research Network:

To identify genetic predictors of therapeutic and adverse drug response using genomewide approaches. Personalized Medicines Picture from CGM Website

Disease Genetic Studies Patients Biological/ Pharmacologic Mechanism Strategies in Genomic Studies Identify Mutant/ Polymorphic Genes Diabetics Controls Genomewide Markers

Bipolar Coronary Artery Disease Crohn’s Hypertension Rheumatoid Arthritis Type 1 Diabetes Type 2 Diabetes Chromosome Number Wellcome Trust, Nature, 2007 Genomewide Association of Seven Common Diseases P= P value -log 10

Cancer Cardiovascular/ Diabetes/ Obesity Inflammatory Disease Aging/ Psychiatric/ Other Physiologic/ Biochem Trait Drug Response/ Toxicity Number of Studies Drug Response/ Toxicity January of 2009, > 200 Genomewide Association Studies Identifying Disease Risk Alleles Fewer than 20 Pharmacogenomic Studies

Pharmacogenomic Studies Patients Biological/ Pharmacologic Mechanism Strategies in Genomic Studies Identify Mutant/ Polymorphic Genes Receive Drug Adverse Reaction Receive Drug No Adverse Reaction Genomewide Markers

Pharmacogenomics: Focus on breast cancer Kathy Giacomini Deanna Kroetz Joan Venticinque

Stratification Pre-Post Menopausal ER/PgR AC q 2 wk 60 mg/m mg/m 2 4 cycles – 8 wk 6 cycles – 12 wk Paclitaxel q 2 wk 175 mg/m 2 4 cycles – 8 wk 6 cycles – 12 wk AC = doxorubicin/cyclophosphamide Target Accrual 4646 pts Pharmacogenetic analysis of predictors of paclitaxel and AC toxicity

Genes ABCB1 ABCC1 ABCC2 ABCG2 CYP1B1 CYP2C8 CYP3A4 CYP3A5 MAPT TP53 SLCO1B3 Endpoints Pharmacokinetics Toxicities Outcomes (DFS, OS) Findings Small Populations Inconsistent results

Riken Center for Genomic Medicine and PGRN Collaboration for Genome Wide Association Study of CALGB Genome wide analysis of CALGB samples using Illumina 610-Quad platform Doxorubicin/Cyclophosphamide Arm (n=919) Paclitaxel Arm (n=1040)

Illumina 610-Quad (≈592K SNPs) (Subject CR >99%, SNP CR >95%; Chr 1-23, MAF > 0.5%, IBD) 1040 Subjects 1029 Subjects/554,450 SNPs QC Principal Components Analysis 859 Genetically Western Europeans 170 Remaining Subjects Time to Event Analysis Replication Study Study Design Logistic Regression Ordinal Regression Regression, Admix Mapping ‘Genetic Caucasian’ PC1,PC2,PC3 all within 2 SDs of mean values from all patients self-declaring as ‘White’

Activities of Daily Living (ADL) Instrumental ADL: Preparing meals, shopping for groceries or clothes, using the telephone, managing money, etc. Self care ADL: Bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not bedridden. Neuropathy Grading Criteria

Grade Grade Grade Grade 3 59 Omitted 4 Maximum grade sensory peripheral neuropathy during treatment or follow-up: 859 subjects

 Ordinal Logistic Regression  Highest grade sensory peripheral neuropathy  Additive genetic model  Covariate: Log cumulative dose/BSA at first instance of maximum grade toxicity. If no toxicity, equal to cumulative dose at end of treatment.  Time to Event Analysis  “Event” - The first occurrence of a grade 2 or higher sensory peripheral neuropathy.  “Time” - Cumulative dose/BSA at event. If no toxicity, equal to cumulative dose at end of treatment.  Additive genetic model

EPHA5 FGD4 PITPNA GRIP1

FZD3

GRIP1 EPHA5 Probability of NOT having an Event Time to Event Analysis

Kobayashi et al. Spine 32: , 2007

FGD4 NDRG1 Probability of NOT having an Event Time to Event Analysis

FGD4 is Implicated in Congenital Neuropathic Disease Am J Hum Genet 81: , 2007

Stendel et al. Am J Human Genet 81: , 2007 Sural nerve biopsy M298R E543fs

FZD3

Endo et al. Mol Cell Biol 28: , 2008

AJHG 2007 AJHG 2009 Gene Ontology Analysis is Being Applied to GWA Studies

 Developed by Jesse Paquette at the Biostatistics and Computational Biology Core (BCB) at the Helen Diller Cancer Center  Tool for visualizing gene-gene networks and pathway enrichment using several publicly available databases   Paquette and Tokuuasu Bioinformatics 26:285-6, 2010 Exploratory Gene Association Networks (EGAN)

 Used top 2500 candidate SNPs (based on P-value)  Illumina’s annotation used for gene assignments  SNP with smallest P-value used for each gene  Taking into account genes with multiple hits and SNPs without gene annotation:  1154 SNPs were analyzed  “Background” Gene Space  Genes represented on the Illumina 610quad considered as all “potential” hits (i.e. the denominator in calculations of pathway “enrichment”) EGAN: CALGB Neuropathy GWA Data

GO ProcessAll Genes40101 “Hits”Enrichment Nervous System Development x Cell Adhesion x Central Nervous System Development x Cell Development x Hemophilic Cell Adhesion x Cell-Cell Adhesion x Neurogenesis x Neuron Projection Development x Generation of Neurons x Cell part Morphogenesis x Synaptic Transmission x Neuron Differentiation x SNP Hits are Enriched in Neuronal Development and Cell Adhesion Pathways

Visualization of Protein-Protein Interactions

 BioBank Japan – no dosing data  Remaining samples  Scottish Ovarian Cancer Trial: 454 women receiving 175 mg/m 2 paclitaxel plus carboplatin  SWOG 0221: Phase III trial of AC + G vs. Q 2 wks AC followed by paclitaxel weekly or Q 2 wks

SNPs for Replication Analyses Number SNPs Cutoff: <1 x Number SNPs Cutoff: <1 x Time to Event848 Ordinal4 (*0)28 (*4) Total1272 (*Number of Shared SNPs)

 Ovarian function  Breast cancer outcomes

Pharmacogenomics: Focus on breast cancer Kathy Giacomini Deanna Kroetz Joan Venticinque