The Gentel researchers identified candidate biomarkers through the literature and sourced high-performance antibodies, eliminating those that showed cross reactivity. The antibodies can then be quality controlled for reproducibility. Because of the small volumes required and the ability to subdivide the array into many individual sample wells, it is possible to generate a large body of data from a single slide. Many samples can be leveraged allowing the characterization of disease-related proteins and the response of regulatory proteins to therapeutic interventions.
Charles Birse, Ph.D., lung biomarker group leader at Celera, reported that the currently available selection of lung cancer biomarkers is inadequate, represented by carcinoembryonic antigen, cytokeratin fragment 19, and neuron specific enolase. Not one of these markers provides the sensitivity and specificity required of a truly accurate diagnostic tool.
This shortage has spurred the company’s search for better serum markers. The huge range of concentrations of different proteins in serum makes the search difficult and requires that the 97% most concentrated proteins such as albumin and immunoglobulin be removed prior to evaluation. But albumin is notorious for binding to other proteins, and its elimination may inadvertently remove possible biomarkers.
So Celera has an extensive program of discovery and validation, screening for shed proteins as well as cell-surface proteins in tissue specimens, cell lines, and serum samples. Using mass spec as a screening method, Celera researchers have identified over 500 possible disease-associated proteins. These investigations narrowed down the candidates that are expressed in the serum of patients, and finally yielded 27 targets as promising lung cancer biomarkers. The Celera researchers are also examining tissues, and Dr. Birse cautions that serum may not be appropriate for the validation of all biomarkers.
Pancreatic Cancer Biomarkers
Pancreatic cancer results in 30,000 deaths per year in the U.S., almost equal to the number of new cases. The five year survival rate is less than 5%, reported Ru Chen, Ph.D., a research scientist in the department of medicine at the University of Washington. Early detection could significantly improve the prognosis of this deadly disease. When pancreatic cancer is detected in the early stage (when the tumor is smaller than 2 cm), the five-year survival rate is about 46%.
Dr. Chen’s screening program is similar to many other cancer biomarker searches described in this article, including evaluation of pancreatic tissue, pancreatic secretions, and serum profiles from patients and matched controls. Proteins from normal and cancerous tissue are differentially labeled, broken into peptides, and affinity separated. This allows protein classification according to whether they are increased, decreased, or remain at the same level in the normal as compared to the malignant tissue.
Dr. Chen and her colleagues identified 500 pancreatic proteins that were unchanged, 95 that were upregulated, and 61 that were downregulated. They then designed a microarray platform in which paraffin specimens from a variety of normal and diseased tissue were mounted on an assembly that scanned them, using standard pathology staining, immunofluorescence staining, and immunohistochemistry.