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 2012

www.clinchem.org/content/article/58/3/599.full

© Copyright 2012 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

BackgroundBackground

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

MethodsMethods 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© 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 Study DesignDesign

© Copyright 2009 by the American Association for Clinical Chemistry

Tissue Protein EvaluationTissue Protein Evaluation

Immunohistochemistry

Samples

FFPE Tissue (N=20 pairs)10 samples, Gleason 5 or 610 samples, Gleason 8 or 9Tumor samples had matched adjacent benign tissue

QTOF MS

Samples

Frozen PCa Tissue (N=2 pairs)Gleason 9, T3aN0+ tumorGleason 7, T3aN0-

© Copyright 2009 by the American Association for Clinical Chemistry

MS/MS Analysis of Tissue ExtractsMS/MS Analysis of Tissue ExtractsSamples 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© 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 AssaysSerum 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© Copyright 2009 by the American Association for Clinical Chemistry

Immunoassay ResultsImmunoassay Results

** Significant Rank Sum Test when comparing advanced prostate cancer sera and control sera (P < 0.05)

Biomarker Vendor Results

APO-C1 AssayPro 8/50 **

C4A Assay Design 4/50

CCL19 R&D Systems 2/50

COMP Immuno-Biology 0/50

CXCL11 R&D Systems 7/50 **

CCXL14 R&D Systems 2/50

CXCL9 R&D Systems 13/50 **

Factor V Aniara 8/50 **

© Copyright 2009 by the American Association for Clinical Chemistry

MS/MS Serum Assay Method (Low Abundance)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

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

MS/MS Assay ResultsMS/MS Assay Results

** Significant Rank Sum Test (P < 0.05) for separating advanced cancer from other groups

Biomarker MS/MS Affinity Col

Results

APOF A 2/50

ASPN B 26/50 **

C1orf64 A 0/50

CDH7 A 11/50

COL2A1 A 5/50

Factor V B 23/50 **

PCSK6 B,C 8/50, 4/50 **

PGLS C 7/50

RPL22L1 C 11/50

© Copyright 2009 by the American Association for Clinical Chemistry

Plots of sequential measurements for Plots of sequential measurements for A) ELISAs and B) MS/MS set A A) ELISAs and B) MS/MS set A

© Copyright 2009 by the American Association for Clinical Chemistry

Plots of sequential measurements for Plots of sequential measurements for MS/MS bead sets B and C MS/MS bead sets B and C

© Copyright 2009 by the American Association for Clinical Chemistry

ConclusionsConclusions

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

© Copyright 2009 by the American Association for Clinical Chemistry

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