Genomics and new molecular diagnostics

Transcription

1 Genomics and new molecular diagnostics

2 The FDA approves the first anti-angiogenic angiogenic drug for cancer, Avastin (bevacizumab) DNA microarray test system, the AmpliChip Cytochrome P450 Genotyping Test, to aid in selecting medications for a wide variety of common conditions An RNA-interference product for age-related wet macular degeneration becomes the first RNAi product to enter a clinical trial Researchers at the University of Georgia successfully produce a cow cloned from the cells of a carcass FDA for the first time approves a drug for a specific race. The drug, NitroMed s BiDil, treats congestive heart failure in self-identified black patients. The company hopes a genetic test can be developed to identify patients likely to benefit, regardless of race The National Institutes of Health begins a 10-year, 10,000-patient study using a genetic test that predicts breast-cancer recurrence and guides treatment. Patients whose cancer is deemed unlikely to recur will be spared chemotherapy. The genetic test, Oncotype DX was developed by the biotech company Genomic Health and is already commercially available HPV vaccine on the market. Newest history of biotechnology

3 Genomics is the study of the genome and the role genes play, individually and collectively, in determining structure, directing growth and development, and controlling biological functions. It consists of two branches: Structural Genomics * The field of structural genomics includes the construction and comparison of various types of genome maps and large-scale DNA sequencing. The Human Genome Project and Plant Genome Research Program are structural genomics research on a grand scale. * In addition to genome mapping and sequencing, the objective of structural genomics research is gene discovery, localization and characterization. Functional Genomics Genomics * Gene sequence and mapping data mean little until determination what those genes do, how they are regulated, and how the activity of one affects others. Functional genomics enables to navigate the complex structure of the human genome and to make sense of its content.

4 Best known genomes Arabidopsis taliana Caenorhabtitis elegans Drosophila melanogaster

5 Best known genomes of vertebrates Xenopus laevis Danio rerio Homo sapiens Mus musculus

6 Genomics Knowing only portions of the DNA sequence of certain genes can provide useful products, even without knowing about the gene s s function or the protein it encodes. For example, new product discoveries based solely on DNA sequence data acquired through structural genomics include: * diagnostic tests for microbial contaminants in food products or donated blood. * diagnostic tests for plant, animal and human diseases. * diagnostic tests to identify genetic susceptibilities to certain diseases. * tests for drug-resistant resistant mutants of HIV and other pathogens. * research tools such as antisense or to block gene expression. * gene-based therapeutics, such as DNA vaccines and gene therapies.

7 Pharmacogenetics - Pharmacogenetics was born during the period of intense interest in clinical genetics in the 1950s, after three discoveries: * It was found that individuals who received the drug isoniazid for the treatment of tuberculosis could be clearly divided into slow and rapid metabolisers of the drug, and that this rate was genetically determined. * PatientsP who had prolonged effects of the anaesthetic agent succinycholine,, had an atypical enzyme, in this case a cholinesterase that was inherited. * Studies of the red blood cells of African American American soldiers who had developed severe anaemia after taking the anti-malarial drug primaquine were found to be deficient in the enzyme glucose-6-phosphate dehydrogenase. This inherited error of metabolism was later found to affect 400 million people worldwide.

8 - Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the Hexose Monophosphate Shunt (HMS), the endogenous source of antioxidant. The HMS pathway ay generates reducing power in the form of NADPH, which maintains an adequate level of reduced glutathione (GSH). - G6PD is present in all cells but is particularly important to red d blood cells, where is required to make NADPH. It protects the -SH groups of hemoglobin and the cell membrane from oxidation by the reactive oxygen species es.. Defects in the HMS pathway due to defective G6PD can lead to inadequate protection against oxidation, resulting in oxidation of sulfhydryl groups, precipitation of hemoglobin, and hemolysis. - Multiple hemolysis episodes in a short time span lead to a condition known as hemolytic anemia (jaundice, dark urine, abdominal and back pain, lowered red blood cell count, and elevated bilirubin). - People who suffer from severe and chronic forms of G6PD deficiency in addition may have gallstones, enlarged spleens, defective white blood cells, and cataracts.

9 Ethnic- and gender-based medicine - Some drugs, including common blood-pressure medicines and antidepressants, exhibit significant ethnically correlated safety and efficacy differences. * BiDil - first drug for use in a specific ethnic group, approved by FDA in BiDil,, a life- saving medication for heart failure in n had failed to beat placebo in a broad population but showed promise in African Americans.

10 Echols and Yancy, Vasc Health Risk Manag

11 Ethnic- and gender-based medicine Echols and Yancy, Vasc Health Risk Manag

12 * Interferon - appears to be less effective in blacks with hepatitis than white patients (19 percent vs. 52 percent response rate). * Cozaar (Losartan)- one of the most common blood pressure drugs has a reduced effect in black patients. * Iressa - it seems that Japanese cancer patients are three times more likely to respond to Iressa, apparently because of a mutation in a gene for the drug s s target, epidermal growth factor receptor. * Aspirin - prevents heart infarction in men but not in women. Men - MI Women - MI P<0.001 P=0.98 Aspirin Placebo Aspirin Placebo

13 Personalized medicine - We are entering the age of personalized medicine in which genetic differences among patients are acknowledged and used to design more effective treatments. A medicine s s effectiveness and safety often varies from one person to the next. - Using data acquired in functional genomics, it will be possible to identify genetic differences that predispose patients to adverse reactions to certain drugs or make them good subjects for other drugs. This tailoring of therapeutics to the genetic makeup of the patient is known as pharmacogenomics. - Some diseases also vary in aggressiveness. For example, some forms of breast cancer are more aggressive than others and require different therapeutic approaches. By identifying the unique molecular markers or different types of cancer, we help physicians choose the correct treatment.

14 Pharmacogenetics - The term pharmacogenetics was first used in 1959 to describe a new discipline based on the observation that genetic factors, at that time variation in the function of a single gene, can modify drug action. - More recently, and following the success of the human genome project, the term pharmacogenomics has come into common usage, being defined simply as how people s total genetic make-up affects their response to medicines. - Median difference in allele frequency between major ethnic groups is between 15% and 20%. This means that very common alleles (those present more than 20% of the population) tend to be shared, whereas rarer alleles may be specific to an ethnic sub-set set of the population. Meyer 2004

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16 CYP2D6 polymorphism and use of codeine - Codeine,, a commonly used analgesic, must be converted from an inactive form to the active form (morphine) by the CYP2D6 enzyme for a therapeutic effect to occur. * Patients with a polymorphism leading to increased production of CYP2D6 are ultra-rapid rapid metabolizers ers of codeine and are more likely to develop adverse effects and toxicity when taking a standard dose of codeine,, including impaired breathing and sedation. - Patients with decreased CYP2D6 production are poor metabolisers and will show little or no conversion of codeine to morphine; they will not experience any pain relief, but will become nauseated due to the higher amounts of codeine in their body. - Frequency of CYP2D6 alleles varies in different ethnic groups: * 7% of Caucasians may have a defective CYP2D6 gene,, resulting in reduced pain relief due to poor metabolism of the drug.

17 CYP2D6 - The CYP2D6 enzyme is involved in the metabolism of around a quarter of all prescribed medicines. - A gene-based microarray test has recently been launched by Roche Diagnostics for the detection of poor, intermediate, extensive or ultra-rapid rapid metabolisers according to variants in the CYP2D6 gene. - It is envisaged that one of the first clinical uses of this test would be in hospital-based psychiatry. A report from the Department of Health suggested that t genetic testing for CYP2D6 gene variants could be cost effective in identifying psychiatric patients who would be prone to severe side effects from antipsychotic drugs allowing better tailoring of drug dose.

18 CYP2D6 polymorphism and use of notriptyline

19 Warfarin - Warfarin is a routinely used oral anticoagulant (750,000 patients in the UK, increase by 10% yearly),, given to prevent blood clot formation in people who have coronary artery disease, or venous thrombosis, particularly after surgery and periods of immobility. - Warfarin is metabolized ed in the liver by a CYP2C9, but a variant CYP2C9 gene alters the rate of its metabolism: * People with the "slower" variant of gene break the drug down more slowly than usual, so require lower doses to achieve the same anticoagulant effect (1-5 5 mg a week instead of 5 mg a day). High doses of anticoagulant can lead to potentially dangerous bleeding (side effect rate: 8-26% in the patients treated for one year). * This variant genes occur at a higher frequency in Caucasians than Afro-Caribbeans or Asians.

20 Warfarin - Physicians routinely start patients on low doses of warfarin,, monitor their blood clotting, and increase the dose gradually until the appropriate level is reached. A 20- to 30-fold variation is commonly seen in the response to drugs. - Possibly, pharmacogenetic testing for CYP2C9 alleles may identify people at risk of warfarin- associated bleeding. Potentially such tests are close to clinical l application although rigorous data showing clinical use and cost effectiveness are not yet available. The recent discovery of the warfarin target gene, VKORC1 indicates the likely complexity of studies needed to obtain such data. - Although a gene test would be useful in determining drug dose, many other factors can have a effect on the response to different drugs, but probably the most important are: * Environmental influences such as diet, alcohol consumption and cigarette smoking. * Diseases,, especially liver and kidney disorders, which effect the metabolism of drugs. * Interactions with other drugs, which can influence rates of drug metabolism. bleeding after warfarin

21 Thiopurine S-methyl-transferase - The thiopurine drugs mercaptopurine and azathiopurine are used clinically as immunosuppressants for Crohn s disease, systemic lupus erythematosus, dermatomyositis,, and severe psoriasis, in renal transplantation and to treat neoplasias,, such as acute lymphoblastic leukaemia. - The predominant metabolic pathway for these medications is by thiopurine S-methyl- transferase (TPMT). - A mutation in the TPMT gene can result in decreased levels of enzyme production and therefore a decreased rate of breakdown of the thiopurine drugs. When treated with mercaptopurine or azathioprine,, patients who inherit a TPMT deficiency accumulate excessive concentrations of the active thioguanine nucleotides in blood cells. This can lead to severe and potentially life-threatening problems with the formation of blood cells (haematopoietic( toxicity). - About 1 in 300 is mutated homozygous, and do not express functional TPMT. These individuals require doses to be reduced to as little as 5 10% 5 of the conventional dose to tolerate therapy. About 10% of the population are heterozygous for this polymorphism and have intermediate levels of TPMT activity requiring only modest dosage e reductions.

22 Thiopurine S-methyl-transferase - Acute lymphoblastic leukaemia patients with one or both mutant TPMT alleles tend to have an improved response to mercaptopurine therapy and better chances of being cured, compared with patients who have two normal alleles. - Clinical diagnostic tests are now available for the detection of inactivating SNPs in the human TPMT gene. However, the routine use of TPMT genotyping to make treatment ent decisions is still limited. To some extent, this is related to the perceived high cost c of genotyping, even though this process has been shown to be cost effective. - The drug label includes information on the prevalence of patients with reduced TPMT activity and states that genotypic and phenotypic testing is available to determine if a patient has homozygous, heterozygous or wild-type TPMT deficiency/activity. Detection of G460A mutation in TPMT gene with PCR RFLP1 method.

23 The FDA approves the first anti-angiogenic angiogenic drug for cancer, Avastin (bevacizumab) DNA microarray test system, the AmpliChip Cytochrome P450 Genotyping Test, to aid in selecting medications for a wide variety of common conditions An RNA-interference product for age-related wet macular degeneration becomes the first RNAi product to enter a clinical trial Researchers at the University of Georgia successfully produce a cow cloned from the cells of a carcass FDA for the first time approves a drug for a specific race. The drug, NitroMed s BiDil, treats congestive heart failure in self-identified black patients. The company hopes a genetic test can be developed to identify patients likely to benefit, regardless of race The National Institutes of Health begins a 10-year, 10,000-patient study using a genetic test that predicts breast-cancer recurrence and guides treatment. Patients whose cancer is deemed unlikely to recur will be spared chemotherapy. The genetic test, Oncotype DX was developed by the biotech company Genomic Health and is already commercially available HPV vaccine on the market. Newest history of biotechnology

24 Microarrays - Microarray technology is transforming laboratory research because it allows to analyze tens of thousands of genes simultaneously. It is currently used to study gene structure and function. Thousands of DNA or protein molecules are arrayed on glass slides to create a microarraym matrix. - Recently, a 12,000-gene microarray allowed researchers to identify the 200 or so genes that, based on their gene expression profiles, distinguish stem cells from differentiated cells.

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26 SNP chips - It has been suggested that a SNP single microarray might predict accurately each individual s drug response, and that the test might only need to be done once in a patient s s life time. - However, more work is required to assess whether this approach will be feasible and, in particular, the number of SNPs that will be required to determine the spectrum of common and rare variations that underlies individual drug response. - At present, there are commercially available prototype chips for SNPs; ; the cost is about one US cent per SNP. However, costs are decreasing rapidly.

27 Personalized medicine Genotyping tests: * AmpliChip CYP450 Genotyping Test (Roche and Affymetrix s), the first DNA microarray test approved by the FDA (2004), is a blood test that allows to select medications and doses of medications for a wide variety of common conditions such as cardiac disease, psychiatric disease, and cancer. * It analyzes cytochrome CYP2D6, which are active in the liver to break down certain drugs. Variations in this gene can cause a patient to metabolize certain drugs more quickly or more slowly than average, or, in some cases, not at all. * Cytochrome CYP2D6, plays an important role in metabolizm of some commonly prescribed drugs including antidepressants, anti-psychotics, beta-blockers, blockers, and some chemotherapy drugs.

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29 Cytochrome P450 enzymes (CYP450) - The cytochrome P450 (CYP450) genes are a family of genes whose products are active in the liver to metabolize many drugs. - There are many different P450 genes, each of which makes a protein that modifies a different sub-set set of drugs. Different polymorphisms have been implicated in increased, decreased or completely absent levels of metabolism. - Amongst the CYP450 enzymes,, particularly important in oxidative metabolism are: * CYP2D6, * CYP2C9, * CYP2C19.

30 - Possible advantages of pharmacogenomics in clinical trial: * stratifying for responders * stratifying for full representation * increased safety * proper dosage high responders poor responders non responser hypermetabolizers extended metabolizers poor metabolizers no effect desired effect increased risk of ADR - accordingly to FDA 100,000 patients die in the USA because of adverse drug reaction - ADR

31 CARGO study and AlloMap Assay - CARGO - cardiac allograft rejection gene expression observational - AlloMap assay - test generated after CARGO, which enables the assessment of immunological stuatus of the patients; - It may help to reduce the number of biopses from the transplanted heart (usually 12 biopses during the first year post-transplant transplant to check for the signs of rejection)

32 Endoglin The MVD counts using anti-cd105 antibody correlated with overall and disease-free survival, in cases where no such correlation was seen using the pan-endothelial marker CD34. Immunohistochemical staining of consecutive cryosections of breast cancer using two mabs to CD105. Mab CLE4 (A)) stained both large and microvessels whereas mab E9 (B)( decorated mainly microvessels of the breast cancer tissue. Shed CD105 levels may be useful as an indicator for disease progression and to identify patients at risk of recurrence and metastasis. In patients with breast cancer, shed CD105 levels were markedly elevated in plasma samples compared with healthy controls, especially in those who subsequently developed distant metastasis.

33 YKL40 Microarray gene analyses have identified the human YKL-40 gene to be one of the most overexpressed genes in glioblastoma multiforme, in papillary thyroid carcinoma, and in chondrosarcoma. YKL-40 belongs to the glycosyl hydrolase family-18, which includes chitinases from various species. It binds chitin but has no chitinase activity. Median serum concentration of YKL-40 in healthy adults is 43 μg/l, with no difference between genders. Level of YKL-40 increases in elderly people. Elevated serum YKL-40 levels are found in some patients with nonmalignant diseases characterized by inflammation or tissue remodeling such as rheumatoid arthritis, severe osteoarthritis, severe bacterial infections, inflammatory bowel disease, and liver disease.

34 YKL40 - a potential prognostic marker YKL-40 is expressed by several types of solid tumors: * breast cancer, * colon cancer, * lung cancer, * kidney cancer, * ovary cancer, * prostate cancer, * uterine cancer, * pancreas cancer, * osteosarcoma, * thyroid cancer, * oligodendroglioma, * glioblastoma, * germ cell tumors. Johansen et al., Cancer Epidemiol Biomarkers Prev,, Immunohistochemical staining for YKL-40 in invasive ductal breast cancer cells.

35 YKL-40 - role in cancers Function of YKL-40 in cancers is not known. It has been suggested to: * play a role in the proliferation of malignant cells, * protect cells from apoptosis, * stimulate angiogenesis and extracelluler matrix remodeling YKL-40 modulates vascular endothelial cell morphology by promoting the formation of branching tubules, stimulating the migration and proliferation of vascular endothelial cells.

36 Serum levels of YKL-40 is increased in some patients with solid cancers, and decreased significantly after curative operation. low YKL40 Patients with breast cancer after operation and chemo-therapy, antiestrogen therapy, or radiotherapy. Increased level of YKL-40 in patients with breast cancer predicted shorter overall survival high YKL40

37 YKL40 - potential drowbacks Not all patients with cancer had elevated serum YKL-40 levels compared with healthy age-matched controls, suggesting that not all tumors secrete YKL-40 or that the protein is secreted at a low level. Serum concentrations of YKL-40 do not have a high sensitivity for primary cancer, and determination of serum YKL-40 cannot be used as a single screening test for cancer. At time of first cancer diagnosis,, 16% to 74% of the patients had elevated serum YKL-40. It seems that serum YKL-40 may be a useful prognosticator prognosticator identifying a subgroup of cancer patients with a poor prognosis, having a subset of tumors with a more aggressive phenotype. However, in patients with advanced cancer at the time of diagnosis,, serum YKL-40 levels were elevated in 39% to 91%. Large studies are needed to evaluate if serum YKL- 40 can be useful as a biomarker for screening of cancer together with a panel of other tumor markers and imaging techniques.

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39 Blockbuster business model - Pharmaceutical industry is dominated by the blockbuster model, in which companies built their operations around a few products.. For example (data for 2002): * AstraZeneca - 3 drugs - $7.7 bln. * Johnson & Johnson - 3 drugs - $4.9 bln. Total - 40 blockbuster drugs - $89.3 bln. - The blockbuster drug is defined as one with peak annual global sales exceeding $1 billion (market definition). - From medical point of view blockbuster drug is: * single compound effective in most or all patients, who have particular condition * labeled for use by the general population * prescribed for a chronic condition (thus, providing long-term sale)

40 Blockbuster business model - In fact the blockbuster drug can be effective e.g. in 40-60% of the general population.. As long as there is no adverse effect in the remaining patients, physicians are able to prescribe them on the trial-and and-error basis. - Clinical trials must be large enough (that means very expensive), to show clear efficacy, even if large proportion of a general population does not respond to the drug. - Success rate in the drug development * 9% selected candidates for preclinical studies * 21% for those which advanced to clinical trial

41 Traditional evolution of fully integrated pharmaceutical company discovery preclinical development clinical testing approval and launch University spinsoff compound as a start-up company Add: * lab space, * lab stuff, * scientific board, * collaborations Add: * regulations, * pilot production * quality control, * administrative staff Add: * manufacturing, * distribution, * sales, * marketing, * additional administrative staff - With exceptions of a few companies the life-sciences start-ups did not have the funding or timely successes to move them to the blockbuster market. - Usually they succeeded by contracting their specialized services or innovative new compounds to the larger players.

42 Blockbuster drugs withdrown from the market because of side effects Vioxx (Rofecoxib, Merck) - bluckbuster drug prescribed for artretic pain, withdrown from the market because of increased risk of stroke and cardiovascular events in some patients. Cerivastatin (Baycol, Lipobay, Bayer) - bluckbuster drug prescribed for lowering cholesterol, withdrown from the marked because of increased risk of muscle breakdown and kidney failure. Rezulin (Troglitazone,, Warner-Lambert) - bluckbuster drug prescribed for insulin resistance, withdrown from the marked because of increased risk hepatotoxicity.

43 Orphan medicines - Medicines that are developed for the treatment of very uncommon diseases. - The EU defines an orphan medicine as one that could treat a disease with a prevalence of less than 5 per of the population, which approximates to cases across the European Union (EU). - As the number of patients who would benefit is too small to be profitable for the pharmaceutical industry, regulatory incentives such as a period of market exclusivity and research grants, currently exist for orphan medicines in the EU, along with tax incentives developed by individual member states. Neglected diseases - Diseases that primarily affect the developing world 1999: There have only been 13 new neglected disease drugs since Multinational companies had very little neglected disease activity. 2005: Four of the top 12 companies now have neglected disease R&D units employing over 200 scientists; three others work on a smaller scale. This activity is driven by non-commercial motives and is conducted under a new no profit-no loss model that provides drugs to developing country patients at cost price.

44 Thank you