Blood is a readily available and rich source of biomarkers that have the potential for use in diagnostics and companion diagnostics development, disease characterization and monitoring, and predictive drug response and toxicity analysis. Serum profiling using a variety of techniques to identify disease- or treatment-related proteins, antibodies, or oligonucleotides is leading to the identification of well-defined biosignatures and patterns of biomarker expression that are playing an important role in research, preclinical testing, patient stratification for clinical trials, therapeutic assessment, and increasingly in commercial diagnostic and prognostic applications and in drug selection.
At the American Association for Cancer Research annual meeting, Exiqon will present data from its diagnostic program aimed at early detection of colorectal cancer (CRC). The company applied its miRCURY LNA™ Universal RT microRNA PCR system to detect a miRNA signature in blood plasma indicative of CRC. The signature was derived from comparisons of profiles of several hundred miRNAs in about 200 patients with CRC and a comparably sized control group.
By the end of 2011, Exiqon expects to complete a validation study that will include about 5,000 patient samples. “It is hoped that this will eventually lead to a blood sample miRNA-based test that will identify patients at high risk of having early-stage (0-II) CRC with significantly greater precision than the currently used—but not very accurate—fecal occult blood test, which also suffers from poor patient adherence,” says Niels Montano Frandsen, Ph.D., product manager at Exiqon.
Patients identified as high-risk would be referred for follow-up testing such as colonoscopy.
Dr. Frandsen believes that despite the challenge of detecting miRNAs due to their small size, they “have all the hallmarks of a new class of powerful diagnostic and prognostic biomarkers of disease, drug efficacy, and toxicity.”
He notes their stability in clinical sample material such as FFPE tissue samples, blood serum and plasma, and urine. “We have found that miRNA levels in plasma are virtually unaffected by sample handling; samples can be left at room temperature for extended periods and thaw/frozen multiple times with no effect.”
Exiqon’s miRCURY system incorporates a universal cDNA synthesis reaction followed by PCR amplification with two miRNA-specific primers, which is possible through the use of LNA technology. The use of SYBR Green enables melt-curve analysis, which allows for verification of the amplification products.
The company currently offers two human miRNA panels and one mouse/rat panel, each containing 370 miRNAs on a 386-well plate, and will soon launch a second mouse/rat panel. In addition, toward mid-year, Exiqon plans to introduce its new Pick-a-Mix customized panels for which customers can specify a set of desired miRNAs and design a panel for a specific disease indication.
VeriStrat from Biodesix is an example of a serum-based test used in the clinic that can predict response to a second-line biotherapeutic treatment option. The test identifies patients who are likely or unlikely to benefit from treatment with erlotinib, an EGFR-TKI in patients with advanced non-small-cell lung cancer (NSCLC). Supporting data was generated following analysis of the BR.21 pivotal trial for erlotinib.
The VeriStrat test is available to clinicians through Biodesix’ CLIA-accredited laboratory. The test classifies serum samples from patients with NSCLC as either “good” (survival following erlotinib) or “poor” based on the results of mass spectrometry and analysis of an eight-peak protein signature. The result is available within 24–72 hours.
In addition to its in-house development and commercialization program, Biodesix collaborates on projects aimed at identifying patterns of serum protein expression that can be developed into predictive tests for use as companion diagnostics for targeted therapies, to predict and monitor treatment response, or to stratify patients for clinical drug testing. The company applies its ProTS® technology platform, based on MALDI-MS, which separates the serum proteome into 300–500 protein peaks.
The accompanying software normalizes and aligns the peaks and identifies signatures that can be used to distinguish between clinical subsets of patients. The algorithms are trained across large numbers of clinical samples and positive and negative controls; the software then applies that knowledge to define protein patterns that can differentiate good from poor responders to a drug against a background of genetic variability and variability in sample preparation and test conditions.
Paul Beresford, Ph.D., vp of business development and strategic marketing at Biodesix, anticipates broad clinical utility for VeriStrat across multiple types of cancer—including colorectal, head/neck, pancreatic, and breast—in which the test appears to detect a tumor/host interaction. The current test is designed to work with a dried serum spot that can be stored and transported at ambient temperature.
The next-generation test will require a blood sample obtained with a pinprick and spotted directly onto a collection card. Via active diffusion the serum component would separate from the red blood cells, yielding a dried serum spot that can then be punched out of the card at the laboratory and the serum extracted.
In a recent Phase II trial of first-line combination therapy with erlotinib and the oral multiple kinase inhibitor sorafenib in patients with advanced NSCLC, VeriStrat analysis was able to classify 48 of 49 serum samples with only one indeterminate result. Median overall survival among the patients identified as “VeriStrat good” (13.7 months) was significantly greater than for the patients classified as poor responders (5.6 months).