Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy.

OBJECTIVES: To determine the expression of prostate-specific membrane antigen (PSMA) before and after androgen-deprivation therapy and to compare PSMA expression with prostate-specific antigen (PSA) expression. METHODS: We studied specimens from 20 patients with prostate cancer undergoing medical or surgical castration or combination androgen-deprivation therapy in whom matched pretreatment and post-treatment tissue specimens were available and 16 patients in whom only a post-treatment specimen was available. The expression of PSMA and PSA in the tissue specimens was determined by immunoperoxidase staining. The extent of staining was calculated by multiplying the percent of antigen-positive tumor cells by the staining intensity to arrive at a stain index for each biomarker. An in vitro study assessed the concentration of PSMA and PSA in extracts of LNCaP cells cultured in the presence or absence of androgen as determined by immunoassays and Western blot analysis. RESULTS: PSMA reactivity was found to be increased in 55% (11 of 20) of post-treatment primary tissues and 100% (4 of 4) of post-treatment metastatic specimens. In contrast, PSA expression was found to be decreased in 70% (14 of 20) of post-treatment primary and 100% (4 of 4) of post-treatment metastatic specimens. Neither type of androgen-deprivation treatment nor tissue sensitivity to androgen deprivation appeared to influence degree of biomarker expression. PSMA was found to be downregulated and PSA upregulated when LNCaP cells were cultured in the presence of testosterone or dihydrotestosterone. CONCLUSIONS: The enhanced expression of PSMA in tissues and LNCaP cells after androgen deprivation suggests that PSMA is upregulated in the majority of prostate carcinomas after androgen treatment. The high expression in metastatic tissues strongly suggests that PSMA may be a clinically useful target for antibody-and genetic-directed therapy of prostate cancer that recurs after androgen deprivation. The mechanism whereby androgens suppress the expression of PSMA, and the association of PSMA with the development of hormone-independent prostate cancers, will require further study.

Deoxyribonucleic acid cytometric analysis of prostate core biopsy specimens: relationship to serum prostate specific antigen and prostatic acid phosphatase, clinical stage and histopathology.

We describe a sampling method of obtaining fresh prostate cells that yields adequate numbers of cells for flow cytometric deoxyribonucleic acid (DNA) analysis and produces histograms of good resolution. Exfoliated cells from 204 prostate biopsy wash specimens obtained by agitation of biopsy cores in saline were fixed and stained for DNA analysis. The mean percentage of hyperdiploid cells was statistically different between the pathologically benign and malignant specimens (p < 0.0001). Hyperdiploid cells of 22% or more exhibited a high degree of specificity for the malignant specimens with only a 1.4% (1 of 69 benign specimens) false-positive rate. However, sensitivity was only 41% (25 of 59 malignant specimens were associated with a flow cytometry analysis of 22% or greater hyperdiploid cells) because of the high false-negative rate (59%, 35 of 59). The percentage of hyperdiploid cells correlated statistically with increasing prostate specific antigen (PSA) levels and approached significance with Gleason grade but did not correlate with prostatic acid phosphatase or clinical stage. When the amount of hyperdiploid cells was 22% or more and serum PSA level was greater than 4.0 ng./ml. a 95% chance of a malignant biopsy was predicted. This result was greater than that predicted by a PSA elevation alone. Only a 5% chance of a malignant biopsy was present for patients with less than 22% hyperdiploid cells and 4.0 ng./ml. or less serum PSA, a decrease over either method separately. This method of DNA assessment permits prospective categorization of tumors by ploidy without interfering with histological assessment. The prognostic importance of ploidy analysis awaits further clinical followup.

Failure of anticytokeratin 18 antibody to improve flow cytometric detection of bladder cancer.

BACKGROUND: Bladder washing specimens containing inflammatory or squamous cells have been difficult to accurately analyze with single-parameter DNA flow cytometric (FCM) methods. METHODS: The anticytokeratin 18 antibody, CK5, was used in a multiparameter assay of 275 bladder washing and voided urine specimens to immunoselect only the bladder transitional cells for DNA analysis. RESULTS: Flow cytometric detection of transitional cell carcinoma was increased by immunoselection of CK5-positive cells in specimens from patients with disease. Unfortunately, a similar increase in hyperdiploid cells in pathologically benign specimens was observed, which resulted in a false-positive rate of 45%. In some instances, multiparameter FCM assays with CK5 could detect aneuploid cell populations not clearly evident by single-parameter analysis. CONCLUSIONS: However, the results from this study of the hyperdiploid cell fraction showed that the increased sensitivity resulting from the use of CK5 was not clinically useful because of the decrease in specificity.

Flow cytometric DNA analysis after immunoselection of bladder tumor cells with monoclonal antibody DU83.21.

Adequate preservation of neoplastic cells and the elimination of interference by inflammatory cells in measuring tumor cell DNA content represent two important objectives necessary for accurate flow cytometric analysis of bladder carcinomas. An experimental model consisting of a mixture of cultured bladder carcinoma cells (T24) and human buffy-coat (BC) cells was used to evaluate various preservatives and an anti-bladder carcinoma monoclonal antibody (MoAb), DU83.21, for separating inflammatory cells (BC cells) from T24 cells. A final concentration of 25% ethanol was found to be the most effective preservative of several tested. After incubation with the MoAb DU83.21 and propidium iodide (DNA stain), the T24 cells could be separated from the BC cells, permitting accurate DNA analysis of the tumor cells. Application of this system to specimens from bladder cancer patients enhanced the detection and DNA analysis of the tumor cell populations.

Flow cytometric study comparing paired bladder washing and voided urine for bladder cancer detection.

Paired bladder washings and voided urines from bladder cancer patients were compared as sources of exfoliated cells for detection of bladder carcinoma by flow cytometry (FCM). Bladder specimens fixed in 25% ethanol within sixty minutes of collection were found to be superior to unfixed bladder specimens. The percentage of specimens with good DNA resolution was greater for bladder washings (67% unfixed, 90% fixed) than voided urines (41% unfixed, 66% fixed). There was no difference in DNA resolution between specimens that remained unfixed less than one day, one day, or two days suggesting that the cells undergo the majority of degradation within a critical period soon after collection. Once fixed, there was no difference in DNA resolution for up to nineteen days, which suggests the feasibility of specimen transport to central FCM laboratories. Eighteen percent of unfixed bladder washings and 33 percent of unfixed voided urine specimens contained an insufficient number of cells (less than 5,000) at the time of analysis compared with 6 percent bladder washings and 17 percent voided urines fixed in 25% ethanol. Flow cytometry and cytology results were concordant in 28/43 (65%) of fixed bladder washings and 9/13 (69%) of fixed voided urine. Voided urine was unreliable in providing consistent FCM data due to the high number of specimens with poor resolution or insufficient cells and is not recommended as a substitute for bladder washing when screening high-risk populations or monitoring patients with past history of bladder cancer.

Light scatter and total protein signal distribution of platelets by flow cytometry as parameters of size.

The use of flow cytometry for determination of platelet size was investigated by measurement of forward-angle light-scatter (FALS) signals and the fluorescent right-angle signals from platelets incubated with the fluorescent protein dye fluorescein isothiocyanate (FITC). Both FALS and fluorescence signals displayed a unimodal log normal pattern of distributions that were almost identical to distributions obtained by resistive particle sizing. As measured on 12 platelet samples from normal individuals, FALS, FITC, and resistive particle size data exhibited a high degree of fit to log normal distributions as shown by the coefficients of determination (R2), which were 0.9962 +/- 0.0035, 0.9966 +/- 0.0056, and 0.9987 +/- 0.0012, respectively. The geometric standard deviation (GSD), reflecting the heterogeneity of the FALS, FITC, and resistive particle size signals, was almost identical: 1.69 +/- 0.03, 1.67 +/- 0.04, and 1.69 +/- 0.02, respectively, for spherical platelets from normal individuals. Platelet FALS signal distributions were compared with Coulter resistive particle size distributions by using platelet samples from 12 normal patients and 27 patients with thrombocytopenia. Significant correlation was found between mean FALS and mean resistive particle size (r = 0.83) and between GSD of FALS and GSD of resistive particle size (r = 0.74) with the platelet samples from the 39 subjects studied. These studies, which document the high degree of correspondence among these three independent measurements of platelet size, based on three entirely different principles, strongly suggest that platelet size is log normal distributed and that the GSD value shown above reflects actual heterogeneity in size. The FALS signal distribution was, however, found to be markedly influenced by changes in platelet shape and internal structure.(ABSTRACT TRUNCATED AT 250 WORDS)