The distribution of immunohistochemically defined neuroendocrine (NE) cells in benign, pre-cancerous and neoplastic prostatic tissues and the prognostic value of these cells in prostate cancer were studied in the radical prostatectomy specimens of 90 patients from whom complete long-term follow-up data were available. The tissue blocks containing all the different Gleason patterns observed in a particular tumor were selected and immunostained. Since chromogranin B stained only a few cells compared to chromogranin A (CgA), NE cells were only defined by their reactivity with CgA. A semi-quantificative CgA score was assessed for all distinct pathological areas. Cox's regression model was used to analyze the influence of final TNM classification (TNM, 1992), Gleason sum score (GSS), age and CgA score on the probability of progression and tumor-specific death. NE cells were demonstrated in all normal prostatic tissues and in most hyperplastic and intra-epithelial neoplastic lesions. CgA staining was seen in 78% of the tumors. CgA scores were not related with Gleason growth patterns, GSS or TNM classification and had no prognostic value. The independent prognostic variables in Cox's regression model were: GSS and pT stage for progression and GSS for tumor-specific survival. Theoretically, NE cells could influence tumor behavior and this discrepancy suggests the need for experimental studies to investigate the role of NE cells in the normal and neoplastic prostate.

BACKGROUND Androgen‐independent growth leads to progressive prostate cancer after androgen‐ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand‐independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS We established and characterized the androgen‐independent FGC‐DCC from the androgen‐dependent LNCaP fast growing colony (FGC) cell line. The androgen‐independent DU‐145, FGC‐DCC, and PC‐3, and the androgen‐dependent LNCaP and PC‐346C cell lines were used to study growth modulation of gastrin‐releasing peptide (GRP), calcitonin (CT), serotonin (5‐HT), and vasoactive intestinal peptide (VIP) by 3H‐thymidine incorporation. Specificity of the growth‐modulating effects was tested with the anti‐GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS Androgen‐independent growth stimulation by neuropeptides was shown in DU‐145 and PC‐346C. 2A11 inhibited GRP‐induced 3H‐thymidine incorporation in DU‐145 and PC‐346C and inhibited proliferation of the FGC‐DCC and PC‐3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP‐induced growth effect in DU‐145 and PC‐346C, whereas oxadiazoloquinoxaline‐1‐one (ODQ) had no effect on 3H‐thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC‐DCC, or PC‐3. CONCLUSIONS GRP‐induced growth of DU‐145 and PC‐346C was specific and cAMP‐mediated. Androgen‐independent growth of FGC‐DCC cells was mainly due to an induction of Bcl‐2 expression and possibly through the activation of an autocrine and NE‐like pathway, as has been shown also for the PC‐3 cell line. Growth induction of non‐NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer. Prostate 42:34–44, 2000. © 2000 Wiley‐Liss, Inc.

Reduced p27(kip1) expression is an independent predictor of poor outcome in prostate cancer, while MIB-1 is not. Decreased expression of CD44s yields additional information in predicting clinical recurrence. These tissue markers may identify patients at risk for disease recurrence after retropubic radical prostatectomy who may benefit from adjuvant therapy.

It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G 0 -phase-arrested cells.Here we further characterized NE differentiation , androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0% , and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgApositive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days , respectively. However , no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G 0 -phase-arrested cells. 2-8 These cells produce various growth-modulating neuropeptides in a paracrine or autocrine way. -15Possible roles for NE cells in the prostate may be regulation of homeostasis and secretion of prostatic fluid, either actively or passively. NE cells can be identified by routine electron microscopy (dense core granules) or by immunohistochemistry with specific antibodies against secreted products, for example, serotonin, 16 or secretion-associated proteins, such as chromogranin A (CgA), [17][18][19] which is a marker for neuroendocrine differentiation. NE cells are considered to be nonproliferating cells and do not express the androgen receptor 20 and are therefore probably unaffected by androgen deprivation. Consequently, they will not undergo apoptosis under such circumstances. Therefore, it is relevant to assess whether or not CgA-positive cells co-express the antiapoptotic oncogene Bcl-2. 21 Moreover, NE cells show a heterogeneous cytokeratin expression pattern as there are basal, luminal, and intermediate NE cell types 16,22,23 and are often found near Bcl-2-positive prostate cancer cells. 24,25The single expression of CgA is not the only requisite for the determination of NE differentiated cells as there exists a regulated secretory pathway (RSP) in NE cells 26 next to the lysosomal and an exocrine constitutive pathway. Along the RSP pathway, secretion and processing of bioactive neuropeptides and growth hormones, such as insulin and glucagon in the pancreas, 27,28 are regulated. The RSP consists of a sequence of processes linked from transcription/translation of various factors ...

The pre-operative prediction of prognostic tumour features in the radical prostatectomy specimen using routine clinicopathological variables remains limited. The present study evaluated the predictive value of the cell-cycle protein p27 kip1 , the proliferation marker MIB-1, and the celladhesion protein CD44s, determined on the diagnostic needle biopsy of asymptomatic men screened for prostate cancer. Of 81 screen-detected prostate cancers, representative biopsy cores and matched radical prostatectomy specimens were immunohistochemically stained for these tissue markers. Conventional pre-operative and post-operative clinicopathological variables were assessed and cancers were divided according to a validated tumour classification model (potentially harmless, clinically significant). Low (<50%) p27 kip1 expression, high (i10%) MIB-1 expression, and low (<25%) CD44s expression were considered adverse prognostic signs.

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Neuroendocrine cells can be demonstrated in normal, hyperplastic and neoplastic prostatic tissues. The products secreted by these cells can be used as tissue and/or serum markers but may also have biological effects. Neuroendocrine cells in prostate cancer most probably do not contain the androgen receptor and are therefore primarily androgen independent. Some of the neuropeptides secreted by the neuroendocrine cells may act as growth factor by activation of membrane receptors in an autocrine-paracrine fashion or by ligand-independent activation of the androgen receptor in neighboring non-neuroendocrine cells. Evidence is accumulating from experiments with tumor models that neuropeptides indeed can influence the growth of prostatic tumor cells. Future research on neuroendocrine differentiation may answer some questions concerning the biological behavior of clinical prostatic tumors.

Androgen deprivation of the NE-differentiated PC-310 model induced the formation of NE-differentiated AR(minus sign) and non-NE AR(+) tumor residues. The NE-differentiated cells actively produced growth factors via an RSP that may lead to hormone-refractory disease. The dormant non-NE AR(+) tumor cells were shown to remain androgen sensitive even after long-term androgen deprivation. In the PC-310 xenograft, time-dependent NE differentiation and subsequent maturation were induced after androgen depletion. The androgen-dependent PC-310 xenograft model constitutes an excellent model for studying the role of NE cells in the progression of clinical prostate cancer.