Application of a Clinical Whole-Transcriptome Assay for Staging and Prognosis of Prostate Cancer Diagnosed in Needle Core Biopsy Specimens Beatrice S.

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Application of a Clinical Whole-Transcriptome Assay for Staging and Prognosis of Prostate Cancer Diagnosed in Needle Core Biopsy Specimens Beatrice S. Knudsen, Hyung L. Kim, Nicholas Erho, Heesun Shin, Mohammed Alshalalfa, Lucia L.C. Lam, Imelda Tenggara, Karen Chadwich, Theo Van Der Kwast, Neil Fleshner, Elai Davicioni, Peter R. Carroll, Matthew R. Cooperberg, June M. Chan, Jeffry P. Simko The Journal of Molecular Diagnostics Volume 18, Issue 3, Pages 395-406 (May 2016) DOI: 10.1016/j.jmoldx.2015.12.006 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Quality control (QC) characteristics of biopsy and radical prostatectomy (RP) samples. A: Total RNA yield in RP samples is greater than in biopsy samples. B: An equal amount of extracted RNA was converted to cDNA, yielding similar amounts of cDNA in biopsy specimens and RPs. C: The three QC parameters (RNA yield, cDNA yield, and microarray probe signal intensity) are not affected by tumor content in the punch of the biopsy core samples. n = 77 (A, RP samples); n = 81 (A, biopsy samples). The Journal of Molecular Diagnostics 2016 18, 395-406DOI: (10.1016/j.jmoldx.2015.12.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Sources of variance in radical prostatectomies (RPs) and biopsy (BX) samples. A: Hierarchical clustering of samples from tumor and benign tissues from BX and RP origins. The two main clusters are generated on the basis of the origin (BX versus RP), containing both benign and tumor samples. Additional clusters are obtained on the basis of the tissue type (tumor versus benign). B and C: Principal component analysis (PCA) shows that sample origin (BX versus RP) contributes the biggest variation and that tissue type (tumor versus benign) contributes less to the total variation. The Journal of Molecular Diagnostics 2016 18, 395-406DOI: (10.1016/j.jmoldx.2015.12.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Expression analysis between radical prostatectomies (RPs) and biopsy (BX). A: High concordance of gene expression levels between BX and RP (r = 0.96). B: Differential expression analysis using median fold difference (MFD) shows that the MFD between RP tumor and RP benign contralateral, and BX tumor and BX benign contralateral, is consistent in terms of directionality. C: The genes differentially expressed between tumor and benign contralateral in BX and RP are overlapping with genes differentially expressed between tumor and benign contralateral in external public data sets. Statistical significance was assessed via bootstrapping (P < 0.05). The Journal of Molecular Diagnostics 2016 18, 395-406DOI: (10.1016/j.jmoldx.2015.12.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Robustness of the Decipher assay between biopsy (BX) and radical prostatectomies (RPs). A: Correlation of Decipher scores from BX and RP samples (r = 0.7). B: Correlation of scores for the Penney et al20 signature from BX and RP samples (r = 0.65). The blue dashed line represents the line of best fit, whereas the dotted blue lines represent the 95% CI. C: Concordance of Decipher category between cancers in RP and BX (Fisher's exact test P = 0.08). Int., intermediate. The Journal of Molecular Diagnostics 2016 18, 395-406DOI: (10.1016/j.jmoldx.2015.12.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 A: The expression of Decipher features is consistent across tissue types between radical prostatectomies (RPs) and biopsy (BX). Most features are overexpressed in tumor compared with benign in RP and BX. A group of features (LASP1, CAMK2N1, and NFIB) expressed at levels similar to the tumor specimens in benign adjacent to tumor (benign close to tumor) but not in benign contralateral to tumor (benign away from tumor). B: The concordance of prostate cancer molecular subtypes between BX and RP cancer samples. The Journal of Molecular Diagnostics 2016 18, 395-406DOI: (10.1016/j.jmoldx.2015.12.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Functional analysis of the field effect signature. A: The field effect signature is distinct from prostate cancer genes differentially expressed between tumor and benign tissues (Memorial Sloan Kettering Cancer Center and German Cancer Research Center).24,25 B: Functional enrichment analysis reveals that the field effect signature is highly enriched with gene categories of RNA splicing, ubiquitin-dependent catabolism, epigenetics, and cellular transport. C: Functional interaction networks generated by STRING of 75 transcription factors whose targets are enriched in the field effect signature on the basis of a chromatin immunoprecipitation enrichment analysis tool.27 D: Density plots of Pearson's correlation between field effect signature and ERG and ETV1 genes, suggesting that field effect is independent of ERG and ETS. The Journal of Molecular Diagnostics 2016 18, 395-406DOI: (10.1016/j.jmoldx.2015.12.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions