N. J. R. George

Loss of HLA class I expression in prostate cancer: Implications for immunotherapy

OBJECTIVES There is currently no reliable predictor of the metastatic potential of apparently localized prostate cancer in an individual patient or satisfactory treatment for patients with advanced disease. One of the factors that may influence tumor progression is the cellular arm of the immune response, and central to this is the human leukocyte antigen (HLA) system, which acts to restrict T-cell recognition of potential tumor antigens. It has been reported in some cancers that down regulation of HLA class I expression by the tumor cells is associated with poor prognosis. In this report, HLA class I and II expression have been investigated in both benign and malignant prostate disease, first to define the extent of altered HLA expression and second to assess whether HLA expression may be related to disease progression. METHODS HLA expression was assessed by immunohistochemistry utilizing a set of monoclonal antibodies that recognize both monomorphic determinants and the commoner HLA class I allelic products. RESULTS In contrast to the normal HLA class I expression of the benign tissue, complete loss of HLA class I expression occurred in 34% of primary prostate cancers and 80% of lymph node metastases. When individual allelic expression was assessed, the minimum estimate of down regulation was 85% in the primary prostate cancers and 100% of the metastases. CONCLUSIONS This investigation has demonstrated a higher rate of HLA class I loss than has been reported in other tumors and would suggest that the immune system may have an important role in the progression of prostate cancer, as well as having implications for the design and success of immunotherapy regimens in advanced disease.

Novel method for the isolation and characterisation of the putative prostatic stem cell

Prostate stem cells, responsible for the development, maturation, and function of the prostate, have been implicated in the aetiology of both benign prostate hyperplasia (BPH) and prostate cancer (CaP). However, research has been hampered by the lack of a definitive stem cell marker. We have adapted the protocol for differential Hoechst 33342 uptake by hemopoietic stem cells to enable isolation of putative stem cells from the prostate.

Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and invasion.

Prostate cancer shows a propensity to form secondary tumours within the bone marrow. Such tumours are the major cause of mortality in this disease. We have developed an in vitro system to study the binding of prostate epithelial cells to bone marrow endothelium (BME) and stroma (BMS). The metastatic prostate cancer cell line, PC3 (derived from a bone metastasis), was seeded onto confluent layers of BME and its binding characteristics compared to human umbilical vein endothelial cells (HUVEC), lung endothelium (Hs888Lu) and BMS. The PC3 cell line showed significantly increased binding to BME (P< 0.05) compared to endothelium derived from HUVEC and lung or BMS with maximal binding occurring at 1 h. Following pre-incubation with a β1 integrin antibody PC3 binding to BME was inhibited by 64% (P< 0.001). Antibodies directed against the integrins β4, α2, α4, α5 and the cellular adhesion molecules P-selectin, CD31, VCAM-1 and sialy Lewis X showed no effect on blocking PC3 binding. Primary prostatic epithelial cells from both malignant (n = 11) and non-malignant tissue (n = 11) also demonstrated equivalent levels of increased adhesion to BME and BMS compared to HUVEC, peaking at 24 h. Further studies examined the invasive ability of prostate epithelial cells in response to bone marrow endothelium using Matrigel invasion chamber assays. In contrast to the previous results, malignant cells showed an increase (1000 fold) in invasive ability, whilst non-malignant prostate epithelia did not respond. We have shown that both malignant and non-malignant prostate epithelial cells can bind at equivalent levels and preferentially to primary human bone marrow endothelium in comparison to controls. However, only malignant prostate epithelia show increased invasive ability in response to BME.

Osteoblast function and osteomalacia in metastatic prostate cancer.

This study has examined the effect of prostate cancer on bone matrix formation and mineralisation by osteoblasts, with special reference to osteomalacia. Sixty-seven patients with prostatic bone metastases underwent transiliac bone biopsy after double tetracycline labelling. Histomorphometric analysis was then undertaken in areas distant from, local to and infiltrated by prostate cancer. In bone free of tumour (n = 45) and bone surrounding metastases (n = 7) both matrix formation and corrected mineral apposition rate were low. By comparison osteoid surface, osteoid volume and mineral apposition rate were markedly increased within metastases (tumor-free bone vs. metastatic bone, p < 0.0001), a finding consistent with a high bone turnover state. Although osteoblast function was disturbed both within metastases and in tumour-free areas, classical osteomalacia was not associated with prostate cancer.

Osteoclast inhibition by pamidronate in metastatic prostate cancer: a preliminary study.

Twenty five hormone manipulated patients with prostate cancer and metastatic bone disease, treated at least 6/12 previously by hormone manipulation, were given intravenous infusions of Disodium Pamidronate (APD) over a 6 month period. Patients received 30 mg weekly for 4 weeks then twice monthly for 5 months. No other treatment was administered during study. Eleven of 17 patients with pain at the start of the study were pain free at the end. Fasting morning calcium excretion and serum osteocalcin fell significantly with Pamidronate (P less than 0.0001) and urine hydroxyproline was lowered in 13/20 evaluable patients at 6 months. Alkaline phosphatase fell in a proportion of patients and five of 17 patients with previously progressive bone scans stabilised (4) or regressed (1) on treatment. Rising acid phosphatase levels were also lowered in five patients. It is concluded that Pamidronate may be effective in palliating bone pain in some patients and has a stabilising influence on abnormally high bone turnover in metastatic prostate cancer. Further controlled studies of the compound are now warranted.

Parathyroid hormone related peptide and receptor expression in paired primary prostate cancer and bone metastases

Parathyroid hormone-related peptide is a regulatory protein implicated in the pathogenesis of bone metastases, particularly in breast carcinoma. Parathyroid hormone-related peptide is widely expressed in primary prostate cancers but there are few reports of its expression in prostatic metastases. The aim of this study was to examine the expression of parathyroid hormone-related peptide and its receptor in matched primary and in bone metastatic tissue from patients with untreated adenocarcinoma of the prostate. Eight-millimetre trephine iliac crest bone biopsies containing metastatic prostate cancer were obtained from 14 patients from whom matched primary tumour tissue was also available. Histological grading was performed by an independent pathologist. The cellular location of mRNA for parathyroid hormone-related peptide and parathyroid hormone-related peptide receptor was identified using in situ hybridization with 35S-labelled probe. Expression of parathyroid hormone-related peptide and its receptor was described as uniform, heterogenous or negative within the tumour cell population. Parathyroid hormone-related peptide expression was positive in 13 out of 14 primary tumours and in all 14 metastases. Receptor expression was evident in all 14 primaries and 12 out of 14 metastases. Co-expression of parathyroid hormone-related peptide and parathyroid hormone-related peptide receptor was common (13 primary tumours, 12 metastases). The co-expression of parathyroid hormone-related peptide and its receptor suggest that autocrine parathyroid hormone-related peptide mediated stimulation may be a mechanism of escape from normal growth regulatory pathways. The high frequency of parathyroid hormone-related peptide expression in metastases is consistent with a role in the pathogenesis of bone metastases.

Primary prostatic epithelial cell binding to human bone marrow stroma and the role of alpha2beta1 integrin.

Prostate cancer selectively metastasises to the bone. To investigate the importance of prostate epithelial cell adhesion to bone marrow cells in this process we examined the binding of human primary prostatic epithelial cells (PEC) to human bone marrow stromal cultures (BMS). We found that PEC derived from both malignant and benign tissue showed greater adhesion to BMS than to benign prostatic fibroblasts (median difference was 340% and 200% respectively), skin fibroblasts or plastic tissue culture plates. Adhesion to BMS grown from the bone marrow of patients with prostatic skeletal metastases was no different from those grown from normal bone marrow. The role of integrin molecules in these cell interactions was determined. Collagen type I and fibronectin were found to increase PEC adhesion whereas vitronectin and laminin did not. Inhibition studies demonstrated that although there was heterogeneity between samples, antibodies against the integrins a2 and b1 consistently inhibited PEC binding to BMS. This result was more marked for PEC derived from malignant tissue. However studies investigating the effects of disintegrins and anti-a3 and anti-a5 integrins indicated that for a percentage of patients these integrins and RGD (arginine, glycine, aspartamine)-dependent binding pathways were also involved. In summary, the results indicate that BMS are adherent to primary PEC derived from both malignant and benign tissue. The integrin a2b1 is a major contributor to this interaction.

Interaction of prostate epithelial cells from benign and malignant tumor tissue with bone-marrow stroma

Metastases of prostate cancer form selectively within the skeleton. To understand this metastatic spread, we studied the ability of prostate epithelial cells to grow and proliferate within the bone marrow, using primary coculture.

E-cadherin and beta-catenin are down-regulated in prostatic bone metastases.

Objective To determine the E‐cadherin and β‐catenin expression phenotype in untreated primary prostate cancer and corresponding bone metastases.

Paradoxical expression of E-cadherin in prostatic bone metastases.

To determine whether the calcium‐dependent cell adhesion molecule E‐cadherin is expressed in metastatic deposits of prostate cancer in bone.