TENB2, a proteoglycan identified in prostate cancer that is associated with disease progression and androgen independence.

TENB2 encodes a putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, which has been identified through the application of a differential display technique to a xenograft model of prostate cancer. Northern analysis and competitive PCR were used to demonstrate significantly increased TENB2 expression (p = 0.0003) on the acquisition of androgen independence in the model system. TENB2 is also overexpressed in clinical prostate carcinoma vs. its benign counterpart (p < 0.0001), with particular prominence in high-grade tumours, and shows a high degree of tissue specificity, being detected on a multitissue Northern array exclusively in brain and prostate material. Studies of recombinant protein expression demonstrate that TENB2 is a chondroitin sulphate proteoglycan. The presence of an EGF and 2 follistatin domains suggests a role in the regulation of growth factor signalling either as a ligand precursor, a membrane-bound receptor or as a binding protein for growth factors. These data are indicative of a significant role for TENB2 in the progression of poorly differentiated tumour types, with implications for prostate cancer detection, prognosis and therapy.

Expression of androgen receptor and growth factors in premalignant lesions of the prostate.

Analysis of growth factors and receptors in putative premalignant lesions of prostatic adenocarcinoma should aid our understanding of their growth pathways. Sixty prostatic TURP (transurethral resection of the prostate) specimens exhibiting atypical adenomatous hyperplasia (AAH) and/or prostatic intraepithelial neoplasia (PIN) lesions were assayed by immunohistochemistry for androgen receptor (AR), epidermal growth factor receptor (EGFR), c-erbB-2, transforming growth factor-alpha (TGF-alpha), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), MIB-1, E-cadherin, and high molecular weight keratin. Expression of these factors in the lesions was compared with that in the co-existing benign prostatic hyperplasia (BPH) or prostatic adenocarcinoma. Strong AR nuclear staining was observed in the luminal cells, but not the basal cells, of BPH and PIN lesions and in all the carcinomas examined. A similar growth factor and receptor profile was demonstrated in the secretory epithelium of high-grade PIN and carcinoma with a tendency to higher expression of membranous EGFR and c-erbB-2 and cytoplasmic TGF-alpha, and lower levels of FGF-2 than in low-grade PIN or BPH glands. Also, increased rates of proliferation, as estimated by MIB-1 stained cells, were observed in high-grade PIN in comparison with low-grade PIN and BPH and were not confined to the basal layer. AAH lesions resembled neither BPH nor carcinoma. Proliferation was virtually absent (MIB-1 expression); both AR and E-cadherin expression was significantly reduced; and, with the exception of FGF-2, all the other growth factors and receptors studied were absent. The results presented would support a premalignant role for high-grade PIN, whilst AAH would appear to represent a quiescent phenotype unlikely to progress to neoplasia.

Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells.

Vascular endothelial growth factor (VEGF) expression was examined by immunohistochemistry in 45 prostatic carcinoma specimens and ten benign prostatic tumours (BPH). The majority of carcinoma specimens exhibited cytoplasmic staining for VEGF and showed a trend of increasing expression with dedifferentiation (2p = 0.003). Immunoreactive VEGF was also seen in the prostatic carcinoma cell lines, the order of staining intensity was PC3 > DU145 > LNCaP. Intense granular cytoplasmic staining for VEGF was observed in neuroendocrine-like cells which were seen focally in many of the prostatic specimens. Consecutive sections were incubated with a chromogranin A antibody to confirm the neuroendocrine phenotype of these cells. A significant correlation (P < 0.0001) between the total number of intensely stained VEGF-positive cells and chromogranin A-positive cells was found. A subpopulation of neuroendocrine-like cells also showed intense immunoreactivity for transforming growth factor alpha (TGF-alpha). A correlation was observed (2p = 0.0092) between the intensity of VEGF and TGF-alpha immunostaining in carcinoma cells which were not of neuroendocrine differentiation. The presence of these two angiogenic factors may aid the neovascularisation of carcinomas and their increased expression in tumour-associated neuroendocrine cells may contribute to a more aggressive phenotype.

Comparative analysis of mRNA and protein expression for epidermal growth factor receptor and ligands relative to the proliferative index in human prostate tissue.

The expression of epidermal growth factor receptor (EGF-R), transforming growth factor alpha (TGFalpha), and epidermal growth factor (EGF) was evaluated in a series of prostate cancer (CaP; n = 55) and benign prostate hyperplasia (BPH; n = 44) specimens using immunocytochemistry (ICC) and Northern blotting. In situ hybridization (ISH), performed on a subgroup of these specimens, proved to be a more informative technique for the assessment of messenger RNA (mRNA) in this heterogeneous tissue. A comparative analysis was made in relation to the proliferative index, assessed using the MIB-1 antibody. Elevated levels of EGF-R and TGFalpha, mRNA, and protein were observed in carcinoma cells compared with benign, secretory epithelium using in situ hybridization and immunocytochemistry. In carcinoma specimens evidence of an autocrine growth loop is provided by a correlation between EGF-R and TGFalpha, mRNA (P < .0001), and protein expression (P < .01). A trend toward increased expression of EGF-R and TGFalpha protein with dedifferentiation and a similar trend in the growth fraction suggest a role in tumor progression. Although there was a correlation between EGF-R and the proliferative index (P < .01), no relationship was found between this latter parameter and TGFalpha immunoreactivity (P > .05), indicating that this growth factor may be linked with other aspects of malignant activity rather than directly stimulating proliferation.

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA.

Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semi-quantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p < or = 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p < or = 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.

Epidermal growth factor receptor expression by northern analysis and immunohistochemistry in benign and malignant prostatic tumours.

Epidermal growth factor receptor (EGFR) expression in 44 benign prostatic hyperplasia (BPH) and 55 prostatic carcinoma specimens has been investigated using Northern blot analysis and immunohistochemistry. The values obtained for the EGFR mRNA in the BPH and carcinoma specimens were not significantly different and in the latter there was no correlation with grade. In the immunohistochemical assays, two antibodies to the external and one to the internal domain of EGFR were used. The former ones stained the basal cell membranes intensely whilst cytoplasmic staining of secretory epithelium was seen in BPH specimens with the latter. In the carcinoma specimens, the intensity of membrane staining correlated with the two external domain antibodies, r = 0.640, P < 0.001, but neither of these correlated with the EGFR mRNA results. All three antibodies demonstrated a trend towards elevated expression of EGFR with dedifferentiation which reached significance only with the internal domain antibody results, P < 0.02. No correlation was observed with tumour EGFR mRNA values and the EGFR immunohistochemical results. The EGFR immunoreaction with the external domain antibody in 14 treated high-grade tumours was comparable to that obtained in 15 untreated anaplastic prostatic tumours. In 5 patients, both pre- and post-treatment samples were available and these exhibited little or no difference in EGFR expression with therapy.

Transforming growth factor beta 1 expression in benign and malignant prostatic tumors.

The expression of transforming growth factor beta 1 (TGF-beta 1) in prostate specimens obtained from patients with benign prostatic hyperplasia (BPH, n = 32) and prostate carcinoma (n = 66) was investigated using Northern blot analysis and immunohistochemistry. Northern blot analysis revealed TGF-beta 1 message (2.5 kb) in virtually all of the samples examined, reflecting the ubiquitous nature of this growth factor. No statistical difference was found between the levels of mRNA detected in benign and malignant tissues due, in part, to the inherent heterogeneity of prostate tissue. Immunohistochemical methods using an antibody to native TGF-beta 1 revealed a novel pattern of immunoreactivity. Staining observed only in certain epithelial cells of benign glands was associated with areas of infection rather than tumorigenesis. Interestingly, intense staining was also seen in polymorphonuclear leukocytes. No correlation was found with the mRNA results, suggesting that this antibody is binding to TGF-beta 1 activated in response to infection rather than detecting sites of synthesis of latent TGF-beta 1.

An immunocytochemical analysis of TGF alpha expression in benign and malignant prostatic tumors.

Transforming growth factor alpha (TGF alpha) expression was analyzed immunocytochemically on formalin-fixed wax-embedded sections obtained from 24 benign prostatic hyperplasia (BPH) specimens and 76 prostatic carcinoma tissues, 3 human prostatic tumor xenografts, normal kidney, and salivary gland. Low amounts of TGF alpha immunopositivity were encountered in the epithelium of BPH glandular tissues, whereas in the prostatic adenocarcinoma samples, a greater heterogeneity and intensity of TGF alpha immunostaining was observed. The most intense staining was exhibited by the least differentiated tumors, although a few of these were weakly stained. Statistical analysis of the relationship of histopathological grade of tumor with TGF alpha expression in the carcinomas showed a significant correlation of these parameters, 0.01 > P > 0.001. The expression of the proliferation markers Ki-67 and PCNA was also analyzed in the carcinoma specimens, and the relationship of these to TGF alpha expression indicated that there was no significant correlation in this series of tumors between increased growth activity and TGF alpha expression (p approximately 0.25 with both markers). The prostatic carcinoma xenografts TEN12 and TEN15 contained low levels of immunoreactive TGF alpha, which was uniformly distributed, whilst heterogeneous immunostaining was observed in the uroepithelial xenograft TEN16. In the normal human kidney, TGF alpha was concentrated in the epithelium of the distal convoluted tubules (DCT) and the collecting tubules (CT), and lower amounts were identified in the proximal convoluted tubules (PCT). As in the prostatic carcinomas, the immunostaining was eliminated by prior absorption of the antibody with pure TGF alpha and not with human or mouse EGF. No crossreactivity of the TGF alpha antibody with salivary EGF was demonstrated. This study concludes that, in prostate carcinoma, the least differentiated tumors more often expressed greater amounts immunoreactive TGF alpha; however, no relationship between TGF alpha expression and cellular proliferation markers was found.

Relationship of proliferating cell nuclear antigen (PCNA) in prostatic carcinomas to various clinical parameters.

Proliferating cell nuclear antigen (PCNA) expression was determined immunohistochemically, using a monoclonal antibody PC10, in 102 prostatic carcinoma samples and in prostate tissue from 21 patients with benign prostatic hyperplasis (BPH). The percentage of cells with stained nuclei ranged from 1% to 58% in the carcinoma specimens and 0% to 10% in the BPH specimens. A semiquantitative scoring system was devised for the degree of PCNA positivity observed in the tumors. Statistical analysis of the PCNA score in relation to the histological grade of the tumors gave a significant positive or negative correlation between these parameters P less than 0.001. No significant correlation between PCNA score was, however, seen with metastatic status, T category (TMN classification) of the primary tumor, or the patient's age at diagnosis. In 65 prostatic cancer patients of known survival, those individuals whose tumors had a PCNA score of +/- (less than 10% of nuclei stained) were compared with those patients whose tumors were either 1+, 2+, or 3+ (greater than 10% of nuclei stained). Life table analysis of the two groups indicated that the patients with the lower PCNA score survived significantly longer than those with the higher PCNA scores, P less than 0.04. Comparison of the Ki-67 expression in frozen sections with the PCNA expression in wax-embedded tissue of 86 prostatic carcinomas was also undertaken. A significant correlation between these two parameters was found, P less than 0.001, although the growth fraction estimated by Ki-67 expression was generally lower than that given by the PCNA scoring system.

Plasma erythropoietin assay in patients with chronic renal failure.

Using the post-hypoxic mouse method of assay, values for erythropoietin in the plasma of patients with chronic renal failure were equal to, or greater than, normal values. Results suggest that the source of erythropoietin may be primarily extrarenal. Normal renal tissue, provided in the assay by the intact mouse, is required for activation of the hormone. There still remains to be explained the enhanced erythropoietic response to haemorrhage or hypoxia that can occur in anephric man.

Red face and reduced plasma volume.

Twenty-five patients who were suspected clinically of having either polycythaemia rubra vera or secondary polycythaemia had no haematological abnormalities apart from a reduction in the plasma volume. Twenty-one were hypertensive. The plasma volume was lowest in those who were receiving treatment with diuretic agents, antihypertensive drugs, or steroid therapy. These cases demonstrate the need for blood volume studies in patients with high values for the blood haemoglobin and the packed cell volume in order to establish a differential diagnosis from myeloproliferative disorder or secondary polycythaemia.