Predicting Drug Efficacy
France Carrier, Ph.D., associate professor of radiation oncology, Marlene and Stewart Greenebaum Cancer Center, University of Maryland, envisions an opportunity to use PCR as the basis for a prognostic tool to predict a patient’s responsiveness to anticancer drugs that inhibit DNA synthesis and, thereby, cell replication. Dr. Carrier discussed his research at CHI’s recent “Biomarker Assays Development” conference.
Specifically, Dr. Carrier has developed a patent-pending technique to measure breaks in genomic DNA that result from anticancer drugs that interfere with DNA synthesis, such as topoisomerase inhibitors. Topoisomerases are enzymes that facilitate DNA replication by creating and then religating nicks in the DNA to alleviate torsional stress as the strands unwind. They preferentially cut DNA at particular sites. Treatment with a topoisomerase inhibitor that compromises the enzyme’s function will result in more DNA breaks, which prevent DNA polymerase from reading and copying the affected strand.
Dr. Carrier points to combining RT-PCR with conventional Stop-PCR as the enabling technological advance for this predictive assay strategy. It provided the sensitivity needed to be able to quantify the level of DNA damage produced by typical therapeutic doses of topoisomerase inhibitors.
Using RT-PCR to amplify genomic DNA from cells exposed to a topoisomerase inhibitor, Dr. Carrier can assess the efficiency of the drug. The more efficient it is, the less amplification there will be of the DNA sequences it targets. Based on the predictive power of this inverse correlation, it may be possible to evaluate in vitro the potential effectiveness of a drug that interferes with DNA synthesis, enabling a more personalized approach to the selection of anticancer drugs and combination therapies.
Dr. Carrier has used the assay to study peripheral blood mononuclear cells (PBMCs) isolated from the blood of treated patients and has demonstrated its utility at therapeutic drug doses. The next step toward developing a clinically applicable predictive tool will be to conduct animal studies designed to compare conventional measures of drug efficacy (tumor shrinkage) with PCR-based quantification of DNA breaks in cells derived from biopsy samples and PBMCs.
Powerful Prognostic Tools
Dave Hoon, Ph.D., director of molecular oncology at the John Wayne Cancer Institute, will also speak at the “Molecular Medicine TriConference”. He will present the results of two Phase III multicenter randomized clinical trials that demonstrate that circulating tumor cells (CTCs) are an independent prognostic factor for disease-free and overall survival in patients with stage 3 and 4 melanoma. Dr. Hoon and colleagues developed a multimarker real-time quantitative reverse transcriptase assay designed to detect and quantify CTCs in the blood of patients with melanoma.
The RT-qPCR assay measures the levels of four melanoma-associated CTC mRNAs: MART-1, GalNAc-T, PAX3, and MAGE-A3. In Phase II trials, the presence of and increasing numbers of these biomarkers, measured serially in patients undergoing combination immunotherapy and chemotherapy, correlated with disease progression, treatment response, and overall and disease-free survival.
“This is a direct blood assay” performed on mRNA isolated from whole cells in patients’ blood samples, says Dr. Hoon. Its advantages include being real-time, probe-based, highly specific, and optimized for the biomarkers selected. The rationale behind designing a multimarker assay was based on the heterogeneity of tumors and CTCs, making it unlikely that a single marker would be predictive, particularly in aggressive disease with different sites of origin.
“Tumor cells that express these markers are more aggressive. It is not the number of CTCs that is prognostic; rather it is the content of the cells,” Dr. Hoon says. The goal is to develop this assay as a blood test for use in predicting which treatment is likely to be most effective following surgery, for determining disease spread, and for monitoring the effectiveness of adjuvant therapy.