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.
• Adverse 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
• Categories - Poor metabolizer
Intermediate metabolizer
Extensive/normal metabolizer
Ultrarapid metabolizer

10. 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.

11. SLC6A4 gene
• Codes 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.

12. HTR2A gene
• Codes for the serotonin 2A receptor.
• Variant rs6313 (common) - Carriers for either cytosine or thymine
polymorphisms.
• Thymine polymorphism - More likely to respond to antidepressants.
• Variant rs7997012 - Increased antidepressant response in patients carrying
a guanine polymorphism.

13. DRD2 gene
• Located on chromosome 11q22 and codes for the dopamine 2 receptor.
• Polymorphism141C Ins/Del - Ins/Ins genotype are more likely to respond antipsychotic
medications.
• Another variant rs2514218 - Homozygous C allele were significantly more likely to respond to
antipsychotics than T allele.
• More akathisia was reported in C homozygotes taking aripiprazole.
• Greater prolactin elevations were demonstrated in T homozygotes taking risperidone.
• Variant rs1079597 (C polymorphism) - Improvement in the negative symptoms of schizophrenia
after a short treatment with amisulpride.

14. CURRENTLY AVAILABLE RESOURCES FOR THE
IMPLEMENTATION OF PHARMACOGENETIC
BIOMARKERS

15. Pharmacogenetic Clinical Practice Guideline
Development Groups
• Clinical Pharmacogenetics Implementation Consortium (CPIC)
- Provides peer-reviewed, evidence-based pharmacogenetic clinical practice guidelines.
- issued 47 guidelines
● Dutch Pharmacogenetics Working Group (DPWG)
- Therapeutic recommendations based on the pharmacogenetic information.
- Published 93 guidelines
● Canadian Pharmacogenomics Network for Drug Safety (CPNDS)
- Conducts systematic literature reviews followed by guideline development using the Appraisal of Guidelines Research
and Evaluation Enterprise (AGREE) instrument.
- Released eight guidelines

16. Internet resources in the pharmacogenetic sector

17. 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

18. EXAMPLES OF IMPLEMENTING PHARMACOGENETIC
TESTING IN CLINICAL SETTINGS

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

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

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

26. 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

27. 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

28. 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)

29. Interpretation PGx Test Results

31. MAJOR CHALLENGES IN THE CLINICAL
IMPLEMENTATION OF PHARMACOGENETICS

32. 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.
• Evidence thresholds used for reporting the gene-drug associations are often not transparent.
• Reporting 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.

33. Rare but Life-Threatening Adverse Drug Reactions
• The 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.
• In 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.

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

35. 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.

36. 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.

37. 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.

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

39. • Extremely 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.

40. POTENTIAL OPPORTUNITIES TO ADDRESS
CURRENT CHALLENGES

41. 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.

42. Number 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)

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

44. 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.

45. 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.

46. FARMAPRICE platform workflow

47. 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).
• Price of sequencing a human genome to fall below $1,000 in 2019.

48. 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:
Naive 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.

49. Cost-effectiveness of PGx treatment

50. • 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.

52. CONCLUSION

53. 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

57. • 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.

58. Pharmacogenomic Databases
• Pharmacogenomics Knowledge Base (PharmGKB)
- (https://www.pharmgkb.org)
- Collects, 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)
- Focused 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.

59. 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