In the future, a visit to your doctor may include genetic prescreening using a microarray to determine if you will be able to metabolize a new prescription drug. Even a visit to the dentist might include microarray screening to determine if oral lesions are benign or malignant.

Rapid advances in our understanding of the human genome and the technologies we use to study it are ushering in a new age of “personalized medicine," in which the diagnosis and treatment of disease can be tailored to a patient’s genetic makeup, delivering the right drug at the right dose at the right time. Here we discuss some of the technologies (Microarrays and Biochips) that are enabling these advances and a few practical examples of their applications (Delivering the Right Drug at the Right Dose at the Right Time).

Part I. Microarrays and Biochips

Pharmacogenomics uses the information gained from the Human Genome Project and advances in high-throughput methods for analyzing DNA and proteins to study how genotype affects the efficacy and toxicity of pharmaceutical compounds. Pharmacogenomics is already yielding benefits. Pharmaceutical companies can prescreen patients before clinical trials so that trials are conducted only on patients who are likely to respond to the pharmaceutical being tested, allowing safer clinical trials on smaller populations (1).

The Human Genome Project has generated a wealth of information, but the real progress comes with the technologies that have developed to understand, manipulate and analyze this information. Much of this technology has centered on the development of microarrays or “biochips” that can measure the expression of thousands of genes or search for the presence of variations in DNA such as single-nucleotide polymorphisms (SNP) or deletions, insertions and expansions of DNA sequences (2). These microarrays enable the rapid assessment of gene expression and will ultimately make a significant contribution as point-of-care testing (3). Additionally microarray analysis of gene expression profiles has been used to distinguish among types of breast cancer and predict cancer outcome (4,5).

DNA microarrays are small glass slides or silicon chips that contain known DNA sequences (probes) spotted in a precise arrangement. The DNA used can be short oligos or longer cDNAs (2,6). Researchers probe the “unknown” sample (e.g., from a patient). The unknown sample often consists of labeled cDNAs generated by reverse transcription from total RNA. Researchers look for hybridization or matches between the experimental sample and the DNA on the array.

DNA microarrays can be used to see which genes are being expressed in a sample. For instance, DNA from a tumor can be screened to look for the expression of genes involved in drug resistance. DNA microarrays can also help to determine if a particular sample contains a specific genetic polymorphism linked to a clinically important outcome.  In addition, DNA microarrays may be useful in detecting virulent bacteria, particularly anerobic bacteria that are difficult to culture in a laboratory or bacteria that are particularly slow growing. Rapid detection of pathogens could greatly improve infectious disease outcomes.

Several companies have created arrays to identify polymorphisms in the genes encoding the cytochrome P450 proteins. These proteins are responsible for metabolizing drugs and other compounds in the body. PPGX has developed a test to identify individuals who have variations of the CYP450 2D6 protein that make them “slow metabolizers” of many popularly prescribed drugs including antidepressants (7). Additionally Affymetrix has produced an array that detects polymorphisms in CYP450 2D6 and 2C19 (7).

Microarrays show great promise, but they still have some obstacles to overcome before they will be widely used. Currently microarrays are costly, and the initial setup costs for a lab to create and process microarrays is significant. There has been some difficulty with data reproducibility, protocols and statistical methods for generating and analyzing microarray data. All of these things will have to be standardized before microarrays can come into widespread clinical use. Additionally, efforts need to be made to educate both physicians and patients about microarray results (6,8).

In addition to DNA microarrays, researchers are also creating protein arrays. Protein microarrays can complement gene expression profiles of DNA microarrays. Protein microarrays will allow researchers to determine if a sample contains an aberrantly processed or modified protein (6,8). The ProteinChip (Ciphergen Biosystems, Inc.) can be used to distinguish pancreatic cancer from other pancreatic diseases based on proteins in serum (3).

Part II: Delivering the Right Drug at the Right Dose at the Right Time

Breast Cancer: Delivering the Right Drug

Breast cancers are actually several different diseases each with its own set of genetic mutations, histology and optimal treatment (8). Some cancers (~25–30%) overexpress the HER2/neu receptor, a tyrosine kinase receptor that transduces cell growth signals in normal cells, and this overexpression is associated with more aggressive cancers (9).

The therapeutic agent Herceptin is a humanized monoclonal antibody specifically targeted against the HER2 receptor and is used to treat metastatic breast cancer. This therapy requires a diagnostic test to confirm the overexpression of the HER2 receptor before it is used. Although the current test relies on detecting protein overexpression using immunohistochemistry (IHC), methods to look at the DNA of the tumor cells are being developed. Measuring the amplification of the HER2 gene using fluorescence in situ hybridization (FISH) may be a better predictor of efficacy. In one study, women who received Herceptin in conjunction with traditional chemotherapy survived 50% longer than those receiving the traditional therapy alone. Those identified by IHC showed only a 24% survival advantage (10,11). Evidence also suggests that some other cancers, such as esophageal or gastric cancers that overexpress HER2, would also respond to therapy with antibodies against EGFRs like Herceptin (12,13).

Currently researchers are developing methods for creating a gene expression profile or transcriptional profile (TP) from breast cancer needle biopsies. The researchers have been able to make TPs from needle profiles and identify profiles that are specific to cancer cell type, expression of HER2 and expression of the estrogen receptor (14).

Acute Lymphoblastic Leukemia: Delivering the Right Dose

One disease in which pharmacogenetics is already being applied is the childhood cancer, Acute Lymphoblastic Leukemia (ALL). ALL is treated with a cocktail of chemotherapeutic agents that include 6-mercaptopurine, 6-thioguanine and azathiopurine. These drugs are broken down by the enzyme thiopurine methyl transferase (TPMT). Approximately 1 in 300 people are homozygotes who produce no functional TPMT (10). For those people the cancer treatment can result in severe toxicity or death. Even heterozygous individuals who produce reduced amounts of the functional enzyme show toxic responses to chemotherapy with these agents (7,15). However, these patients can be treated with doses 10 to 15 times less than the standard regimen.

Researchers at St. Jude’s Children’s Hospital in Memphis, TN, and at the Mayo Clinic in Rochester, MN, are prescreening patients to determine if they have functional or nonfunctional TPMT. The dosage of the components in the chemotherapeutic cocktail are then tailored precisely to the child’s molecular makeup—personalized prescribing.

Detecting Body Rhythms: Delivering at the Right Time

Many genes undergo changes in expression levels with specific periodicity; genes that cycle in a 24-hour period are said to follow circadian (daily) rhythms. Ueda et al. (16) used microarray analysis to extract genes expressed in liver from the mouse genome that show circadian expression under both light/dark and total dark conditions. Since the liver is the organ primarily responsible for metabolizing pharmaceuticals, understanding when specific genes in the liver are expressed at their highest and lowest levels may allow scientists to determine the best time in the circadian cycle to administer a drug to an individual in order to maximize efficacy and minimize toxicity.

Bed-side microarray screening may not be a reality tomorrow or the next day, but certainly the first decade of the third millenium will see increased perscribing based on genotype as pharmaceutical companies continue to develop therapies targeted to work best in specific gene expression environments.

Literature Cited

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15. US Department of Energy (2004) Pharmacogenomics. Human Genome Project Information accessed Sept. 30, 2004.

16. Ueda, H. et al. (2004) Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 101, 11227–32.