title: Gene-expression analysis of gleason grade 3 tumor glands embedded in low- and high-risk prostate cancer.
abstract: The Gleason score (GS) of prostate cancer on diagnostic biopsies is an important parameter for therapeutic decision-making. Biopsy GS under-estimates the actual GS at radical prostatectomy in a significant number of patients due to samplingartifact. The aim of this study was to identify markers that are differentially expressed in Gleason grade 3 (GG3) tumor glands embedded in GS 4 + 3 = 7 and GS 3 + 3 = 6 prostate cancer using laser capture microdissection and RNA sequencing.GG3 tumor glands embedded in nine GS 3 + 3 = 6 and nine GS 4 + 3 = 7 prostate cancers were isolated by laser capture microdissection of frozen radical prostatectomy specimens. After RNA amplification and RNA sequencing, differentially expressed genes in both GG3 components were identified by a 2log fold change > 1.0 and p-value < 0.05. We applied immunohistochemistry on a tissue micro-array representing 481 radical prostatectomy samples for further validation on protein level.A total of 501 genes were up-regulated and 421 down-regulated in GG3 glands embedded in GS 4 + 3 = 7 as compared to GS 3 + 3 = 6 prostate cancer. We selected HELLS, ZIC2 and ZIC5 genes for further validation. ZIC5 mRNA was up-regulated 17 fold (p = 8.4E-07), ZIC2 8 fold (p = 1.3E-05) and HELLS 2 fold (p = 0.006) in GG3 glands derived from GS 4 + 3 = 7. HELLS expression of ≥ 1% occurred in 10% GS < 7, 17% GS 7 and 43% GS >7 prostate cancer (p < 0.001). Using a cut-off of ≥ 1%, protein expression of ZIC5 was present in 28% GS < 7, 43% GS 7 and 57% GS > 7 cancer (p < 0.001). ZIC2 was neither associated with GS nor outcome in our validation set. HELLS was independently predictive for biochemical-recurrence after radical prostatectomy (HR 2.3; CI 1.5-3.6; p < 0.01).In conclusion, HELLS and ZIC5 might be promising candidate markers for selection of biopsy GS 6 prostate cancer being at risk for up-grading at prostatectomy.
authors.affiliation: Departments of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: A Marije
authors.initials: AM
authors.lastname: Hoogland
authors.affiliation: Departments of Urology, Erasmus Medical Center, Rotterdam, The Netherlands.|Department of Bioinformatics, University of Applied Sciences Wildau, Wildau, Germany.
authors.forename: René
authors.initials: R
authors.lastname: Böttcher
authors.affiliation: Departments of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: Verhoef
authors.affiliation: Departments of Urology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Guido
authors.initials: G
authors.lastname: Jenster
authors.affiliation: Departments of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Geert J L H
authors.initials: GJ
authors.lastname: van Leenders
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D001706:Biopsy; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D061346:Laser Capture Microdissection; D008297:Male; D009367:Neoplasm Staging; D011237:Predictive Value of Tests; D011468:Prostatectomy; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D015854:Up-Regulation
chemical_list: D014408:Biomarkers, Tumor
keywords: RNA sequencing; immunohistochemistry; laser capture microdissection; prostate cancer