Candidate Serum Biomarkers for Prostate Adenocarcinoma Identified by mRNA Differences in Prostate Tissue and Verified with Protein Measurements in Tissue and Blood E.W. Klee, O.P. Bondar, M.K. Goodmanson, R.B. Dyer, S. Erdogan, E.J. Bergstralh, H.R. Bergen III, T.J. Sebo, and G.G. Klee March © Copyright 2012 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Background  Improved tests are needed for detection and management of prostate cancer  Hypothesized that  Differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer  Blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation

© Copyright 2009 by the American Association for Clinical Chemistry Methods  Identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue  Confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera  Analyzed tissue extracts with tandem mass spectrometry (MS/MS)  Measured blood concentrations using:  Immunoassays  MS/MS of trypsin-digested, immunoextracted peptides

© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Processes used to identify novel candidate biomarkers and measure proteins in tissue and blood. LCM, laser-capture microdissection. Study Design

© Copyright 2009 by the American Association for Clinical Chemistry Tissue Protein Evaluation  Immunohistochemistry Samples FFPE Tissue (N=20 pairs)  10 samples, Gleason 5 or 6  10 samples, Gleason 8 or 9  Tumor samples had matched adjacent benign tissue  QTOF MS Samples Frozen PCa Tissue (N=2 pairs )  Gleason 9, T3aN0+ tumor  Gleason 7, T3aN0-

© Copyright 2009 by the American Association for Clinical Chemistry MS/MS Analysis of Tissue Extracts  Samples electrophoresed on SDS-PAGE gels and proteins digested in-situ.  Peptide identification by mass spectrometry using LTQ Orbitrap Hybrid Mass Spectrometer  Results: Identified 5 candidate markers, 3 with increased concentration in tumor samples:  ASPN  PGLS  RPL22L1

© Copyright 2009 by the American Association for Clinical Chemistry Table 1. Immunohistochemistry (13 markers). C, prostate cancer tissue; N, adjacent normal tissue; 0, no staining; 1, weak staining; 2, moderate staining; 3, heavy staining. P-values from one-tail sign test.

© Copyright 2009 by the American Association for Clinical Chemistry Serum Protein Assays  Immunoassays where available  MS/MS  Extract Glycoproteins with Lectins  Trypsin Digest and Immuno extract peptides  Samples: Frozen aliquots of serum and EDTA  Cases: 50 men with advanced prostate cancer  Controls: 26 men with recent prostate biopsies −13 PCa cancer-free −13 with low-grade PCa

© Copyright 2009 by the American Association for Clinical Chemistry Immunoassay Results ** Significant Rank Sum Test when comparing advanced prostate cancer sera and control sera (P < 0.05) BiomarkerVendorResults APO-C1AssayPro8/50 ** C4AAssay Design4/50 CCL19R&D Systems2/50 COMPImmuno-Biology0/50 CXCL11R&D Systems7/50 ** CCXL14R&D Systems2/50 CXCL9R&D Systems13/50 ** Factor VAniara8/50 **

© Copyright 2009 by the American Association for Clinical Chemistry MS/MS Serum Assay Method (Low Abundance)  Synthesized 49 peptides representing 25 biomarkers  Immunized rabbits for peptide antibodies  Extracted target peptides  First depleted high abundance proteins  Then digested blood to make peptides  Then used rabbit antibodies to extract peptides  Measured peptides using Mass Spectrometry (MS/MS)

© Copyright 2009 by the American Association for Clinical Chemistry

MS/MS Assay Results ** Significant Rank Sum Test (P < 0.05) for separating advanced cancer from other groups BiomarkerMS/MS Affinity ColResults APOFA2/50 ASPNB26/50 ** C1orf64A0/50 CDH7A11/50 COL2A1A5/50 Factor VB23/50 ** PCSK6B,C8/50, 4/50 ** PGLSC7/50 RPL22L1C11/50

© Copyright 2009 by the American Association for Clinical Chemistry Plots of sequential measurements for A) ELISAs and B) MS/MS set A

© Copyright 2009 by the American Association for Clinical Chemistry Plots of sequential measurements for MS/MS bead sets B and C

© Copyright 2009 by the American Association for Clinical Chemistry Conclusions  7 novel biomarkers were identified as statistically significant in discriminating protein concentrations in blood from men with advanced prostate cancer compared to controls:  APOC1, ASPN, COMP, CXCL11, CXCL9, F5, and PCSK6  4 additional novel biomarkers were identified with increased serum concentration values in at least 10% of advanced prostate cancer samples compared to controls:  CDH7, COL2A1, PGLS, and RPL22L1

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