Autoantibodies are present in many conditions such as cancer, autoimmune disorders, and transplant rejection. This affords an opportunity to develop diagnostic and prognostic assays, as well as to monitor disease progression and treatment effectiveness. There are several challenges, however.
“Only a fraction of patients develop autoantibodies to a given protein,” indicates Lisa Freeman-Cook, Ph.D., senior manager, R&D, Life Technologies.
“Thus, identification of multiple biomarkers in patient samples is important. We have developed functional protein microarrays (ProtoArrays®) that may be used to screen complex biological mixtures such as serum to detect autoantibodies directed against any of the 9,000 proteins on the microarray.”
The quality of the protein on the array is an important factor. “The ProtoArray contains proteins that are expressed in insect cells and purified under native conditions. These more closely recapitulate appropriate post-translational modifications and allow a more native protein conformation.”
Dr. Freeman-Cook says the utility of the ProtoArray for identifying biomarkers was demonstrated recently by investigators looking for novel autoantigens in rheumatoid arthritis, prostate cancer, and Alzheimer disease. But the next step is validation of identified biomarkers.
Validation studies typically assess candidate markers against an increasing number of independent patient samples. Thus throughput becomes important along with accurate measurements of sufficient sensitivity, dynamic range, and reproducibility to differentiate responders from nonresponders.”
Since the ProtoArray was not designed for high throughput, the company decided to employ Luminex xMAP technology.
“We developed the ProtoPlex™ Immune Response Assay featuring Luminex xMAP technology. No one before has paired these two technologies. The idea is to first utilize the ProtoArray to discover biomarkers of interest. Then, Luminex technology can be used for validation.”
The Luminex xMAP technology employs spectrally distinguishable fluorescent beads to which antigens of interest are coupled. “Cross-reactivity issues have not been a problem. As a result, we are able to accomplish higher levels of multiplexing (>80-plex) per assay with any antigen. We can process up to 500 samples per day with very low CV’s (<10%) and using only a few micoliters of patient serum.”
The company markets ProtoArray products and provides ProtoArray and ProtoPlex custom services. “We feel that combining these two technologies now not only supports biomarker discovery for a wide variety of diseases, but also allows validation with the familiar Luminex technology,” Dr. Freeman-Cook remarks.
Most biomarker analyses are generated by excising tumor samples from patients. A noninvasive means, however, is assessing biomarkers in body fluids, such as urine. One drawback to the use of body fluids is the low concentration of analytes in the sample, notes Anthony P. Shuber, CTO, Predictive Biosciences.
“Biological fluid-based assays can be very powerful. But the presence of biomarkers at very low levels has been a hindrance. To date, there have been no biological fluid-based diagnostic tests that have sensitivity comparable to tissue-based assays.”
According to Shuber, Predictive Biosciences is utilizing next-generation sequencing to detect biomarker mutations.
“We have been focusing on mutations in the fibroblast growth factor receptor three (FGFR3) gene, which are present in 30–50% of bladder cancer patients. The presence of FGFR3 mutations correlates with a lower rate of bladder cancer recurrence. Next-gen sequencing as a diagnostic platform has a great deal of appeal, because it allows sensitivity greater than qPCR.”
The company collects a small sample of urine from patients and then subjects it to next-generation sequencing to determine the presence of mutations.
“No one really knows what percentage of mutated DNA exists in body fluid, although small amounts of 1% have been detected. We have developed a new urine-based assay that uses ultra-deep next-generation sequencing on amplicon-generated FGFR3. We found that this assay reliably detects a mutation when it is present in as little as .02% of the total amount of DNA in urine.”
Shuber says it is unlikely that every clinical lab will make the investment in a next-generation sequencing apparatus. “Where this is likely to go is via samples obtained at a point of care and sent to a lab that specializes in next-gen sequencing. Clearly, there is a place in the clinical diagnostic laboratory for next-gen sequencing. We are certainly moving in that direction.”
More than 250,000 women are diagnosed in the U.S. with breast cancer every year. A majority of patients (~170,000) are estrogen receptor positive (ER+), and subsequently receive a regimen that includes anti-estrogen therapeutic agents such as tamoxifen or aromatase inhibitors.