Abstract: PD65-04
Sources of Funding: This work was supported by the Urology Care Foundation's Resident Research Award (H.J.C.), NCI grant numbers. U54CA143803, CA163124, CA093900 and CA143055 (K.J.P.), and by the Prostate Cancer Foundation (K.J.P.).

Introduction

Circulating tumor cells (CTCs) have great potential as circulating biomarkers for solid malignancies. Currently available assays for CTC detection rely on epithelial markers with somewhat limited sensitivity and specificity. Here we report that the staining pattern of nucleolin, a common nucleolar protein in proliferative cells, separates CTCs from white blood cells (WBCs) in men with metastatic prostate cancer.

Methods

Whole peripheral blood from three men with metastatic prostate cancer was processed with the AccuCyte CTC system (RareCyte, Inc., Seattle, WA). Slides were immunostained with DAPI, anti-pan-cytokeratin, anti-CD45/CD66b/CD11b/CD14/CD34, and anti-nucleolin antibodies and detected using the CyteFinder system. DAPI nucleolin co-localization and staining pattern wavelet entropy was measured with novel image analysis software.

Results

33,718 DAPI-positive cells were analyzed with the novel imaging software, of which 45 (0.13%) were known CTCs based on the established AccuCyte system criteria. Nucleolin staining pattern for segmentable CTCs demonstrated greater wavelet entropy than that of WBCs (median wavelet entropy: 6.86 x 10⁷ and 3.03 x 10⁶, respectively; p-value = 2.92 x 10^-22; approximated z-statistic: 9.63). Additionally, the total nucleolin staining of CTCs was greater than that of WBCs (median total pixel intensity: 1.20 x 10⁵ and 2.55 x 10⁴ integrated pixel units, respectively; p-value = 2.40 x 10^-21; approximated z-statistic: 9.41).

Conclusions

Prostate cancer CTCs displayed unique nucleolin expression and localization as compared to WBCs. This finding has the potential to serve as the basis for a sensitive and specific CTC detection method.

Funding

This work was supported by the Urology Care Foundation's Resident Research Award (H.J.C.), NCI grant numbers. U54CA143803, CA163124, CA093900 and CA143055 (K.J.P.), and by the Prostate Cancer Foundation (K.J.P.).