Epigenetic qPCR requires only about 2 mL of blood, and samples can be frozen and shipped—giving it a huge logistical advantage for large, multicenter clinical trials. “For about half of the top 20 pharma, we provide this as an immune-monitoring service for clinical trial samples,” Dr. Hoffmueller said.
Molecular signatures are sought out to distinguish among seemingly similar maladies, their discovery performed by looking at samples from patients with known infirmities and outcomes and comparing what can be garnered with data about the patients themselves. Nothing—from lifestyle choices to birth order to ancestral genetics and epigenetics—is off the table. It can take enormous datasets, sophisticated algorithms, and vast computational resources to tease out correlations that may prove useful in the clinic.
Take breast cancer, a disease now considered to be a collection of many complexes of symptoms and signatures—the dominant ones being labeled Luminal A, Luminal B, Her2, and Basal—that suggest different etiologies and offer different prognoses. Yet even these labels are lately considered too simplistic for understanding and managing a woman’s cancer.
Studies published in the past year have looked at somatic mutations, gene copy number aberrations, gene expression abnormalities, protein and miRNA expression, and DNA methylation, coming up with a list of significantly mutated genes—hot spots—in different categories of breast cancers. Targeting these will inevitably be the focus of much coming research.
“We’ve been taking these large trials and profiling these on a variety of array or sequence platforms. We think we’ll get not only prognostic drivers (which are only good if you know what to do with them) but also predictive markers for taxanes and monoclonal antibodies and tamoxifen and aromatase inhibitors,” explained Brian Leyland-Jones, Ph.D., director of Edith Sanford Breast Cancer Research. “We will end up with 20–40 different diseases, maybe more.”
Edith Sanford Breast Cancer Research is undertaking a pilot study in collaboration with The Scripps Research Institute, using a variety of tests on 25 patients to see how the information they provide complements each other, the overall flow, and the time required to get and compile results.
Laser-captured tumor samples will be subjected to low passage whole-genome, exome, and RNA sequencing (with targeted resequencing done in parallel), and reverse-phase protein and phosphorylation arrays, with circulating nucleic acids and circulating tumor cells being queried as well. “After that we hope to do a 100- or 150-patient trial when we have some idea of the best techniques,” he said.
Dr. Leyland-Jones predicted that ultimately most tumors will be found to have multiple drivers, with most patients receiving a combination of two, three, or perhaps four different targeted therapies.
Reduce to Practice
Once biomarkers that may have an impact on therapy are discovered, it is not always routine to get them into clinical practice. Leaving regulatory and financial, intellectual property and cultural issues aside, developing a diagnostic based on a biomarker often requires expertise or patience that its discoverer may not possess.
Andrew Gribben is a clinical assay and development scientist at Randox Laboratories, based in Northern Ireland, U.K. The company utilizes academic and industrial collaborators together with in-house discovery platforms to identify biomarkers that are augmented or diminished in a particular pathology relative to appropriate control populations. Biomarkers can be developed to be run individually or combined into panels of immunoassays on its multiplex biochip array technology.
“Individual biomarkers alone may not be specific enough to support a diagnostic claim,” he noted.
Specificity can also be gained—or lost—by the affinity of reagents in an assay. The diagnostic potential of Heart-type fatty acid binding protein (H-FABP) abundantly expressed in human myocardial cells was recognized by Jan Glatz of Maastricht University, The Netherlands, back in 1988. Levels rise quickly within 30 minutes after a myocardial infarction, peaking at 6–8 hours and return to normal within 24–30 hours. Yet at the time it was not known that H-FABP was a member of a multiprotein family, with which the polyclonal antibodies being used in development of an assay were cross-reacting, Gribben related.
Randox developed monoclonal antibodies specific to H-FABP, funded trials investigating its use alone, and multiplexed with cardiac biomarker assays, and, more than 30 years after the biomarker was identified, in 2011, released a validated assay for H-FABP as a biomarker for early detection of acute myocardial infarction.