3.1.1. Task 1: Demonstrate the sensitivity of quantitative SRM-MS assays in detection of target biomarkers. The initial detection of the biomarkers will focus on human prostate tissue. The Agency will search for the same markers in blood and urine only if they are successfully detected in the prostate cancer tissue or the normal prostate tissue. A) Demonstrate the ability to quantitatively measure approximately 50 prostate cancer biomarkers at a concentration as low as 1-50 ng/ml first, in human prostate and prostate cancer tissue and then, only for detectable biomarkers also in plasma and urine using LC-SRM-MS platform using: 1. Triple quadrupole mass spectrometer with a novel dual ion-funnel interface 2. Tandem IgY14-SuperMix immunoaffinity depletion. B) Comparison of the limit of detection (LOD), limit of quantification (LOQ) and absolute concentration estimates between SRM and commercial ELISA for 5 selected markers. C) Evaluation of the multiplex power of SRM assays. D) Improvement of the limit of detection of the biomarkers to a range of 100- 1000 pg/ml ?in human plasma and urine through the application of technologies, but are not limited to, as listed below: 1. SPIN (sub-ambient pressure ionization with nanoelectrospray) source. 2. Multi-ESI (electrospray ionization) emitters. 3. Synchronized ion-funnel trap for trap and release ions to increase sensitivity. 4. Penta-quadrupoles for further enhance specificity of the assay. E) Development of alternative approach (a backup plan) for multiplex detection of biomarkers based on affinity enrichment of specific peptides coupled with mass spectrometry. Overall, the approach would be based on antibody capture of individual tryptic peptides from a digest of the target tissue (e.g., prostate and prostate cancer and plasma from a case and control). Antipeptide antibodies will be developed against a selected signature of tryptic peptides of a chosen biomarker. After the tryptic digestion of the target tissue and addition of known amounts of stable isotope?labeled calibrator peptide, both added and sample-derived versions will be enriched, and the relative amounts of the biomarker will be measured by SRM-MS. Task 1 Requirement: 1. Identification of biomarkers detectable at concentrations as low as 100-1000 pg/ml in prostate cancer tissue and in normal prostatic tissue. 2. Based on the tissue detectable biomarker, detection of candidate prostate cancer biomarkers at concentrations of 100 ? 1000 pg/ml in human plasma and urine. 3. Demonstration of multiplex ability for at least 25 distinct biomarkers in the same run, using either platform, and achieving a sensitivity of at least 1 ng/ml. 4. Development of an alternative approach (a backup approach) based on affinity enrichment of specific peptides coupled with SRM-MS to detect biomarkers at concentration of 100-1000pg/m. 3.1.2. Task 2: Develop isoform-specific SRM assays for quantifying proteolytic processing products (e.g. multiple products of PSA), alternative splicing isoforms and gene fusion products in human urine or plasma. Task 2 Requirement: Demonstrated ability to use SRM to quantitatively distinguish between two distinct isoforms in at least two of the three categories (proteolytic processing isoforms, alternative splicing isoforms, gene fusion isoforms). 3.1.3. Task 3: Quantitative detection of approximately 50 selected prostate cancer markers in blood and urine samples using LC-SRM-MS. The initial verification will focus on cancer biomarkers which were detected in the prostate cancer (tissue). Validation of the selected biomarkers should be carried in a case control study in two steps;the first should be a pre-Validation (~200 samples). Samples will be provided through collaboration with the EDRN GU Collaborative group and if necessary supported by EDRN statisticians from the EDRN Data Management and Coordination Center (DMCC). In addition, it is anticipated that at this stage of the work there will be additional EDRN Biomarkers Reference Laboratory, which will be trained to reproduce the PNNL protocol for the selected markers, which will independently analyze least 10%-15% of selected specimens. The EDRN laboratory will not be funded by this contract. This step will allow the NCI to evaluate whether the sensitivity and specificity of the best panel of biomarkers. Task 3 Requirement: pre-Validation of a panel of biomarkers and establishment of their concentration in blood and urine of cases and controls. 3.1.4. Task 4: Clinical validation of the biomarkers (in collaboration with the EDRN). Validation study based on the best panel/s (for subtypes of the cancer) using (~1000 samples. Samples will be provided through collaboration with the EDRN GU Collaborative group and if necessary supported by EDRN statisticians from the Data Management and Coordination Center (DMCC). In addition, it is anticipated that at this stage of the work there will be additional EDRN Biomarkers Reference Laboratory, which will be trained to reproduce the PNNL protocol for the selected markers, which will independently analyze least 10%-15% of selected specimens. The EDRN laboratory will not be funded by this contract. Task 4 Requirements: Clinical validation of quantitatively detectable panel or panels of biomarkers for early and diagnosis of prostate cancer/s in collaboration with EDRN and the EDRN DMCC. 3.2. Phase 2: Development of additional multiplex detection of biomarkers by SRM-MS for early detection and diagnosis of cancers of the pancreas and breast and validation of their clinical utility. The list of required tasks for phase 2 is similar to that for prostate cancer. Phase 2 Tasks'Requirements: Analytical and Clinical validations of quantitatively detectable panel or panels of biomarkers for early detection and diagnosis of cancers of the pancreas and breast in collaboration with the NCI, the EDRN investigators and the EDRN DMCC.