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Detection and Characterization of Disseminated Tumor Cells from the Bone Marrow in Localized Prostate Cancer Patients undergoing Radical Prostatectomy

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Sources of Funding: This work is supported by NCI grant nos. U54CA143803, CA163124, CA093900, CA143055 to K.J.P as well as the Prostate Cancer Foundation, the Patrick C. Walsh Fund, and a gift from the Stutt family. E.E.vdT is supported by the Cure for Cancer Foundation. H.J.C. is supported by the Urology Care Foundation's Resident Research Award.

Introduction

Disseminated tumor cells (DTC) are prostate cancer cells that escape the primary lesion and enter the bone marrow (BM) niche, representing a first step towards conventionally detectable metastasis. It is unknown how frequently this occurs in clinically localized prostate cancer. We detected and characterized these cells by measuring gene expression of prostate-specific markers from BM samples taken at the time of radical prostatectomy (RP)._x000D_

Methods

5 mL of BM were harvested at RP for 36 clinically localized patients. A whole cell extract was assayed with the AdnaTest ProstateCancer Select kit (Qiagen). Reverse transcription (SensiScript RT kit, Qiagen) and real-time qPCR quantified expression of RPL13A (control, ribosomal protein), EPCAM (epithelial), NKX3.1 and HOXB13 (prostate-specific), and AR-FL (androgen receptor full length). Prostate markers known to be less sensitive or specific were also assayed in a subset of patients (TMPRSS2-ERG, AR-V7, PSA, and PSMA). DTC detection was defined as prostate-specific marker expression in the BM. Quality control was performed with Sanger sequencing. The associations of PSA and Gleason score (GS) with DTC detection were evaluated with the Mann-Whitney U Test and Fisher&[prime]s exact test respectively._x000D_

Results

DTCs were detected via NKX3.1 expression in 30/36 patients (83%). HOXB13, AR-V7, and TMPRSS2-ERG were not detected in any sample. 100% of patients were EPCAM+, consistent with the known non-specific expression of EPCAM in the BM. AR-FL was also non-specifically expressed in 67% of NKX3.1+ and 83% of NKX3.1- patients. There was a trend toward DTC detection associating with higher PSA and GS, with 100% of NKX3.1- patients having low-risk PSA<10, and only one with primary GS >3 (17%, 1/6). Conversely 47% (14/30) of NKX3.1+ patients had primary GS≥4, and 27% (8/30) had PSA>10. Yet, this was not statistically significant (GS p=0.367, PSA p=0.302), and DTCs were detected across all Gleason scores._x000D_

Conclusions

DTCs were detected based on NKX3.1 positivity in a large portion of clinically localized prostate cancer patients at all Gleason scores. Ongoing investigation with healthy patient BM will clarify whether NKX3.1 is truly prostate-specific, and if its expression associates with clinico-pathologic outcomes._x000D_

Funding

This work is supported by NCI grant nos. U54CA143803, CA163124, CA093900, CA143055 to K.J.P as well as the Prostate Cancer Foundation, the Patrick C. Walsh Fund, and a gift from the Stutt family. E.E.vdT is supported by the Cure for Cancer Foundation. H.J.C. is supported by the Urology Care Foundation's Resident Research Award.

Authors
Stephanie Glavaris
Emma van der Toom
Michael Gorin
James Verdone
Changxue Lu
Jun Luo
Kenneth Pienta
Heather Chalfin
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