With the announcement of the completion of the draft sequence of the human genome in 2001, three pathways of activity seemed likely to be followed.1,2 First, there were technical issues to resolve. The sequence was not totally complete – it needed sequence error correction and selected verification – and interpretations of data then required revision. This important activity has recently led to a reduction in the estimates of the total number of genes in our genome to the surprisingly small number of approximately 25,000.3 Now nearing completion, the full revised sequence of the human genome and its interpretation will be essential to its further use.4,5

Second, the information generated as part of the Human Genome Project (HGP) would need to be compared to other information known about human diversity and would form the basis of a whole new series of comparative studies essential to developmental and evolutionary biology as well as anthropology. It was clear, for instance, that the complete set of human proteins when compared to the genomic set was substantially larger. Therefore, mechanisms for amplifying genomic information including messenger RNA (mRNA) splicing, translational and post-translational variations, and epigenetic mechanisms were likely to be important. In addition, proposals for the minimum set of genes required for a functional living genome flow directly from comparative genomics and the results of HGP.6 New insights into human diversity and our role in the biological world continue to be aided by progress in genomics.

Finally, there needed to be direct clinical applications of the work associated with producing the human genomic sequence. Methods for the isolation, manipulation, and analysis of genetic material were improved as part of HGP. Previously unknown genes and modifying deoxyribose nucleic acid (DNA) sequences were discovered. Important bioinformatic tools were created to help mine genomic information and make appropriate associations with clinical data. In some cases, these developments would supplement already applied laboratory methods and clinical information. In other situations, they appeared to be “disruptive” innovations and discoveries that were likely to create new types of clinical laboratory practice.7 This post- project clinical activity is critical for deriving near-term benefits from the basic knowledge generated as part of HGP.

This brief framework provides understanding as to how progress in genomics will impact clinical laboratory practices by reviewing:

* tests and methods that are currently being ordered;

* some important new procedures and assays that are or will likely become prominent parts of laboratory menus in die next few years;

* the types of new equipment, skills, and ancillary services required to deliver clinical genomics in a high-quality manner; and

* some practical suggestions about how clinical laboratories, with their differing goals, sizes, and resources, might prepare for continuing innovation and application of molecular biology and genomics.

Clinical laboratory genomics: a framework

Technically, genomics and genomic testing imply the assessment of many or all elements within the genome that modify a trait or condition of interest. Therefore, genetic testing traditionally involved the assessment of a single genetic locus that was relevant. In the current clinical laboratory milieu, aside from molecular assessment of multiallelic genes (e.g., DNA testing for cystic fibrosis) and certain new oncology tests, most testing is genetic; assays include biochemical, cytogenetic, and molecular methods.

Biochemical genetic testing is the most common. Genotype is inferred from measures of protein products and clinical history, if available. It is represented by traditional assays for hormones or other analytes as part of combination assays for fetal risk assessment during pregnancy (approx 2.5 million per year) or the assessment of risk in newborns (about 4 million per year via Guthrie spots). A variety of immunohistochemistry (IHC) methods are applied to cytology and microscopic pathology samples to assess the expression of proteins that may characterize the presence or activity of clinically important genes. IHC has been most prominently utilized in detecting and subclassifying malignant cells and tissues – for example, in characterizing the presence of products of the estrogen receptor gene on breast-cancer cells.

Cytogenetics is applied primarily in the assessment of fetuses and in the practice of oncology. Aniniocentesis with karyotyping of fetal cells represents a commonly ordered test (about 300,000 to 400,000 per year). The search for gross and cryptic chromosomal changes in bone marrow samples, solid tumors, and other sources of tumor cells (e.g., blood, urine, stool, aspirates) is also well- established and common. Fluorescence in situ hybridization (FISH) has increased the speed and utility of chromosomal analyses, and the volume of tests ordered is growing.

Molecular analyses are most commonly used to detect carriers of cystic-fibrosis-associatcd risk alleles who are considering pregnancy (about 1 million per year) and to evaluate whether individuals with recurrent deep-vein thrombosis or pulmonary emboli have the gene associated with Factor V Leiden (about 200,000 per year). Molecular quantification and typing of HIV and HCV viruses are increasingly used to confirm or monitor infection and determine treatment strategies. A large number of other molecular tests, each individually far less common, are used to diagnose or identify risk associated with genetic variants. When somatic mutations occur in cancers, their presence can be used as a monitor of cancer burden or therapeutic response (e.g., the BCR-ABL fusion gene in chronic myelogenous leukemia).

Molecular methods include direct sequencing of DNA, hybridization with probes in a manner sensitive enough to detect even single nucleotide variants (single nucleotide polymorphisms or SNPs), or detection of mutations in DNA sequences by physiochemical properties, all usually after amplification of DNA using the polymerase chain reaction (PCR) that allows for highly sensitive assays.

Expansion of clinical laboratory genomics

With few exceptions, the use of molecular tests or techniques in general clinical practice is limited. Similarly, the characterization of the human host experiencing disease risk or an illness (the response to environmental agents or therapeutic interventions, the identification of high/low responders to a specific therapy, and those likely to experience side effects – the field of pharmacogenomics), while an attractive model, has yet to be proven as broadly clinically useful. In fact, the most likely early applications of clinical genomics and its methods will be enhancements of current high-volume testing; tests that help reduce adverse events will likely be introduced gradually.

For instance, new highly sensitive biochemical analyses when coupled with accurate diagnostic imaging will enhance strategies for fetal screening during the early stages of pregnancy. The deployment of tandem mass spectroscopy platforms will result in newborns being screened for many biochemical analytes associated with rare genetic metabolic disorders. Molecular arrays or chips that allow rapid and reproducible detection of genes or their expression in target specimens will begin to supplement standard pathological determinations in oncologic, inflammatory, and cardiovascular diseases. Similar platforms constructed to detect or subclassify infectious agents or human-host responsiveness (pharmacogenomics) will also slowly come into use.

For all of these visions for the future of genetic medicine to be realized, many hurdles will have to be overcome. Primarily, these approaches must be shown to result in improved clinical outcomes in a cost-effective manner. Additionally, providers of these tests will be substantially burdened with imperatives for improving physician and patient education in order to capitalize more fully on these new testing technologies. Technology-assessment processes will have to improve so that new technologies and tests will continue to flow from the research setting to the clinical lab to patients with reasonable speed and demonstrated specificity and sensitivity. The expansion in molecular testing within the clinical lab will undoubtedly continue as long as technical, economic, legal, and ethical issues can be resolved.

Developments in clinical laboratory genomics

Nucleic acid amplification techniques

Most DNA-based genetic testing utilizes amplification of DNA using PCR. PCR can be used to directly identify alterations in DNA sequence, as in microsatellite instability testing. To detect or monitor RNA, reverse transcription of RNA to DNA followed by PCR amplification (RT-PCR) can be used. Real-time quantitative PCR is useful for monitoring changes in RNA transcripts, especially low- abundance transcripts. This technology is also very useful for pathogen quantification and SNP genotyping.8

Additionally, these techniques can be applied to quantify gene expression levels (quantitative reverse-transcription PCR or QPCR) as determined by mRNA transcript levels. In DNA microarray-based amplification (on-chip PCR), amplification is performed directly on the surface of a glass chip, and the products are visualized by fluo\rescence scanning of the chip rather than by electrophoretic separation.9 On-chip PCR can be used to identify SNPs in human genomic DNA or for detection and identification of pathogens. There are a number of esoteric methods for nucleic acid amplification. Rolling circle replication with PCR has been used for high- throughput SNP detection.10 Whole genome amplification has also been accomplished using multiple-displaced amplification, an extension of rolling circle replication techniques.11 NonPCR based DNA detection methods are also being developed. These technologies hopefully will overcome the limitations of the polymerase enzyme. One recently published technique uses nanotechnology to develop point-of-care medical diagnostics. This technology utilizes gold nanoparticles, magnetic separation, and chip-based DNA detection and purportedly can detect as few as 10 DNA molecules in a sample.12

Chromosome imaging

Traditional karyotyping utilizes dyes to identify chromosomes by banding pattern. Spectral karyotyping utilizes fluorescent, chromosome-specific probes to label each chromosome uniquely. An interferometer is used for detection. This technology is useful for genomewide detection of structural chromosome changes such as translocations that can be difficult to detect utilizing traditional karyotyping.13

FISH has been utilized in research for over two decades and was approved by the Food and Drug Administration (FDA) for prenatal diagnosis applications in 1997.14 FISH has become part of other clinical laboratory assays. The method is straightforward: fluorescently labeled DNA probes arc hybridized to a metaphase chromosome spread or interphase nuclei of specimen cells that have been fixed on slides. Binding of the probe to the chromosome can then be visualized using epi-fluorescent microscopy. FISH is more expensive to perform than standard IHC techniques, but is more sensitive and specific. Consequently, in some cases, FISH has been recommended as a reflex for indeterminate results using IHC rather than being used as the primary screening tool.15 Microdeletions and other subtle structural changes of chromosomes can be reliably detected by FISH and linked to clinical syndromes (e.g., developmental delay).

Conventional comparative genome hybridization (CGH) provides information on the number of copies of chromosomes throughout the genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads.16 The hybridization is detected with two different fluorochromes, and the fluorescence ratio is measured along the chromosome using a laser microarray scanner and digital imaging analysis. A gain or amplification in the target genome is visualized by an excess of one fluorochrome, while a deletion or loss in the target genome is visualized by an excess of the other.

Array comparative genome hybridization is a more powerful technique that detects high-level amplification and homozygous deletions in small genomic regions.17 This technique uses large insert clones, such as bacterial artificial chromosomes (BACs) of genomic DNA, as probes and uses differentially labeled test and control DNA as targets. Array technology has been used to detect high-resolution copy number changes in breast, renal, and bladder cancer.18 Using the genomic clone sequence data, this powerful technique can lead quickly to the identification and cloning of genes associated with disease for use in diagnostics and targeted therapeutics. Array technology promises to revolutionize the field of medical cytogenetics by providing molecular karyotyping without the need to culture cells or stain chromosomes. Another advantage of array technology is that assays can be repeated or duplicated – something that is not typical with single gene mutation detection methods.

Genomics technologies

Functional genomics, which involves the study of genes within the human genome, the determination of a gene’s function, and the identification of allelic polymorphisms associated with disease risk, is of most interest to the clinical lab. The ultimate goal, however, is to understand how genetic information is processed through mRNA (transcriptomics) to proteins (proteomics) to metabolites (metabolomics) to biological function or dysfunction. Systems biology is often used as a synonym for functional genomics – a description of the genomic and epigenomic influences on a trait and their interactions with environmental variants. Pharmacogenomics exploits the information on genetic variation to optimize drug efficacy, reduce drug toxicity, and optimize therapeutic treatments.

Solid-phase microarray technology involves tethering a known DNA sequence or probe to a solid substrate. The probes usually consist of small oligonucleotides, protein nucleic acids (PNAs), or complementary DNAs (cDNAs). DNA targets, commonly in the form of fluorescently labeled cDNA or genomic DNA fragments, are then hybridized to the probe. Once hybridization of the target DNA to the probe occurs, the array is digitally scanned. The data collected is then analyzed for hybridization patterns and fluorescent intensity.19 DNA microarray technology provides data on DNA sequence variation (mutations and polymorphisms) and gene expression levels. In expression studies, the target DNA may be a mixture of normal and perturbed cell transcripts that are differentially labeled during cDNA synthesis. Expressed sequence tags can also be used as target DNA. Different fluorescent signals are recorded for spots hybridizing to either one of the probe types or to both probes.20 Gene expression profiles can identify up-regulated and down- regulated genes, which are then targets for novel therapeutics. cDNA arrays have also been used to classify pathological subgroups of specific disorders. When polymorphisms are identified, genomic DNA targets are used with oligos or PNA probes, which define allelic differences. Oligonucleotide arrays have been termed DNA chips.21 Recent advances in microarray technology involve the use of liquid- phase array technology. One example would be bead-based multiplexing, which allows multiple analytes to be assayed in the same well.

Proteomics technology

Proteomics seeks to understand the structure, function, and expression of all proteins encoded by a given genome. This encompasses protein expression profiles as a function of age, state, and environment. Additionally, post-translational modifications and protein-macromolecule interactions can also be studied. Proteomics attempts to determine the relative abundance of proteins in a given tissue, protein-protein interactions, and protein structure. The bedrock technique of proteomics is the separation of proteins by two- dimensional poly-acrylamide gel electrophoresis (2 D-PAGE). This technique uses isoelectric focusing to separate proteins in one dimension by their charge, followed by gel electrophoresis to separate proteins by their molecular mass. With advances in separation technology and fluorescent protein dyes, more than 10,000 proteins can be resolved by 2D-PAGE.22 Gel excision of protein spots of interest followed by in-gel enzymatic digestion allows for peptide fingerprinting by mass spectrometry.

Mass spectrometry involves the ionization of peptide fragments followed by the detection of the mass and charge of individual fragments. Peptide mass fingerprinting compares the mass-to-charge ratios observed for each peptide fragment to the mass-to-charge ratio predicted for known gene sequences. Statistical analysis of the measure of fit is essential in determining good matches. There are a number of mass spectrometers in use, and each varies on the sample preparation required and the appropriate application. Structural and sequencing data can be obtained from tandem mass spectrometry (MS/MS), where peptide massto-charge ratio is determined by one mass spectrometer, then the sample is further fragmented by collision-induced dissociation, followed by passage of the fragments through a second mass spectrometer. Upstream liquid chromatography (LC) separation technologies aim to mimic 20-PAGE in realizing protein separation based on a number of separation methods. LC-MS/MS seeks to provide structural and sequence data while allowing sample automation. Mass spectroscopy, coupled with bioinformatic software that allows sensitive pattern recognition, has recently been used to detect diagnostic protein patterns linked to early ovarian cancer.23

Proteomic analysis is also moving toward automated chip technology using protein arrays. Protein arrays use protein probes that in solution retain the ability to interact specifically with other proteins or molecules. Protein arrays can be used to identify protein-protein interactions, enzyme-substrate interactions, and antibody-antigen interactions.24

Current pharmacogenomic tests

Chronic myelogenous leukemia (CML) is one of the most common forms of leukemia. Most cases of CML result from a chromosome abnormality whereby DNA from chromosome 9, which contains most of the proto-oncogene c-abl, is translocated onto chromosome 22 within the breakpoint cluster region (BCR) gene. This results in a gene fusion constitutive for expression of a protein with tyrosine kinase activity. This activity affects intracellular signaling pathways that result in unregulated cell proliferation. Molecular diagnosis of CML utilizes QPCR.25 FISH can also be used to visualize the translocated chromosomes. The oral drug imatinib (Gleevec) was specifically designed as a selective inhibitor of the BCR-ABL tyrosine kinase and has demonstrated therapeutic superiority over conventional drug therapy.26 Testing may monitor responsiveness to Gleevec or the development of therapeutic resistance.

The Her-2/neu proto-oncogene is active in 25% to 37% of breast cancers. Overexpression of the Her-2 human epidermal growth factor receptor protein on cellsurfaces stimulates uncontrolled tumor growth and is associated with poor clinical outcome. Her-2 can be detected through IHC or FISH methods. A recent paper recommended IHC as the method of choice with reflex to FISFI for indeterminate outcomes.15 Only patients where Her-2 is detected should receive Herceptin since the therapy is expensive, unproven in other patients, and is found to be associated with mild to severe heart failure in about 12 % of patients treated.27

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common hereditary cause of colon cancer, accounting for about 2 % to 5 % of all colon cancer cases. It is caused by mutations in any of at least five DNA mismatch repair genes, and DNA tests are available for the most common genes. The HNPCC syndrome predisposes a person to developing colon cancer at a young age.28 Presymptomatic and predispositional testing in families has been conducted. Since approximately 90% of tumors from HNPCC patients show microsatellite instability, testing for microsatellite instability alone can be a good guide to the necessity for further molecular characterization. Gene sequencing can then be used to identify the precise mutation. Mutational data, along with DNA microsatellite instability testing results, can then be used to identify first-degree relatives with HNPCC. Once HNPCC has been implicated from clinical data, IHC testing can be conducted on tumor tissue for confirmation of diagnosis.

Thiopurines, thioguanine, and mercaptopurine are commonly used anticancer therapeutics. The thiopurine methyltransferase (TPMT) catalyses the methylation of thiopurines. The TPMT gene is polymorphic, and one in 300 patients is deficient in enzyme activity.29 At standard doses, this can lead to toxic accumulation of thiopurines, which can be fatal. Three mutations account for the majority of mutant alleles, and genetic testing is available. Children with leukemia who receive these medications are routinely screened for these deficiency genes.

Future pharmacogenomic testing

Adverse drug reactions result in 6.7% of hospitalizations and 0.32% of mortalities.50 Pharmacogenomics has the potential to reduce these numbers by characterizing patients’ genetic variances in genes responsible for their responses to drugs. The genes most associated with this process are encoding receptors, metabolic enzymes, and metabolite transport proteins. These very same genes have been implicated in environmental toxin susceptibility and cancer predisposition.

Drug efficacy is directly related to the binding of the drug molecule to cell surface receptors. For example, it has been demonstrated that patients expressing high levels of betaadrenergic receptors are more responsive to beta-agonists and antagonists. Conversely, those expressing low levels of this receptor require higher drug levels to achieve a comparable pharmacological effect; this can lead to an adverse drug reaction. Once inside the cell, the drug is metabolized by a number of enzymes catalyzing alterations in the molecular structure of the therapeutic drug. One class of metabolic enzymes is the cytochrome P450 superfamily that comprises more than 40 isozymes. These enzymes metabolize a large number of drugs, carcinogens, small molecules, mutagens, and carcinogens by modifying parent molecule functional groups. Polymorphisms in one gene, CYP2D6, can result in a homozygous recessive inactive genotype that cannot convert codeine into the active metabolite morphine. This genotype occurs in about 6% of the population.31 CYP2D6 has at least six polymorphisms, and these produce phenotypes (ultrarapid, extensive, intermediate, and slow) that have been shown to impact the metabolism of many drugs. The FDA is currently considering a P450 molecular array.

Metabolites may also be altered by the addition or modification of side groups. Acetylation is an important modification of many drugs. The N-acetyl transferase (NAT) enzymes function both for metabolism and aromatic amine detoxification pathways. NAT2 polymorphisms result in slow, intermediate, and fast acetylator types. Patients who are slow acetylators are at increased risk for adverse drug reactions. Additionally, slow acetylators have been shown to be at an increased risk for bladder cancer and an even greater risk if they are cigarette smokers.32 Cigarette smoke contains aromatic amines that arc known carcinogens. The glutathione S-transferasc (GST) pathway is involved in antioxidant defense. GST polymorphisms can he used to predict response and toxicity to some chemotherapies. Additionally, deletions in several GST genes have been associated with increased risk for cancer.31 Monoclonal antibodies for a given metabolic enzyme can be used in conjunction with other techniques to determine the role of specific genes in pathways impacting drug metabolism, detoxification, and cancer susceptibility. Transport proteins function to deliver drugs to appropriate targets and to exclude xenohiotics. Several transport protein mutations are associated with disease, most notably the cystic fibrosis transmembrane regulator (CTFR) gene mutations associated with cystic fibrosis.

Conclusions

The data generated by the Human Genome Project, coupled with advances that genomic and proteomic technologies suggest, will foster significant changes in the practice of clinical laboratory medicine. Enhancements to current tests, new platforms, and new bioinformatic demands are already emerging. For all of these new methods and procedures to come to practice, however, significant challenges will have to be surmounted. Some are practical; genomics- based testing must be demonstrated to improve clinical outcomes in a cost-effective manner.33 But a significant number of regulatory, educational, and legal issues need research and clarification before clinical laboratory genomics is universally applied.

A key challenge is for inventors of tests and methods to prove the value of their inventions. There have been few studies of the cost-effectiveness of genomics-based testing.31, 34 In 1968, the World Health Organization established principles for mass screening for disease. These include effective drug therapies for the prevention of disease risk, the need for early diagnosis for effective prevention, and the feasibility of presymptomatic diagnosis through genetic testing.35 Using cost-effectiveness analysis, Flowers and Veenstra concluded that pharmacogenomics strategies are likely to be cost-effective when the polymorphism is prevalent in the population and has a high degree of penetrance, genetic testing is highly sensitive and specific, the disease state involves outcomes with high morbidity or mortality, and the treatment involves significant outcomes or costs. They conclude that, based on these criteria, oncology appears to be the most appropriate area for pharmacogenomics applications.

An example of a specific cost-effectiveness analysis is the one performed on human leukocyte antigen (HLA) B*5701 genotyping in preventing abacavir hypersensitivity.34 Abacavir is a nucleoside analog that is a potent inhibitor of HIV reverse transcriptase when used alone or in combination with other drugs. Approximately 4% to 8% of patients utilizing abacavir, however, develop hypersensitivity reactions that can lead to life-threatening hypotension. The HLA B*5701 has been identified as a highly penetrant genetic risk factor for ahacavir hypersensitivity in Caucasians. This suggests that pre- prescription genotyping for this allele would reduce the incidence of abacavir bypersensitivity. A decision analytic model was used to determine the cost-effectiveness of genotyping for HLA 13*5701. It was determined that pre-prescription genotyping would be cost- effective over a broad range of clinical assumptions and would reflect the significant expense of HIV therapeutics.

An additional cost concern is the infrastructure required to adopt genotnics-bascd testing in the clinical setting. Genomics- based technologies utilize platforms with instrumentation requirements distinct from those currently used in the clinical setting. Whether chip readers, liquid phase array instrumentation, mass spectrometers, real-time PCR instrumentation, or any other instrumentation is needed, additional capital expenditures will be required. Additional lab personnel training must also be anticipated. Therefore, the transition of clinical medicine to genomics-based testing will involve infrastructure demands and expenses that need to be considered when determining cost- effectiveness.

Finally, clinical laboratory genomics, like the entire field of human genomics, is developing at a rapid pace. The information needed for proper laboratory practice as well as that delivered to patients and physicians is evolving quickly. Smaller laboratories may not be able to deliver a broad menu of high-quality tests in a cost-efficient manner. Laboratories engaging in this testing must expect significant and continuing major capital and reagent costs and need to be capable of almost continuous technology assessment, often with only scant clinical trial data available. Hidden costs of launching a test – like the legal fees associated with researching and negotiating rights for patented tests or processes – must be considered. While these uncertainties and other challenges are daunting, the promise of an explosion of new tests and information as part of clinical laboratory genomics continues to be compelling.

The expansion in molecular testing within the clinical lab will undoubtedly continue as long as technical, economic, legal, and ethical issues can be resolved.

New insights into human diversity and our role in the biological world continue to be aided by progress in genomics.

Hidden costs of launching a test – like the legal fees associated with researching and negotiating rights for patented tests or processes – must be considered.

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By Paul R. Billings, MD, PhD, and Matthew P. Brown, PhD

Paul R. Billings, MD, PhD, and Matthew P. Brown, PhD, are affiliated with the Center for Molecular Biology and Pathology at Laboratory Corporation of America Holdings in Research Triangle Park, NC.

Copyright Nelson Publishing Dec 2004