Gene-expression analysis of gleason grade 3 tumor glands embedded in low- and high-risk prostate cancer
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A. Marije Hoogland1, René Böttcher2,3, Esther Verhoef1, Guido Jenster2, Geert J.L.H. van Leenders1
1Departments of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands
2Departments of Urology, Erasmus Medical Center, Rotterdam, The Netherlands
3Department of Bioinformatics, University of Applied Sciences Wildau, Wildau, Germany
A. Marije Hoogland, email: [email protected]
Keywords: prostate cancer, RNA sequencing, laser capture microdissection, immunohistochemistry
Received: December 30, 2015 Accepted: April 25, 2016 Published: May 13, 2016
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.
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