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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

Detection of microscopic disseminated disease in prostate cancer has largely been attempted with the study of circulating tumor cells (CTCs). Little is known about the character and clinical significance of disseminated tumor cells (DTCs) that have reached the bone marrow (BM), and prior study has relied on epithelial markers. Yet, it is now known that such markers are also expressed on normal erythroid precursor cells in the BM, thus we developed an assay for prostate-specific DTC identification.

Methods

BM aspirates from 12 men with localized and 2 men with metastatic prostate cancer were collected at time of radical prostatectomy (RP) or clinic visit respectively, and processed with the AccuCyte system (RareCyte, Inc., Seattle, WA). Slides were immunostained with DAPI (nuclear), anti-pan-cytokeratin (epithelial), anti-CD45/CD66b/CD11b/CD14/CD34 (white blood cell), and HOXB13 and NKX3.1 (prostate-specific). DTCs were required to have positive prostate channel staining and no white blood cell signal. The DTC count was adjusted for the starting volume of BM and the number of slides stained and spread over.

Results

DTCs were present in 83% (10/12) of patients at time of RP, with range 0 to 3592 cells/4 mL sample (average 375, median of 39.5). Two patients with metastatic prostate cancer had 52 and 105 DTCs/4mL BM (Table 1). Notably, only 4% of all DTCs were epithelial marker positive (CK+), and only 50% (6/12) of patients had any CK+ DTCs (Fig. 1).

Conclusions

The RareCyte system is a promising selection-free system for DTCs detection that does not rely on epithelial markers. Here we report on a novel assay to distinguish DTCs from other cells in the bone marrow using the presumed prostate-specific markers HOXB13 and NKX3.1. The majority of prostate-specific marker positive DTCs did not express the epithelial marker CK. Analyses of the association of DTC count with clinicopathologic variables are ongoing.

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.