Independent validation of candidate breast cancer serum biomarkers identified by mass spectrometry

Background: We previously selected a panel of 3 breast cancer biomarkers (BC1, BC2, and BC3) from serum samples collected at a single hospital based on their collective contribution to the optimal separation of breast cancer patients and noncancer controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The identities and general applicability of these markers, however, were unknown. In this study, we performed protein expression profiling on samples obtained from a second hospital, included a greater number of ductal carcinoma in situ (DCIS) cases, and performed purification and identification of the 2 confirmed markers. Methods: Using a case-control study design, we performed protein expression profiling on serum samples from the National Cancer Institute (Milan, Italy). The validation sample cohort consisted of 61 women with locally invasive breast cancer, 32 with DCIS, 37 with various benign breast diseases (including 13 atypical), and 46 age-matched apparently healthy women (age range, 44-68 years). Validated biomarkers were purified and identified with serial chromatography, 1-dimensional gel electrophoresis, in-gel ASP-N digestion, peptide mass fingerprinting, and tandem mass peptide sequencing. Results: The BC3 and BC2 expression patterns in this sample set were consistent with the first study sample set. BC3 and BC2 were identified to be complement component C3a _desArg and a C-terminal-truncated form of C3a_desArg, respectively. Conclusions: Evaluation of biomarkers in independent sample sets can help determine the broader utility of candidate markers, and protein identification permits understanding of their molecular basis. C3a _desArg appears to lack specificity among patients with benign diseases, limiting its utility as a stand-alone tumor marker, but it may still be useful in a multimarker panel for early detection of breast cancer.