Biomarkers as Companion Diagnostics Tools to Determine Therapy/Drug Efficacy

Biomarker discovery has been a major by-product of genomic sequencing since the completion of the Human Genome Project. For cancer disease, development of biomarkers for early detection and screening has been the highest priority at Early Detection Research Network (EDRN) in the National Cancer Institute (NCI).

Diagnostic measurement of cancer disease progression is essential to successful cancer disease management. The importance of biomarker to drug development has recently gained momentum, with the realization that integration of biomarkers through the different phases of drug development can yield safer drugs with enhanced therapeutic efficacy in a cost-effective manner. Good biomarker development potentially can help pharmaceutical companies to find targets in discovery much faster and to design clinical trials in a more effective and efficient way.

Meanwhile, personalized healthcare (PHC) is being driven by scientific and technological innovation and better understanding of disease heterogeneity. PHC is seen as the future of medicine, especially in oncology. Laboratory test that predicts individual response to therapy is now available, permitting the choice of the right drug for the right patient. The trial and error approach is being eliminated. Thus PHC will increasingly drive drug development strategy but strong industry buy-in is necessary. Unfortunately, the process involved in identifying and validating biomarkers, which are integral tools in the development of PHC and developing a commercial diagnostic test is lengthy with a low success rate. Examples of commercial biomarker tests include Oncotype DX biomarker test and MammaPrint test for breast cancer.

PHC means drugs and drug doses are made safer and more effective because they are chosen according to an individual’s genetic makeup. Targeted therapy is a familiar component of PHC. Examples of the five US FDA approved anti-cancer drugs that target mutation genes include:

1. Herceptin, which is a humanized antibody approved for treatment of HER2-positive metastatic breast cancer,
2. Gleevec, a protein tyrosine kinase inhibitor that is intended for patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML),
3. Erbitux, an epidermal growth factor receptor (EGFR) inhibitor for colorectal cancer patients and Iressa which is an EGFR inhibitor indicated for patients diagnosed with non-small cell lung cancer, and
4. Tarcevas, which is also an EGFR inhibitor, is currently indicated for the treatment of patients with advanced or metastatic non-small cell lung cancer.

Targeted dosing is also a component of PHC. Examples include irinotecan, a chemotherapeutic drug for colorectal cancer and warfarin. Dosing could be guided by knowledge of CYP2C9 and VKORC1 genotypes, and 6-mercaptopurine (6-MP), a chemotherapeutic drug used in acute lymphoblastic leukemia where dosing should be based upon the thiopurine methyltransferase (TPMT) genotype of the patient.

Much of the inter-individual variability in drug response is attributable to the presence of single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes and drug transporters. The pharmacogenetics of these drug-metabolizing enzymes in the Asian populations is different from those in the Caucasian and African populations. Conceptually dose requirements of certain drugs may not be optimal for Asian populations.
Companion diagnostics
Recent therapeutic advances are based on agents that specifically target the products of the genes mutated in cancer cells. Development of companion diagnostics tests for these agents can simplify the drug discovery process, make clinical trials more efficient and informative, and be used to individualize the therapy of cancer patients.

Three requirements for effective companion diagnostic tests are:
1. Availability of patient sample material suitable for testing,
2. A set of mutations that are predictive of a therapeutic response, and
3. An accurate and cost-effective technique for identifying mutations.

Current methods used in companion diagnostics include: Immunohistochemistry to detect target protein expression, Cytogenetics to detect translocations and amplifications in mitotic cells, Fluorescent in situ hybridization (FISH) to detect translocations and amplifications, quantitative PCR (qPCR) to detect gene translocations or amplifications, DNA sequencing to sequence the entire PCR product, indirect mutation analysis methods, which include single-stranded conformation analysis, high-performance liquid chromatography, denaturing gradient gel electrophoresis or mismatch cleavage, mutation-specific assays which are hybridization-based methods and include allele specific amplification, single base extension, oligonucleotide ligation, and microarrays with labeled DNA or RNA from clinical specimens which are hybridized to thousands of genes.

Method selection for companion diagnostics depends on type of mutations to be detected, type of samples and cost effectiveness. Direct or indirect sequencing methods are for target gene with multiple mutations. Mutation-specific method is for identification of predefined mutations. FISH or qPCR is for increased copy number of the target gene, direct sequencing is for primary or micro-dissected tumor. Mutation-specific assays or indirect mutation analysis methods are for clinical samples containing non-neoplastic cells. Cost effectiveness is also an issue, although companion diagnostic testing will generally constitute only a small fraction of the total cost of treatment with a mutation targeted agent.

Companion diagnostics are now an essential component of the management of CML patients. The Gleevac test is a companion diagnostic test used to identify specific gastrointestinal stromal tumors (GIST) and chronic myeloid leukemia (CML). After the test confirms a positive result, the drug is given to the patient. Gleevec has provided the prototypical example of the principle that a targeted therapy should be gene specific rather than disease specific.

Although Gleevec is the prototype for targeted therapy with small molecules, Herceptin is actually the first clinically approved drug directed against a mutated target. Herceptin is a humanized monoclonal antibody (mAb) generated from a mouse mAb reactive with HER-2. The agent was developed after it was recognized that HER-2 gene amplification was both a prognostic marker for breast cancer and a target for therapy. Approximately 25% of breast cancer patients harbor HER-2 amplification and this is associated with poor prognosis. With the use of Herceptin and the availability of companion diagnostic tests, this genetic alteration has allowed certain patients with HER-2 amplification to have better prognoses than patients without amplification. For measuring amplification of HER-2, the US FDA has approved both immunohistochemistry (IHC) and in situ hybridization (ISH)-based tests to determine the status of HER-2.

Herceptin is a model for the importance of pharmacogenomics. It was not initially developed using genetic testing but it has come to achieve exciting survival benefits via the utilization of genetic testing. Herceptin is effective in the 20-30% of breast cancer patients who over-express HER2/neu, a protein that makes breast cancer cells grow quickly. Herceptin has high efficacy when patients are screened for the presence of HER2/neu. In April 2007, the European Union approved for Herceptin in combination with hormonal therapy (aromatase inhibitor) for the treatment of patients with advanced breast cancer that is both HER2-positive and hormone receptor-positive. This is the first combination of targeted therapies to be approved for the treatment of breast cancer.
Challenges
Companion diagnostics development has many challenges. Examples include the reluctance of drug companies to restrict the use of their drugs through biomarker tests, uncertain regulatory environment, reimbursement for diagnostic products and the difficulties of developing companion diagnostics, especially for EGFR-targeted drugs The approval of Herceptin, requiring the co-development of a test for HER2/NEU to qualify patients for treatment, has inspired many companies to try similar co-development strategies.

Better communication between diagnostic companies and the pharmaceutical industry is seen as a key to accelerate development of pharmacogenomics. However, technical problems in companion diagnostics development are plenty. There is technical issue of IHC relating to the quality, reproducibility and accuracy of the IHC technique. Specimen procurement and specimen analysis also contribute to assay result variability.

Thus, the following give rise to assay variability:
1. Specimen pretreatment in the operating room,
2. Tissue processing including fixation conditions,
3. Slide preparation including antigen retrieval,
4. IHC reagents, including antibody validation, antibody incubation times and temperatures,
5. IHC analysis including detection method and automation for slide-staining, automated microscopes and data analysis software, and
6. IHC interpretation including systems and methods for standardization.

FISH also has technical issue which gives rise to many false positive and false negative assay results. Technical problems of FISH are also related to sample handling, detection and tissue heterogeneity.

Economics of stratification, seen as a key driver for PHC, represents another issue for companion diagnostics development. Therapeutic stratification creates greater patient benefits in targeted populations and may create commercial incentives. The initial commercial pushback on segmenting the market is changing. The regulatory world is leading the change. The need for companion diagnostic in the drug label for patient selection is well recognized.

However, the process aligning regulatory filings of new drugs on the one hand and companion diagnostics on the other hand is not straightforward. Rarely are the biomarker gene and therapy linked at an early stage. Biomarkers for the diagnostics are typically discovered in a Phase II clinical trial. The goal is to determine why there are responders. Historically, pharmaceutical and diagnostics companies are trying to put together an assay to be validated in Phase III, from information collected only a short time before, in Phase II. Ideally, the biology of particular targeted therapies and their associated patterns of disease are understood in very early development and will be reviewed through the entire drug development process.

Patients enrolled in Phase I trials will have blood drawn and tissue samples taken, and putative biomarkers evaluated. As the drug moves through Phase II, those markers will be refined. A decision will have to be made by the time Phase III begins regarding whether or not to use those biomarkers to select patients for enrollment in the definitive trial.

In summary, companion diagnostic development will gradually move toward the earlier phases of the drug discovery process. The FDA is also trying to help by providing general guidelines. On new agents, however, there are few agreed-upon guidelines for performing companion diagnostic studies because the field of companion diagnostics is so new. Workflows for drug and companion diagnostic assay development are also highly complex. The challenge is to integrate across the pharmaceutical and diagnostic activity chains.

Opportunities for companion diagnostics
To date, there are many drugs (receptors and signaling pathways) that have entered clinical trials. The percentage of patients responding to the new inhibitors is often below 30%. A wide variety of drugs in late preclinical and early clinical development is currently being targeted to disease-specific gene and protein defects that will require co-approval of diagnostic and therapeutic products by regulatory agencies.

Conclusion
The search for predictive biomarkers should begin during pre-clinical research. Ideally, predictive marker candidates should have been identified by the time early clinical development starts. Predictive markers can help to focus clinical development leading to more efficient drug development. Co-development of appropriate diagnostic test is critical.

Clinical trials need to be designed and powered to also answer diagnostic questions. Validation, standardization and quality assurance of diagnostic assays and sample generation/handling is vital. Finally, cooperation of all stakeholders – patients, academic institutions, regulators, pharmaceutical and diagnostic companies.