Pharmacogenetic testing in clinical settings

Incorporation of pharmacogenomic testing with clinical trials has multiple advantages. The two most important concerns for new drug development are efficacy and safety.

1. Pharmacogenetic testing in clinical settings Dr. Mohit Kher Pharmacology LHMC

2. OUTLINE Introduction PK & PD genes Developmental groups Resources Examples of implementation List of VIPs Testing approaches and interpretation Challenges Conclusion

3. INTRODUCTION In 1959, Vogel coined the term pharmacogenetics. A dverse reactions – Primaquine ( anemia ), succinylcholine ( apnea ) and isoniazid ( peripheral neuropathy ). Pharmacogenetics is often a study of the variations in a targeted gene , or group of functionally related genes for variability in drug response. Pharmacogenomics is the use of genetic information to guide the choice and dose on an individual basis.

4. Goals of pharmacogenetics Maximize drug efficacy Minimize drug toxicity Predict patients who will respond to intervention Aid in new drug development

5. Pharmacokinetic Targets The CYP 1 to 3 families are involved in phase I drug metabolism. E.g.  CYP1A2, CYP2B6, CYP3A4/5, CYP2C9, CYP2C19 and CYP2D6 C ategories - Poor metabolizer Intermediate metabolizer Extensive/normal metabolizer Ultrarapid metabolizer

6. Pharmacodynamic Targets The evidence behind PD gene testing is less clear than PK gene testing. Common PD genes: SLC6A4 serotonin transporter HTR2A serotonin receptor DRD2 dopamine receptor COMT receptor HTR2C serotonin receptor.

7. SLC6A4 gene C odes for the serotonin reuptake transporter. 3 possible genotypes based on a patient having either 2 short (S) alleles, 2 long (L) alleles, or a combination of the 2 (S/L).  L/L genotype are more likely to respond SSRIs and do so more quickly.

8.   HTR2A gene  Codes for the serotonin 2A receptor. V ariant rs6313 (common) - Carriers for either cytosine or thymine polymorphisms.  T hymine polymorphism - More likely to respond to antidepressants . V ariant rs7997012 - I ncreased antidepressant response in patients carrying a guanine polymorphism.

9. DRD2 gene L ocated on chromosome 11q22 and codes for the dopamine 2 receptor . Polymorphism 141C Ins/Del -   Ins/Ins genotype are more likely to respond antipsychotic medications. Another variant rs2514218 - H omozygous C allele were significantly more likely to respond to antipsychotics than T allele. M ore akathisia was reported in C homozygotes taking aripiprazole . G reater prolactin elevations were demonstrated in T homozygotes taking risperidone . V ariant rs1079597 (C polymorphism) - I mprovement in the negative symptoms of schizophrenia after a short treatment with amisulpride .

10. CURRENTLY AVAILABLE RESOURCES FOR THE IMPLEMENTATION OF PHARMACOGENETIC BIOMARKERS

11. Pharmacogenetic Clinical Practice Guideline Development Groups Clinical Pharmacogenetics Implementation Consortium (CPIC) - P rovides peer-reviewed, evidence-based pharmacogenetic clinical practice guidelines. - issued 47 guidelines ● Dutch Pharmacogenetics Working Group (DPWG) - T herapeutic recommendations based on the pharmacogenetic information. - P ublished 93 guidelines ● Canadian Pharmacogenomics Network for Drug Safety (CPNDS) - C onducts systematic literature reviews followed by guideline development using the Appraisal of Guidelines Research and Evaluation Enterprise (AGREE) instrument. - R eleased eight guidelines

12. I nternet resources in the pharmacogenetic sector

13.   Fewer known resources Mayo Clinic portal - “ AskMayoExpert ” educational materials St. Jude Children’s Research Hospital – Tracking the website-integrated gene or drug information Ubiquitous Pharmacogenomics (U-PGx): e-learning platform

14. EXAMPLES OF IMPLEMENTING PHARMACOGENETIC TESTING IN CLINICAL SETTINGS

15. TPMT and NUDT15 Variants in Patients Receiving 6-Mercaptopurine

16. HLA-B ∗ 15:02 Testing in Carbamazepine-Induced Severe Cutaneous Adverse Reactions

17.   List of PharmGKB VIPs for which guidelines or drug labels recommend changes to medical management on the basis of clinical genetic testing results

18. Various clinical genetic testing approaches for Pharmacogenes Approach Advantages Disadvantages Real-time PCR (RT-PCR) with Taqman probes Efficient: Amplification and interrogation occur in one step Identifies only variants of known significance Identifies only variants in target genes Cannot discover novel variants Complicating result interpretation in rare cases Restriction Fragment Length Polymorphism (RFLP) analysis Low cost—good for Population health/clinical applications Lower sensitivity—will detect 90%–95% of variants, versus 99% Slow and cumbersome The technology for RFLP testing has remained largely unchanged for the past two Decades

19. Approach Advantages Disadvantages Amplichip CYP450 (Roche) test ( first FDA approved  pharmacogenetic test, 2004) Relatively low cost Efficient—high throughput analysis Good for clinical laboratory settings Able to detect SNPs Low discovery power Lower sensitivity—will detect 90%–98% of variants, rather than 99% Sanger sequencing Sanger sequencing is Gold standard for verification of variants Can be more cost effective for small number of samples Slower and relatively more cumbersome More expensive—particularly for large sample sizes

20. Multiplex PCR + Next Generation Sequencing Advantages Disadvantages Exome sequencing (ES)/genome sequencing (GS) High discovery power Less cost-effective and more time-consuming Identifies variants of unknown significance (VUS) Amplicon sequencing Requires smaller amounts of DNA Limited discovery potential Single-molecule real time (SMRT) sequencing assay Good performance on identifying splicing Isoforms Expensive and lower accuracy compared to short-read sequencing Nanopore sequencing Easy to integrate into clinical setting—palm size portable equipment Low capital cost and Fast turnaround time for results Less efficient (lower throughput capacity)

21. Interpretation PGx Test Results

22. MAJOR CHALLENGES IN THE CLINICAL IMPLEMENTATION OF PHARMACOGENETICS

23. Quality of Evidence and Clinical Relevance Homozygous or heterozygous patients for the long form of the serotonin transporter genes ( SLC6A4 and HTTLPR ) in the promotor region may have a marginally better response to SSRI over patients who are homozygous for the HTTLPR short form . The life-threatening nature of copy-number duplications in CYP2D6 makes RCTs unnecessary for codeine-induced infant respiratory depression and death due to maternal use. If the variant is known to be functionally related to the development of toxic in vivo concentrations of the drug, an observational case-control study would be a more ethical study design . E vidence thresholds used for reporting the gene-drug associations are often not transparent . R eporting only the number of studies that found a gene-drug association but excluding important study details limits the value of the testing. Reporting that studies have conflicting results without a quality assessment of each study also limits test value.

24. Rare but Life-Threatening Adverse Drug Reactions T he incidence of carbamazepine-induced SCARs is as low as 0.05‰ and for infrequent ADRs, recruiting a sufficient number of patient ADR cases to identify biomarkers of clinical relevance can be difficult. I n Taiwan, Hong Kong, Singapore, and Thailand , HLA-B ∗ 15:02 testing is paid for by the government before carbamazepine is prescribed. In Europe , the cost of HLA-B ∗ 15:02 genotyping is not covered in most countries, likely due to the low incidence of this variant.

25. Time Lag from Basic Science to Translation It takes an average of 17 years for a research discovery to be implemented in medical practice. O nly one-third of research evidence from basic science is successfully applied in clinical settings.

26. Time Lag from Test Order to Result Not all care facilities or laboratories can quickly perform genotyping. Once the genotyping is performed, the results must be interpreted and resulting recommendations must be created.

27. Insufficient Pharmacogenetics Education According to 2 nationwide surveys in the United States , only a minority of physicians (10.3%) and pharmacists (14.1%) reported that they felt adequately informed about the availability of pharmacogenetic testing and its applicability to their patients’ treatment. Neither subject is covered in detail in medical school curricula in the Unites States at present.

28. Complexity of Pharmacogenetic Results Important pharmacogenetic biomarkers, such as CYP2D6 or CYP2C9 , are highly polymorphic , which makes interpretation more difficult. Heterogeneous data from different testing sources also increases the difficulties associated with clinical implementation and decision making. Evidence sufficiency should be based not only on the number of trials showing similar results but also on the quality of the data and the number of independent population replications.

29. Economic Impact of Pharmacogenetic Testing C ost-effectiveness also depends on health economy of the country as well as the prevalence of the specific pharmacogenetic biomarkers. T esting for genetic variants of CYP2C9 and VKORC1 is covered by Medicare in the United States. Conditions for coverage : P atients have not been previously tested for these alleles, have received fewer than five days of warfarin, and are enrolled in a prospective RCT.

30. E xtremely low incidence of SCARs, poor policy adherence, and a high cost of HLA-B ∗ 15:02 screening for carbamazepine meant such screening was not cost-effective in Hong Kong. A cost-effectiveness analysis from Malaysia demonstrated that HLA-B ∗ 15:02 screening for carbamazepine is likely to worsen clinical and economic outcomes. Similar limitations in cost-effectiveness have also been observed for HLA-B ∗ 58:01 testing prior to allopurinol initiation in Singapore and Malaysia.

31. POTENTIAL OPPORTUNITIES TO ADDRESS CURRENT CHALLENGES

32. Building Capacity for Evidence-Based Pharmacogenetics As of March 2020 , the FDA has included pharmacogenetic biomarkers in the drug labels of 278 therapeutic agents. These 278 medications involve 18 therapeutic areas . According to US federal government’s clinical trials website, as of March 4, 2020 , 452 clinical trials categorized as pharmacogenomic(s)/pharmacogenetic(s) with an active, recruiting, or completed status.

33. N umber of US Food and Drug Administration–approved drug labels in each therapeutic area and the respective percentage of the total number of such labels (n = 278)

34. N umber of clinical trials studying pharmacogenomics or pharmacogenetics in each therapeutic area and the respective percentage of the total number of such trials (n = 452)

35. Active Surveillance and International Collaborations Well-trained clinical surveillors can provide rigorous data thorough characterizations of clinical phenotypes. High quality phenotyping (e.g., clinical characterization of the ADR and assessment of causality) makes it more likely that clinically valid pharmacogenetic biomarkers can be identified from relatively small sample sizes. The CPNDS , for example, is a network of 14 paediatrics and 18 adult academic health centres across Canada and internationally that uses active surveillance to identify patients who experience serious ADRs and matched controls, collect DNA, and conduct pharmacogenomic analyses. RegiSCAR , a international network that brings together multidisciplinary scientists for the centralized collection of clinical data and biological samples (e.g., plasma, lymphocytes, DNA, and skin) from SCAR patients.

36. Integrate Pharmacogenetics into Electronic Health Records Clinical decision support systems (CDSS) are an efficient and effective tool to facilitate the translation from pharmacogenetic data to patient management. CDSS are frequently classified as knowledge-based or non-knowledge based.  In knowledge-based systems , rules are created, with the system retrieving data to evaluate the rule, and producing an action or output. Rules can be made using literature-based, practice-based, or patient-directed evidence. CDSS that are non-knowledge based still require a data source, but the decision leverages artificial intelligence (AI), machine learning (ML), or statistical pattern recognition, rather than being programmed to follow expert medical knowledge.

37. FARMAPRICE platform workflow

38. Advances in Genomic Technologies Next-generation sequencing (NGS) has gradually replaced the traditional Sanger dideoxy terminator sequencing. While Sanger sequencing is usually used to sequence relatively small fragments of DNA (up to 900 base pairs in length) in one gene at a time, NGS is a massively parallel, high-throughput approach used to sequence millions of fragments simultaneously. NGS data may be available within hours. Despite NGS being much faster and comprehensive than traditional sequencing, current technologies remain limited by false-positive results in approximately 1% of cases, especially in complex genomic regions (e.g., HLA and G/C-rich regions). P rice of sequencing a human genome to fall below $1,000 in 2019 .

39. Machine Learning A novel application of artificial intelligence, may be an efficient way to solve complex problems with large and diverse data source. Several machine learning models: N aive Bayesian model - A classification algorithm to rank and predict gene-drug adverse reactions. HUME - A multiphase algorithm to identify causal pharmacogenomic relationships in gene & drug pathways. MOLI (multi-omics late integration) - A method to improve the accuracy of drug response prediction.

40. C ost -effectiveness of PGx treatment

41. The cost of genetic testing quoted by the reviewed studies ranged between US$33 and US$710 with a median value of US$175. The cost of genetic testing can range from under $100 to more than $2,000 , depending on the nature and complexity of the test. Prices were on average higher in the United States and Canada than other regions of the world.

42. CONCLUSION

43. REFERENCES Verbelen , M., Weale , M. E., & Lewis, C. M. (2017). Cost-effectiveness of pharmacogenetic-guided treatment: are we there yet?.  The pharmacogenomics journal ,  17 (5), 395–402. https://doi.org/10.1038/tpj.2017.21 Gross T, Daniel J. Overview of pharmacogenomic testing in clinical practice.  Ment Health Clin . 2018;8(5):235-241. Published 2018 Aug 30. doi:10.9740/mhc.2018.09.235 Malsagova KA, Butkova TV, Kopylov AT, et al. Pharmacogenetic Testing: A Tool for Personalized Drug Therapy Optimization.  Pharmaceutics . 2020;12(12):1240. Published 2020 Dec 19. doi:10.3390/pharmaceutics12121240 Sutton RT, Pincock D, Baumgart DC, Sadowski DC, Fedorak RN, Kroeker KI. An overview of clinical decision support systems: benefits, risks, and strategies for success.  NPJ Digit Med . 2020;3:17. Published 2020 Feb 6. doi:10.1038/s41746-020-0221-y

44. The 2000 ACCE (analytical validity, clinical validity, clinical utility, and associated ethical, legal and social implications) project by the US Office of Public Health Genomics (OPHG) aims at the evaluation of genetic tests in the Centers for Disease Control and Prevention. Later, the OPHG assigned the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) working group, which expanded and refined the ACCE model. EGAPP supports the development of the systematic assessment of available data regarding the validity of genetic tests and their usefulness in clinical practice, works out recommendations for healthcare professionals and evaluates the widely used genetic tests .

45. Pharmacogenomic Databases Pharmacogenomics Knowledge Base ( PharmGKB ) - (https://www.pharmgkb.org) - C ollects, curates, and disseminates updated pharmacogenomic information. - As of February 26, 2020, PharmGKB has information for 683 drugs, 147 drug biotransformation pathways, 139 clinical guideline annotations, and 750 drug label annotations for drug labels . ● Pharmacogene Variation Consortium (https://www.pharmvar.org) - F ocused on the human cytochrome P450 (CYP) genes. ● Other pharmacogenomic databases - Human Leukocyte Antigen (HLA) and Adverse Drug Reaction (ADR) Database, Clinical Genome Resource, ClinVar , and Ubiquitous Pharmacogenomics.

46. Various clinical genetic testing approaches for Pharmacogenes Real-time PCR (RT-PCR) with Taqman probes Restriction Fragment Length Polymorphism (RFLP) analysis Amplichip CYP450 (Roche) test ( first FDA approved  pharmacogenetic test) PCR + Sanger sequencing For exome sequencing (ES)/genome sequencing (GS) Amplicon sequencing Technology: Single-molecule real time (SMRT) sequencing assay Nanopore sequencing