Measurement of the serum level of prostate specific antigen (PSA) and a well-established role in monitoring patients with known prostate cancer. Widespread use of PSA as a screen for prostate cancer, while controversial, has resulted in increased and earlier cancer detection. Widespread use of PSA as a screen for prostate cancer, while controversial, has resulted in increased and earlier cancer detection. Currently, there is an urgent need for a test that will distinguish benefit from treatment and which patients can avoid unnecessary aggressive therapies. Promising new developments in basic research on the glycobiology of cancer have provided a plausible mechanism by which specific glycan alterations to adhesion molecules on the surface of prostate cancer cells modulate the aggressiveness of the cancer. Furthermore, the presence of these glycan structures on PSA from a human prostate cancer cell line has been reported. The goal of this application is the development and refinement of a glycoform assay for PSA that is capable of high throughput analysis using serum from individual patients. Successful development of this assay will accomplishment of three specific aims: 1) determination of the patient-to- patient variation in glycoform content of PSA, 2) draw preliminary correlations between tumor marker glycosylation pattern and patient clinical status, and 3) elucidation of the initial structural detail of the PSA glycan structures identified in specific aims 1 and 2.
The specific aims will be accomplished by applying a novel capillary electroporesis (CE) approach to detect PSA glycovariants using nanogram or subnanogram amounts of glycoprotein. In contrast to the traditional resolution based approach to glycoform analysis, this pilot project will use capillary electrophoresis peak mode mobility determinations to identify PSA molecules with variant glycans. CE analysis will be performed so that the component glycoforms are not resolved but so that each individual contributes to the mobility of the peak mode in proportion to its amount and mobility. Replicate mobility determinations allows statistical analysis. The identification of a statistically significant difference in mobility indicates an underlying structural difference. Reduction of that mobility difference by specific recombinant glycosidases will provide evidence of the structural basis of statistically confirmed mobility differences. The strength of this approach over more standard methodologies is that there is no theoretical limit to optimization of mobility value precision. The degree of structural detail that can be resolved between different PSA glycoforms is dependent on the precision of mobility value determinations. The parameters important to mobility value precision that will be optimized include:: capillary wall equilibration, sample matrix control, buffer stability, ion depletion, sample evaporation, temporal, temporal resolution, sample stability, PSA- serpin complexation, temperature control, sample injection, same plug length, and sample concentration. The optimized assay will be used to analyze a prospective series of serum PSA specimens for significant changes in sialylation and/or N-glycan branching. These findings will be correlated with the patient's clinical status.
