Specific detection of prostate cancer cells in urine by multiplex immunofluorescence cytology

Abstract

Prostate cancer biomarkers are enriched in urine after prostatic manipulation, suggesting that whole cells might also be detectable for diagnosis. We tested multiplex staining of urinary sediments as a minimally invasive method to detect prostate cancer. Urine samples were collected from 35 men who had prostatic massage (attentive digital rectal examination) in a urology clinic and from 15 control men without urologic disease and without massage, for a total of 50 specimens (27 cancer-positive cases and 23 cancer-negative cases). LNCaP prostate cancer cells spiked into urine were used for initial marker optimization. Urine sediments were cytospun onto glass slides and stained. Multiplex urine cytology was compared with conventional urine cytology for cancer detection; anti-alpha-methylacyl-CoA racemase antibody was used as a marker of prostate cancer cells, anti-Nkx3.1 as a marker of prostate epithelial cells, anti-nucleolin as a marker of nucleoli, and 4'-6-diamidino-2-phenylindole to highlight nuclei. Prostate cancer cells were successfully visualized by combined staining for alpha-methylacyl-CoA racemase, Nkx3.1, and nucleolin. Of the 25 informative cases with biopsy-proven prostate cancer, 9 were diagnosed as suspicious or positive by multiplex immunofluorescence urine cytology, but only 4 were similarly judged by conventional cytology. All cases without cancer were read as negative by both methods. The multiplex cytology sensitivity for cancer detection in informative cases was 36% (9/25), and specificity was 100% (8/8). In conclusion, we have successfully achieved multiple staining for alpha-methylacyl-CoA racemase, Nkx3.1, nucleolin, and 4'-6-diamidino-2-phenylindole to detect prostate cancer cells in urine. Further refinements in marker selection and technique may increase sensitivity and applicability for prostate cancer diagnosis.

Figure 1. Immunofluorescence staining of LNCaP prostate cancer cell cytospins

LNCaP cytospin preps were stained with: (A) anti-Alpha-methylacyl-CoA racemase (AMACR) antibody detected in green; (punctuate cytoplasmic staining pattern); (B) anti-Nkx3.1 (nuclear pattern) and (C) anti-Nucleolin (nucleolar pattern); (D) Combined fluorescent staining for AMACR, Nkx3.1, and nucleolin. In all cases nuclei are co-stained blue with the DNA-binding dye DAPI. Original magnification=400X.

Figure 2. Multiplex staining of urine sediments obtained from clinical urine specimens

Panels A,C,E and G: combined fluorescent staining for AMACR (green, cytoplasmic), Nkx3.1 (green, nuclear), and nucleolin (red, sub-nucleolar) strongly suggests the presence of exfoliated prostate cancer cells in these urine specimens. Panels B,D,F and H: same specimens stained by the Papanicolau procedure.