Part 2 Study Day PHARMACOGENETICS

1. Part 2 Study DayPHARMACOGENETICS Samantha Butler 7th January 2010

2. Introduction The term "pharmacogenetics" dates to 1962 when Dr. Werner Kalow published "Pharmacogenetics: Heredity and the Response to Drugs." Pharmacogenetics is now used interchangeably with the newer term "pharmacogenomics."

3. Definition/History Pharmacogenetics is the study of how the actions of and reactions to drugs vary with the patient's genes The role of genetics in pharmacogenetics was initially limited to particular examples but has exponentially increased more recently with the advent of molecular pathology testing and the future requirement of personalised medicine

4. Neonatal Diabetes - a good example Patients with permanent neonatal diabetes usually present within the first three months of life and require insulin treatment. Activating mutations in the Kir6.2 gene (KCNJ11) were shown to cause early onset diabetes, typically diagnosed in the first six months of life (Gloyn et al. N Eng J Med 2004:350; 1838-1849). These patients had de-novo mutations and so did not have diabetic relatives, however, around 10% showed generation to generation transmission with an autosomal dominant inheritance ~ 20% of patients had neurological features, typically developmental delay and in some cases epilepsy. Beta-cell auto antibodies seen in Type 1 diabetes were not detected. They usually presented with very high glucose values and occasionally ketoacidosis However, these patients did show an insulin secretory response to intravenous tolbutamide (a sulphonylurea) suggesting this might offer a new treatment approach. Patients have since been successfully treated with oral sulphonylurea tablets (stopping insulin completely with better control and less hypos) despite being apparently insulin dependent.

5. MSI status and response to 5FU Patients with colon cancers demonstrating high-frequency MSI (MSI-H) have a stage-independent improved overall survival compared to pts with microsatellite stable (MSS) The evidence for significantly improved clinical outcomes in patients with MSI tumours is substantial However, the utility of MSI status as a predictive marker for response to chemotherapy remains uncertain, small no. studies etc. Despite this the routine determination of MSI status is now advocated as a method to identify patients of more favourable prognosis Advances in treatment for CRC have been made for 5- fluorouracil (5FU), oxaliplatin and irinotecan all chemotherapeutic treatments MSI tumours however do not respond well to 5FU-based chemotherapy treatment in stage II/III CRC, again though this is controversial (Richman et al. Annals of Oncology 21 (2010)

6. BRAF and MSI • BRAF mutation V600E is seen as a negative prognostic marker only in microsatellite STABLE tumours • BRAF mutation status is however used to stratify MSI tumours • Can discriminate sporadic MSI tumours (MSI/BRAF mutant) from HNPCC (MSI/BRAF wild-type • Recently though MLH1-hypermethylation thought to be a better indicator than BRAF V600E • It is now commonly believed that better classification of MMR status is needed as well as looking at targeted treatments • This can then predict the risk of relapse, to screen for HNPCC/Lynch syndrome and to possibly modify follow up

7. Personalised medicine At the present time, most drug treatment is done by trial and error; Physicians prescribe medication, and the patient tries the drug. The drug may work, or it may not. It may cause adverse effects, or it may be safe. If the drug does not work, the dose is increased. If it causes harmful or unpleasant effects, a new drug is tried until, finally, the right drug is found. In some cases this procedure may take weeks or even months. In other cases, drugs are carefully tested, and appear to be safe and effective. Only after they are approved for general use are reports of serious adverse effects that did not appear in the initial studies documented. This can occur if there is a rare gene that affects the way in which the drug acts, or the way in which the drug is metabolised (= good or poor metabolisers)

8. Personalised Medicine More recently, discoveries have shown that genes can determine aspects of each individual, down to the level of the enzymes produced in the liver. Since these enzymes determine how quickly a drug is removed from the body, they can make major differences in the way people respond to drugs. There are also gender differences that are likely to be linked to genetics and women often respond differently than men to drugs at the same dose levels. e.g women are more likely to have a good response to specific antidepressant drugs that act as serotonin specific reuptake inhibitors (SSRIs, including Prozac and Paxil) than they are to the older group of tricyclic antidepressants (Elavil and Tofranil). Women also have a greater response to some narcotic pain relieving drugs than do men, but get less relief from some non-narcotic pain medications. Women may show a greater response to some steroid hormones than men do, but have a lower level of response to some anti-anxiety medications than men.

9. e.g. CYP2D6/CYP2C19 • P450 haem-containing enzymes involved in oxidative metabolism of drugs and xenobiotics • CYP2D6 metabolises ~1/4 of all prescribed drugs (e.g. antidepressants and antipsychotics) • At least 70 variants (duplications, deletions, splicing, missense variants) result in 4 phenotypes • Poor metabolisers, intermediate metabolisers, extensive metabolisers and ultra-matabolisers • CYP2C19 metabolises compounds from the classes of anticonvulsants, anticoagulants etc • 2 major variant alleles (1 splice site, 1 premature stop codon) result in enzyme deficiency and have different population frequencies

10. Personalised medicine and race Race may also affect the way people respond to some medications. In many cases, race implies specific genetic factors that are generally, but not always, found among members of specific ethnic groups. e.g. the angiotensin II inhibitor enalopril (Vasotec), which is used to lower blood pressure, works better in Caucasians than in Blacks.

11. Pharmacogenetics simplified One of the simplest examples is the gene that influences body weight. Since many drugs are soluble in body fat, people with large amounts of fat will have these drug deposited into their fat stores. This means that there are lower levels of the drug that can reach the actual organs on which they work.

12. Pharmacogenetics and cancer Cancer treatments are one of the areas where specific targeted therapies would be beneficial Although there are many areas where there already exists the ability to identify specific subsets of cancers by molecular markers, much work needs to be done on refining treatments For example validated molecular markers could contribute to colorectal cancer (CRC) patient management but until recently, none has been routinely used However molecular pathology testing is being used in a number of areas; The use of molecular oncology for the diagnosis and monitoring of leukaemia's The identification of hormone responsive breast cancers to specific drugs e.g. lapatanib/herceptin The identification of KRAS/EGFR mutations in CRC, lung and other cancers The use of FISH/MLPA/array-CGH to diagnose 1p19q oligodendragliomas

13. Molecular Oncology Fluorescence In-Situ Hybridisation (FISH) and molecular genetic testing techniques are used to assist in the diagnosis and classification of leukaemia's/pre-leukaemia's, solid tumours, and related disorders. They provide prognostic information for use in risk stratification, and enable assessment of disease status post treatment/transplantation including stem cell transplantation Around half of the cases are diagnostic cases, with the remainder being referred for a wide range of follow up studies and treatment monitoring But whilst it is known that particular sub-types of disease respond better/worse to chemotherapy/radiation it is hoped in the future that specific drug treatments will be used to target specific molecular sub-types in these patients with better outcomes

14. Chronic myeloid leukaemia • CML can be caused by Philadelphia chromosome translocation of 9 and 22 causing a fusion of the BCR and ABL genes = BRC-ABL • There is a drug that blocks binding site for ATP on the ABL kinase domain preventing tyrosine kinase activity • Mutations are seen in the kinase domain • Different therapy required

15. Oestrogen receptors, progesterone receptors and Her2 • There are three receptors known to fuel most breast cancers; oestrogen receptor, progesterone receptor and Her2/neu • The most successful treatments for breast cancer target these receptors – to see what stimulates the growth of breast cancer cells • Hormone receptor positive cancers rely on oestrogen and/or progesterone to grow and those known as HER2 positive rely on HER2 proteins to grow • There are a subtype of cancer that is generally diagnosed upon the lack of three receptors, –ve for all three that are classed as triple negative patients • These have been shown to have BRCA 1 and 2 mutations and their breast cancer is particularly aggressive • Triple –ve patients are typically responsive to radiotherapy and some chemotherapies but not receptor targeted treatments e.g. Herceptin (EGFR inhibitor) down regulates HER-2 expression and tyrosine kinase inhibitors (TKIs) inhibit all members of Her2 family (lapatanib)

16. EGFR • Non- small cell lung cancer • Treatment by tyrosine kinase inhibitors e.g. Gefiternib • Most that respond to treatment have somatic mutations in the kinase domain of EGFR but later may acquire resistance to treatment • 95% of mutations in exons 18-21 • Those that did not respond did not have mutations

17. KRAS • Mutations in the KRAS oncogene are frequently found in the human cancers • They are common in colorectal cancer, pancreatic cancer and others • ~40% of CRC may carry a mutated KRAS gene • Activating mutations suggest that the patient is unlikely to respond favourably to treatment with small molecule or antibody treatment targeted at EGFR (KRAS lies downstream in EGFR signalling pathway) • Small molecule drugs = erlotinib, gefitinib • Antibody treatments = cetuximab, panitumumab • Therefore activating mutations of the KRAS oncogene can be used as a predictor of resistance to such treatments

18. 1p19q Oligodendragliomas • Annual incidence approximately 1/260,000 • Approximately 5% of primary brain tumours, 10% of gliomas, young patients: median age of onset is 35 years • Symptoms: seizures, headache, nausea, ataxia, dizziness, altered mental state • In recent years it has been recognised that the cytogenetic abnormality of 1p19q deletion is particularly associated with oligodendroglial tumours • The presence of 1p19q deletion is associated with better prognosis and response to treatment • Thus testing for 1p19q deletion has been recommended, particularly in the management of anaplastic oligodendrogliomas (WHO grade III) • MGMT hypermethylation is also a good prognostic factor (greater chemosensitivity) • Suggested correlation between MGMT hypermethylation and 1p19q status (96%, n=39) • Useful for all gliomas (except those positive for 1p19q deletion)

19. PARP = Breakthrough example Triple negative breast cancer tumours with BRCA1/BRCA2 mutations Already known that these tumours do not respond well to hormone targeted treatments PARP, or poly(adenosine-disposphate-ribose) polymerase inhibitors kill cancer cells but spare identical normal cells that lack cancer-related alteration, such as those of mutated BRCA1 and BRCA2 In cells that carry BRCA1 and BRCA2 mutations, 1 of the 2 major DNA repair methods, BER which repair double stranded breaks is non-functional. However, the other major repair method, base-excision repair (BER) compensates for that loss. PARP-1 inhibition disables that base-excision repair. Thus, the PARP-1 enzyme is a target that, once hit and inhibited, leads to cell death. Findings of phase 1 trial with the PARP-1 inhibitor olaparib (AstraZeneca), indicate that the agent has antitumor activity in breast, ovarian, and prostate cancers associated with BRCA1 and BRCA2 mutations. Also looking at PARP for uterine and Brain tumours

20. Further advances; LongQT/ cardiomyopathies/atrial fibrillation • Search for new anticoagulants • Although Wafarin is highly effective it is difficult to use and can get bleeding • 1/5 of all strokes due to atrial fibrillation • “Dabigatran” is new drug close to being used and has no increased bleeding • Pharmacogenetics can be used to predict best drug response • Different populations metabolise Warfarin differently etc.

21. Summary Although the study is still new, pharmacogenetics promises to offer great benefits in drug effectiveness and safety. With increasing understanding of how genes determine the way people respond to drugs, it will be possible to select drugs and doses based on a greater understanding of each individual patient. This promises more effective drug therapy, with greater safety and fewer treatment failures.

22. The future of molecular pathology testing There had been a lot of work carried out looking at who is carrying out what within the NHS pathology laboratories Who should be testing? Private labs have been offering tests e.g. CYP2D6 and CYPC19 since 1999 Astra Zenecea produce Iressa (Gefitinib) and pay for EGFR tests and need result in 5 days with a good competitive price Cardiff, Sheffield, Aberdeen, Manchester, Birmingham test all test for EGFR Other laboratories look at KRAS mutations and 1p19q loss as well as offering screening for triple negative breast cancers However Haematology and virology are leading the molecular diagnostics field at present There is going to be a greater need for testing and the recommendations are that molecular diagnostic testing should be undertaken in regional centres or larger laboratories with greater integration of clinical and pathology services Royal College of Pathologists – The future provision of molecular diagnostic services for acquired disease in the UK – October 2010 and Bio-medical diagnostics a service re-design for genetics and pathology maximising the capacity of new technologies – Human Genomics Strategy Group (HGS)