1. André Tartar atartar@me.com

2. The Traditional Treatment Paradigm Symptoms Diagnosis Treatment Dosing Nonspecific Broad Nonselective One-size-fits-all Phenotype Trial and Error

3. Biomarkers can be:  Anatomical  Histological  Imaging  Genes  mRNA  Proteins  Metabolites Essentially, some aspect of biology that has predictive power Evidence-Based Medicine

4. Biomarkers can be used for:  Diagnosis  Presence of disease or identify at-risk patients  Staging of disease (severity)  Prognosis of disease (metastases)  Stratification of patients  Responders/non-responders  Monitoring/predicting Toxicity  Monitoring therapeutic or pharmacodynamic effect

5. A patient, perhaps one you have known for years, who is overweight and does not exercise regularly, shows up in your office with an analysis of his whole genome at multiple single-nucleotide polymorphisms (SNPs). His children, who were concerned about his health, spent $1,000 to give him the analysis as a holiday gift. Letting the Genome out of the Bottle Will We Get Our Wish? NEJM N Engl J Med 2008; 358:105-107January 10, 2008January 10, 2008

6. ILLUMINA JANUARY 2014 http://www.illumina.com/technology/next-generation-sequencing.ilmn

8. The ultimate goal of pharmacogenomics is delivering the right dose of the right medicine to the right patient

9. 85% of patient responses to a drug are linked to genetic factors. DNA based tests: increase efficacy and safety; pharmacogenomics (pharmacology + genomics) Inter-individual variations Efficacy and adverse reactions Environmental factors Climat, Diet, Disease organisms or toxic substances, Lifestyle factors, Weight, Age, Sex, Other medicines Genetic factors 3 billions nucleotides Inter-individual Variations ~ 3 millions Genomic and PGx

10. Genetic Factors influence drug responses via 3 routes: By affecting the processing of the drug by the body By influencing the risk of acquiring certain diseases By modifying the proteins that are targeted by the drug PHARMACO -KINETICS DISEASE PATHWAYS PHARMACO DYNAMICS More than 120 FDA-approved drugs have pharmacogenomics information in their labeling (Dec. 2013)

11. 1/ PGx of Metabolic Enzymes Clinical PGx By affecting the processing of the drug by the body

12. PK factors determining an individual’s drug reaction Poor metabolisers Hyper metabolisers Extended metabolisers Drug cleared too quickly NO EFFECT DESIRED EFFECT Drug slow to be cleared from the body RISK OF ADVERSE DRUG REACTION

13. Evolution of Clinical Pharmacogenomics Phenotyp e Probe drug Metabolite: parent drug ratio Individual genes PCR-based Current Standard GWAS Increasing use Robust findings Sequencin g Future? Implementation

14. Thiopurine Metabolism Phenotypic analysis

15. Thiopurine Metabolism 15 CH 3 Active metabolite Inactive metabolites Important enzyme

16. 20 years ago it was reported that patients can be separated into three groups on the basis of the level of TPMT activity in their red cells. When patients with low levels of TPMT activity received standard doses of thiopurines, they had a greatly increased risk of life-threatening, drug-induced myelosuppression. Azathioprine 6-mercaptopurine Azathioprine used to treat neoplasias is converted to 6-mercaptopurine in vivo.

17. Level of TPMT activity in red cells among 298 randomly selected white adult blood donors In the case of TPMT, a blood sample could be obtained and the enzymatic activity measured directly, The fact that TPMT is expressed in an easily accessible cell facilitated the introduction of this test. If for instance an hepatic enzyme like CYP2D6 would have been involved a probe drug had to be administered and a urine sample collected.

18. Evolution of Clinical Pharmacogenomics Phenotyp e Probe drug Metabolite: parent drug ratio Individual genes PCR-based Current Standard GWAS Increasing use Robust findings Sequencin g Future? Implementation

20. Thiopurine Metabolism Genotypic Analysis

21. The human TPMT gene: TPMT*1 is the most common allele, and TPMT*3A is the most common variant allele among white subjects From TPMT Phenotype to Genotype

22. When mercaptopurine (MP) treatment is given at conventional doses - TPMT-deficient patients (v/v pink) show a markedly (tenfold) higher systemic exposure to active thioguanine nucleotides (TGN) - Heterozygous patients (wt/v blue) show about twofold higher TGN concentrations. The higher concentrations of TGN translate into a significantly higher frequency of haematopoietic toxicity

24. Cytochrome P450 Genotypic Analysis

25. Drug Metabolizing Enzymes The liver is the major site of drug metabolism.

27. The AmpliChip tests are based on Affymetrix microarray technology To address the relevant genetic variations, each array contains over 15,000 different probes complementary to sense and anti-sense P450 genomic DNA. Probes range in length from 18mer to 22mer * labeled DNA target Oligonucleotide probe * * * * * CYP450 2D6 & 2C19

29. Cytochrome P450 2D6 Genotypic Analysis

30. Genotype-Phenotype Relationship of CYP2D6 polymorphism PM (5-10%) URM (5-10%) EM (65-80%) IM (10-15%) or Phenotype Frequency (Caucasians) Genotype

31. 31

32. Cytochrome P450 2C19 Clopidogrel

33. n engl j med 360;4 nejm.org january 22, 2009 Active metabolite Clopidogrel (prodrug)

34. 162 healthy subjects receiving clopidogrel - Pharmacokinetic: Plasma concentrations of the active metabolite of clopidogrel were measured by HPLC-MS. - Pharmacodynamic : absolute reduction in maximal platelet aggregation from baseline in response to 20 μM of ADP. n engl j med 360;4 nejm.org january 22, 2009

35. Experimental: Genomic DNA was extracted from whole-blood. Genotyping was performed after amplification by means of a PCR assay involving two primers for the major variant alleles CYP2C19*2, CYP2C19*3 and CYP2C19*4, CYP2C19*5, CYP2C19*17. Method: We tested the association between functional genetic variants in CYP genes, plasma concentrations of active drug metabolite, and platelet inhibition in response to clopidogrel in 162 healthy subjects. Results: In healthy subjects who were treated with clopidogrel, carriers of at least one CYP2C19 reduced-function allele (approximately 30% of the study population) had a relative reduction of 32.4% in plasma exposure to the active metabolite of clopidogrel, as compared with noncarriers (P<0.001).

36. Effects of at least one reduced-function allele in five genes encoding cytochrome P-450 on the pharmacokinetic and pharmacodynamic responses to clopidogrel

37. Primary efficacy outcome: death from cardiovascular causes, myocardial infarction, or stroke

38. Background Pharmacogenetic determinants of the response of patients to clopidogrel contribute to variability in the biologic antiplatelet activity of the drug. The effect of these determinants on clinical outcomes after an acute myocardial infarction is unknown. Methods 2208 patients presenting with an acute myocardial infarction receiving clopidogrel therapy. Relation of allelic variants of genes modulating clopidogrel absorption (ABCB1), metabolic activation (CYP3A5 and CYP2C19), and biologic activity (P2RY12 and ITGB3) to the risk of death from any cause, nonfatal stroke, or myocardial infarction

39. Estimated Rates of Death from Any Cause, Nonfatal Myocardial Infarction, or Stroke, according to the number of CYP2C19 loss-of-function alleles.

40. Initiation of antiplatelet therapy based on cytochrome P450 2C19 genotype in patients with coronary disease following percutaneous coronary intervention

41. Evolution of Clinical Pharmacogenomics Phenotyp e Probe drug Metabolite: parent drug ratio Individual genes PCR-based Current Standard GWAS Increasing use Robust findings Sequencin g Future? Implementation

42. One in every 1200 bases may be different in any two humans. This variation, which is called a polymorphism, is largely responsible for differences between how humans respond to drugs. Several types of mutations are associated with these variations. SNPs Mutations in the Genome

43. In addition to modifying the function expressed protein, mutations can also affect the level of gene expression when they occur in regulatory or promoter sequences or in the exon/intron boundary. Mutations in the Genome

44. A genome-wide association study (GWAS), is an examination of many common SNPs in different individuals to see if any variant is associated with a trait. These studies normally compare the DNA of two groups of participants: people with the disease (cases) and similar people without (controls). In contrast to methods which specifically test one or a few genetic regions, the GWA studies investigate the entire genome. A Manhattan plot depicting strongly associated risk loci. Each dot represents a SNP, with the X-axis showing genomic location and Y-axis showing association level.Manhattan plotSNPassociation level

45. The Illumina Omni Express array is a 12-sample BeadChip microarray. It includes a total of 730,525 genome-wide markers and targets a minor allele frequency of 5% Price: $199.00

47. The EMA decision-making tree for in vitro studies prior to human exposure and Phase I studies

49. The European Medicine Agency’s decision-making tree for Phase I and Phase II studies.

50. Evolution of Clinical Pharmacogenomics Phenotyp e Probe drug Metabolite: parent drug ratio Individual genes PCR-based Current Standard GWAS Increasing use Robust findings Sequencin g Future? Implementation

51. May 09 2011 NEW YORK (GenomeWeb News) – Illumina today said that it has lowered the cost of its human whole-genome sequencing services to: $5,000 per genome for projects of 10 samples or more, $4,000 for projects of 50 samples or more. May 09 2011 NEW YORK (GenomeWeb News) – Illumina today said that it has lowered the cost of its human whole-genome sequencing services to: $5,000 per genome for projects of 10 samples or more, $4,000 for projects of 50 samples or more.

52. 19 Novembre 2013: http://www.nejm.org/doi/full/10.1056/NEJMp1314561?query=featured_home

53. ILLUMINA JANUARY 2014 http://www.illumina.com/technology/next-generation-sequencing.ilmn

54. 2/ Patients and Pharmacogenomics By influencing the risk of acquiring/ developping certain diseases

55. Risk Assessm ent Screening/ Diagnosis PredictiveMonitoring Predispo sition for developi ng disease Prognostic Early detection Predict probable disease course Predict likely response to a drug Monitor treatment efficacy/ disease recurrence Healthy Asymptom atic disease Chronic disease/ Cured Patient Stratification / Therapy Selection Symptomatic disease Patients and PGx: Biomarkers Therapy adaptation

56. Breast Cancer Genotypic Analysis of recurrence risk

57. Oncotype DX is a 21-gene assay that:  quantifies the likelihood of disease recurrence in early-stage breast cancer  assesses the likely benefit from certain types of chemotherapy.

59. Low RS<18 Int RS18-30 High RS≥31 0 10% 20% 30% 40% n = 353 n = 134 n = 164 % Increase in DRFS at 10 Yrs (mean ± SE) Absolute Increase in DRFS at 10 Years Benefit of Chemotherapy Depends on Recurrence Score

60. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227972/pdf/onc457.pdf

61. Guidelines for hormone receptor (HR)–positive early breast cancer recommend addition of adjuvant chemotherapy for most women, leading to overtreatment, which causes considerable morbidity and cost. The cost-effectiveness of recurrence score (RS)–guided treatment using 21-gene assay (Oncotype DX) was compared with treatment guided by the Adjuvant! Online program (AOL). A validated software program Adjuvant! Online (AOL), projects outcomes at 10 years based on clinico-pathological features and therapy. It is freely available on the Internet and is now widely used to assist in the adjuvant treatment decision-making process. Cost of the 21-gene assay, Oncotype DX, was based on the manufacturer’s suggested retail price of 4102 CAN $ ($3,650 USD).

62. All patients start in the risk classification state where they undergo risk assessment by AOL with or without RS reclassification Treatment strategies are also determined at this state. 1- Patients receive chemotherapy if they are considered high risk by RS reclassification (strategy 1) or high risk by AOL assessment only (strategy 2). Patients enter the chemotherapy state for the next 6 months. 1

63. They may experience no, minor, or major toxicity or may die from complications related to chemotherapy. 2- After 6 months of chemotherapy, they transit to the recurrence- free state. 2

64. 3- Patients who are deemed low risk by RS reclassification (strategy 1) or low risk by AOL assessment only (strategy 2) proceed directly to the recurrence-free state and complete 5 years of tamoxifen 3

65. During each cycle, these patients may remain disease-free or develop distant recurrence. 4- Once patients develop distant recurrence, they remain in the distant recurrence state until they die from breast cancer. 4

68. By modifying the proteins that are targeted by the drug PHARMACO DYNAMICS 3/ Drug Discovery and Development and Pharmacogenomics

69. Pharmacogenomics of drug target

70. Co-developement of NCE and Test Herceptin: Co-developed with Herceptest

78. HER2-positive testing (a) by IHC and (b) by FISH.

79. FFPE: formalin-fixed paraffin- embedded

81. KRAS testing for Erbitux in colorectal cancer Retrospective Analysis

82. 82 Erbitux and K-ras ERBITUX binding blocks EGFR signal transduction and inhibits downstream intracellular signalling Mutation of the K-ras gene results in a constitutively activated K-ras protein, which signals WITHOUT prior EGFR-mediated signalling The anti-tumor effects mediated by EGFR blockade are bypassed by mutated K-ras

83. Erbitux, retrospective Analysis: 572 patients with colorectal cancer were randomly assigned to receive cetuximab plus best supportive care or best supportive care alone. Tumor samples, obtained from 394 of 572 patients were analyzed to look for activating mutations in exon 2 of the K- ras gene. Improved survival with cetuximab was only observed in non mutated (wild type) K-ras group.

84. Erbitux

85. Prognostic vs. Predictive Markers Prognostic marker Influences clinical outcomes independent of therapy used Predictive marker Influences clinical outcomes with a specific therapy Markers may correlate with prognosis and treatment outcome ER, HER2 in breast carcinoma KRAS, BRAF in colorectal carcinoma MGMT in glioblastoma

86. Conclusions Industry perspectives on personalized medicine http://www.nature.com/nrd/journal/v11/n3/pdf/nrd3677.pdf

87. Percentage of company (21) pipeline relying on biomarker data Fewer projects (10% or less) had identified specific target populations or companion diagnostics Nature Reviews Drug Discovery 11, 178 (March 2012)

88. Number of times respondents mentioned personalized medicines for particular therapeutic areas Nature Reviews Drug Discovery 11, 178 (March 2012)

89. Scientific potential and economic attractiveness for companion diagnostics development across therapeutic areas.

94. 94 Erbitux and KRAS ERBITUX binding blocks EGFR signal transduction and inhibits downstream intracellular signaling Mutation of the KRAS gene results in a constitutively activated KRAS protein, which signals WITHOUT prior EGFR-mediated signaling The anti-tumor effects mediated by EGFR blockade are bypassed by mutated KRAS