Adoption of adjuvant bisphosphonates for early breast cancer into standard clinical practice: Challenges and lessons learnt from comparison of the UK and Australian experience.

International guidelines recommend adjuvant bisphosphonates (BPs) for post-menopausal women with early breast cancer to reduce recurrence and mortality. However, globally, wide variation exists in their adoption. In the UK, adjuvant BPs were a recommendation in the breast cancer Clinical Reference Group service specification and were included as a priority for implementation by the national oncologists group UK Breast Cancer Group in November 2015, promoting national uptake, guidance and funding arrangements. In 2018, adjuvant BPs were recommended by the UKs National Institute for Health and Care Excellence. In Australia, adjuvant BPs are still 'off-label' and do not receive national reimbursement or endorsement. To date there has been no research into the prescribing habits of these agents in Australia. With the aim to gather data on adjuvant BPs prescribing practices, online surveys were developed and disseminated to breast oncologists in both countries between December 2018 and June 2019. Almost all of the UK oncologists prescribed adjuvant BPs, demonstrating that education, endorsement from professional bodies, presence of national guidelines and funding decisions have been critical to implementation. In contrast, only 48% of the Australian responders prescribed adjuvant BPs, while 83% reported that they would prescribe them if funding was available. Lack of local protocol guidance was also seen as a major barrier. This study was intended to assess the pathway taken for adjuvant BP implementation in the UK and how it might inform changes in Australian practice and also guide other countries with similar issues with the ultimate aim of improving the care of women with early breast cancer globally.

Adjuvant bisphosphonates in early breast cancer: consensus guidance for clinical practice from a European Panel.

Bisphosphonates have been studied in randomised trials in early breast cancer to investigate their ability to prevent cancer treatment-induced bone loss (CTIBL) and reduce the risk of disease recurrence and metastasis. Treatment benefits have been reported but bisphosphonates do not currently have regulatory approval for either of these potential indications. This consensus paper provides a review of the evidence and offers guidance to breast cancer clinicians on the use of bisphosphonates in early breast cancer. Using the nominal group methodology for consensus, a systematic review of the literature was augmented by a workshop held in October 2014 for breast cancer and bone specialists to present and debate the available pre-clinical and clinical evidence for the use of adjuvant bisphosphonates. This was followed by a questionnaire to all members of the writing committee to identify areas of consensus. The panel recommended that bisphosphonates should be considered as part of routine clinical practice for the prevention of CTIBL in all patients with a T score of <-2.0 or ≥2 clinical risk factors for fracture. Compelling evidence from a meta-analysis of trial data of >18,000 patients supports clinically significant benefits of bisphosphonates on the development of bone metastases and breast cancer mortality in post-menopausal women or those receiving ovarian suppression therapy. Therefore, the panel recommends that bisphosphonates (either intravenous zoledronic acid or oral clodronate) are considered as part of the adjuvant breast cancer treatment in this population and the potential benefits and risks discussed with relevant patients.

Oestrogen receptor positive breast cancer metastasis to bone: inhibition by targeting the bone microenvironment in vivo.

Clinical trials have shown that adjuvant Zoledronic acid (ZOL) reduces the development of bone metastases irrespective of ER status. However, post-menopausal patients show anti-tumour benefit with ZOL whereas pre-menopausal patients do not. Here we have developed in vivo models of spontaneous ER+ve breast cancer metastasis to bone and investigated the effects of ZOL and oestrogen on tumour cell dissemination and growth. ER+ve (MCF7, T47D) or ER-ve (MDA-MB-231) cells were administered by inter-mammary or inter-cardiac injection into female nude mice ± estradiol. Mice were administered saline or 100 µg/kg ZOL weekly. Tumour growth, dissemination of tumour cells in blood, bone and bone turnover were monitored by luciferase imaging, histology, flow cytometry, two-photon microscopy, micro-CT and TRAP/P1NP ELISA. Estradiol induced metastasis of ER+ve cells to bone in 80-100 % of animals whereas bone metastases from ER-ve cells were unaffected. Administration of ZOL had no effect on tumour growth in the fat pad but significantly inhibited dissemination of ER+ve tumour cells to bone and frequency of bone metastasis. Estradiol and ZOL increased bone volume via different mechanisms: Estradiol increased activity of bone forming osteoblasts whereas administration of ZOL to estradiol supplemented mice decreased osteoclast activity and returned osteoblast activity to levels comparable to that of saline treated mice. ER-ve cells require increased osteoclast activity to grow in bone whereas ER+ve cells do not. Zol does not affect ER+ve tumour growth in soft tissue, however, inhibition of bone turnover by ZOL reduced dissemination and growth of ER+ve breast cancer cells in bone.

Human breast cancer bone metastasis in vitro and in vivo: a novel 3D model system for studies of tumour cell-bone cell interactions.

Bone is established as the preferred site of breast cancer metastasis. However, the precise mechanisms responsible for this preference remain unidentified. In order to improve outcome for patients with advanced breast cancer and skeletal involvement, we need to better understand how this process is initiated and regulated. As bone metastasis cannot be easily studied in patients, researchers have to date mainly relied on in vivo xenograft models. A major limitation of these is that they do not contain a human bone microenvironment, increasingly considered to be an important component of metastases. In order to address this shortcoming, we have developed a novel humanised bone model, where 1 × 10(5) luciferase-expressing MDA-MB-231 or T47D human breast tumour cells are seeded on viable human subchaodral bone discs in vitro. These discs contain functional osteoclasts 2-weeks after in vitro culture and positive staining for calcine 1-week after culture demonstrating active bone resorption/formation. In vitro inoculation of MDA-MB-231 or T47D cells colonised human bone cores and remained viable for <4 weeks, however, use of matrigel to enhance adhesion or a moving platform to increase diffusion of nutrients provided no additional advantage. Following colonisation by the tumour cells, bone discs pre-seeded with MDA-MB-231 cells were implanted subcutaneously into NOD SCID mice, and tumour growth monitored using in vivo imaging for up to 6 weeks. Tumour growth progressed in human bone discs in 80 % of the animals mimicking the later stages of human bone metastasis. Immunohistochemical and PCR analysis revealed that growing MDA-MB-231 cells in human bone resulted in these cells acquiring a molecular phenotype previously associated with breast cancer bone metastases. MDA-MB-231 cells grown in human bone discs showed increased expression of IL-1B, HRAS and MMP9 and decreased expression of S100A4, whereas, DKK2 and FN1 were unaltered compared with the same cells grown in mammary fat pads of mice not implanted with human bone discs.

Macrophages as potential targets for zoledronic acid outside the skeleton-evidence from in vitro and in vivo models.

PURPOSE: Multiple cell types of the tumour microenvironment, including macrophages, contribute to the response to cancer therapy. The anti-resorptive agent zoledronic acid (ZOL) has anti-tumour effects in vitro and in vivo, but it is not known to what extent macrophages are affected by this agent. We have therefore investigated the effects of ZOL on macrophages using a combination of in vitro and in vivo models. METHODS: J774 macrophages were treated with ZOL in vitro, alone and in combination with doxorubicin (DOX), and the levels of apoptosis and necrosis determined. Uptake of zoledronic acid was assessed by detection of unprenylated Rap1a in J774 macrophages in vitro, in peritoneal macrophages and in macrophage populations isolated from subcutaneously implanted breast cancer xenografts following ZOL treatment in vivo. RESULTS: Exposure of J774 macrophages to 5 µM ZOL for 24 h caused a significant increase in the levels of uRap1A, and higher doses/longer exposure induced apoptotic cell death. DOX (10 nM/24 h) and ZOL (10 µM/4 h) given in sequence induced significantly increased levels of apoptotic cell death compared to single agents. Peritoneal macrophages and macrophage populations isolated from breast tumour xenografts had detectable levels of uRap1A 24 h following a single, clinically achievable dose of 100 µg/kg ZOL in vivo. CONCLUSION: We demonstrate that macrophages are sensitive to sequential administration of DOX and ZOL, and that both peritoneal and breast tumour associated macrophages rapidly take up ZOL in vivo. Our data support that macrophages may contribute to the anti-tumour effect of ZOL.

The metastatic microenvironment of breast cancer: clinical implications.

Metastasis to bone, and indeed potentially to other sites, results from the numerous interactions between cancer cells, haematopoietic stem cells and normal bone cells within the bone marrow microenvironment. These interactions are in turn influenced by multiple endocrine, paracrine and physical factors. Bone-targeted treatments may modify the course of the disease via both direct and indirect inhibitory effects on this "vicious cycle" of growth factor and cytokine signalling between tumour and bone cells. Improvements in both disease free (DFS) and overall survival in women with early breast cancer have been demonstrated in several large randomised adjuvant trials of oral clodronate and intravenous zoledronic acid. The evidence for a beneficial impact on disease outcome is particularly strong in patients with low levels of reproductive hormones, including pre-menopausal women receiving ovarian suppression therapy and those who have passed through menopause at the time of diagnosis. A recent meta-analysis of postmenopausal women treated with adjuvant bisphosphonates showed an 18% improvement in DFS (hazard ratio [HR] = 0.82; 95%CI 0.74-0.92, 2P = <0.001), with reductions in relapse rates not only in bone but also at extra-skeletal and loco-regional sites. These exciting findings are beginning to change clinical practice.

Location matters: osteoblast and osteoclast distribution is modified by the presence and proximity to breast cancer cells in vivo.

Bone metastasis is a common incurable complication of breast cancer affecting around 70% of patients with advanced disease. In order to improve outcomes for these patients, the cellular and molecular mechanisms underlying bone metastasis need to be established. The majority of studies to date have focused on end-stage disease and little is known about the events taking place following initial tumour cell colonisation of bone. Here we report the results of a longitudinal study that provides detailed analysis of the spatial and temporal relationship between bone and cancer cells during progression of bone metastasis. Tumour growth in bone was initiated by intra-cardiac inoculation of MDA-MB-231-GFP breast cancer cells in immunocompromised mice. Differentiating between areas of bone in direct contact with the tumour and areas distal to the cancer cells but within the tumour bearing bone, we performed comprehensive analyses of the number and distribution of osteoclasts and osteoblasts. Tumour colonies were detectable in bone from day 10, while reduced trabecular bone volume was apparent from day 19 onwards. Cancer-induced changes in osteoblast and osteoclast numbers differed substantially depending on whether or not the cells were in direct contact with the tumour. Compared to naïve controls, areas of bone in direct contact with the tumour had significantly reduced osteoblast but increased osteoclast numbers, whereas the reverse was found in distal areas. Our data demonstrate that tumour cells induce substantial changes in the bone microenvironment prior to the appearance of bone lesions, suggesting that early therapeutic intervention may be required to oppose the tumour-induced changes to the microenvironment und thus tumour progression.

Seed, soil and secreted hormones: potential interactions of breast cancer cells with their endocrine/paracrine microenvironment and implications for treatment with bisphosphonates.

The process of formation of metastasis is undoubtedly inefficient, with the majority of disseminated tumour cells perishing in their metastatic environment. Their ability to survive is determined by their intrinsic abilities, with emerging evidence of the importance of cancer stem cells possessing self propagating potential, but also the interaction with the premetastatic niche, which may either help or hinder their formation into micrometastasis, thus influencing recurrence and survival in breast cancer patients. Use of the bone targeted agents bisphosphonates in the adjuvant setting has been extensively studied in large clinical trials, and demonstrated an interesting interplay with the endocrine microenvironment, with postmenopausal women or premenopausal women receiving ovarian suppression therapy gaining a survival advantage compared to pre/perimenopausal women. The interaction between the endocrine hormones and the paracrine TGFß growth factors may provide an explanation for the differences seen according to ovarian function in the response to bisphosphonates. In this review the evidence of interplay between ovarian endocrine hormones, TGFß paracrine growth factors and bisphosphonates will be presented, and subsequent influence on breast cancer cells in the bone pre-metastatic niche hypothesised.

A single administration of combination therapy inhibits breast tumour progression in bone and modifies both osteoblasts and osteoclasts.

We have previously shown that repeated sequential administration of doxorubicin, followed 24 h later by zoledronic acid, inhibits tumour growth in models of established breast cancer bone metastasis. As breast cancer patients only receive zoledronic acid every 3-4 weeks, the aim of the current study was to establish the anti-tumour and bone effects of a single administration of doxorubicin/zoledronic acid combination therapy in a bone metastasis model. MDA-MB-231-GFP cells were injected i.c. in 6-week-old nude mice. On day 2, animals received PBS, doxorubicin (2 mg/kg i.v.), zoledronic acid (100 µg/kg s.c.) or doxorubicin followed 24 h later by zoledronic acid. Anti-tumour effects were assessed on days 15/23 by quantification of apoptotic and proliferating cells and changes in expression of genes implicated in apoptosis, proliferation and bone turnover. Bone effects were assessed by µCT analysis, bone histomorphometry and measurement of serum markers. A tumour-free control group was included. Combination treatment reduced bone tumour burden compared to single agent or PBS control and increased levels of tumour cell apoptosis on day 15, but this was no longer detectable on day 23. Animals receiving zoledronic acid had increased bone density, without evidence of tumour-induced lesions. Bone histomorphometry showed that zoledronic acid caused a decrease in osteoblast and osteoclast numbers and an increase in osteoclast size, in both tumour-free and tumour-bearing animals. Our data show that although zoledronic acid modifies the bone microenvironment through effects on both osteoblasts and osteoclasts, this does not result in a significant anti-tumour effect in the absence of doxorubicin.

Multidrug resistance in breast cancer: from in vitro models to clinical studies.

The development of multidrug resistance (MDR) and subsequent relapse on therapy is a widespread problem in breast cancer, but our understanding of the underlying molecular mechanisms is incomplete. Numerous studies have aimed to establish the role of drug transporter pumps in MDR and to link their expression to response to chemotherapy. The ATP-binding cassette (ABC) transporters are central to breast cancer MDR, and increases in ABC expression levels have been shown to correlate with decreases in response to various chemotherapy drugs and a reduction in overall survival. But as there is a large degree of redundancy between different ABC transporters, this correlation has not been seen in all studies. This paper provides an introduction to the key molecules associated with breast cancer MDR and summarises evidence of their potential roles reported from model systems and clinical studies. We provide possible explanations for why despite several decades of research, the precise role of ABC transporters in breast cancer MDR remains elusive.

Combined therapies of bone disease with bisphosphonates.

Bisphosphonates are standard treatment for cancer-induced bone disease, a common feature of many advanced malignancies. Traditionally used to inhibit bone turnover and reduce the risk of skeletal-related events, there is now increasing pre-clinical evidence that these agents may also affect tumour burden and disease progression. In particular, combining bisphosphonates with chemotherapeutic agents has been demonstrated to cause substantially increased anti-tumour effects compared to giving the single agents. Clinical studies are in progress to determine whether adding bisphosphonates to standard anti-cancer therapy results in improved outcome for patients. Here we give an overview of the key pre-clinical studies of anti-tumour effects of bisphosphonates, alone and in combination with other agents, and introduce some of the ongoing clinical trials that aim to determine the clinical relevance of bisphosphonates in combination therapy.

Combined effects of the bisphosphonate, zoledronic acid and the aromatase inhibitor letrozole on breast cancer cells in vitro: evidence of synergistic interaction.

BACKGROUND: Aromatase inhibitors are widely used in the treatment of oestrogen receptor-positive post-menopausal breast cancer. These patients may also be receiving the bisphosphonate, zoledronic acid (ZA) to prevent bone loss or reduce skeletal morbidity in the setting of advanced disease. The potential biological interaction of these two drugs in breast cancer has not been assessed. METHODS: Aromatase-expressing breast cancer cells were treated with letrozole and ZA either simultaneously or in sequence, and the resulting apoptosis was assessed by staining with Hoechst 33342 and propidium iodide and examined using a fluorescent inverted Leica DMIRB microscope and a UV filter. RESULTS: We found that letrozole and ZA induce levels of apoptosis in breast cancer cells in vitro that are significantly greater compared with treatment with each drug alone. However, this potentially, synergistic relationship is drug-sequence dependent, occurring only when cells are treated with letrozole, followed by ZA. The converse sequence, or administering drugs simultaneously, induces levels of apoptosis no greater than each drug alone. CONCLUSION: Owing to the enhanced anti-tumour efficacy of sequential drug administration, our findings may indicate that, for post-menopausal women who require treatment with letrozole, ZA should also be considered.

Anti-tumour effects of bisphosphonates--what have we learned from in vivo models?

Bisphosphonates are extensively used to treat cancer-induced bone disease in a range of solid tumours and multiple myeloma, where they reduce the incidence of skeletal related events and improve patients' quality of life. Recent reports indicate that bisphosphonates may also prevent recurrence of breast cancer at peripheral sites, suggesting that these drugs may have anti-tumour effects outside the skeleton. Anti-tumour effects of several bisphosphonates have been reported in a range of tumour cell types in vitro. These positive results have subsequently been supported by investigations of effects of bisphosphonates on tumour growth in vivo, both in bone and at peripheral sites. A reduction of tumour burden and also in cancer-induced bone disease has been reported following bisphosphonate treatment in several model systems, including breast and prostate cancer, osteosarcoma and multiple myeloma. In addition, bisphosphonates have been shown to significantly reduce growth of human tumour cells (including breast, prostate, lung and mesothelioma) implanted subcutaneously in immunocompromised mice. However, the majority of in vivo studies showing a reduction in bone disease and reduced tumour burden have used high doses and frequent administration of bisphosphonates, and the clinical relevance of these data have therefore been the subject of considerable debate. Bisphosphonates may hold greater promise as anti-tumour agents when used in combination with cytotoxic drugs, and several in vivo studies have reported substantial increased inhibition of tumour growth and improved survival when bisphosphonates have been added to standard chemotherapy regimens. This review will summarise the published data on anti-tumour effects of bisphosphonates from in vivo models, alone and in combination with other anti-cancer agents, and highlight the main lessons learned and future challenges in this field.

Mechanisms and pathways of bone metastasis: challenges and pitfalls of performing molecular research on patient samples.

The molecular mechanisms underlying the development of bone metastases in breast cancer remain unclear. Disseminated tumour cells (DTCs) in the bone marrow of breast cancer patients are commonly identified, even in early stage disease, but their potential to initiate metastases is not known. The mechanism whereby DTCs become overt metastatic tumour cells (MTCs) is therefore, an area of considerable interest. This study explored the analysable yield of genetic material from human biopsy samples in order to describe differences in gene expression between DTCs and bone MTCs. Thirteen breast cancer patients with bone metastases underwent a CT-guided bone metastasis biopsy and a bone marrow biopsy. Tumour cells were enriched and gene expression profiling was conducted to identify differentially expressed genes. The analysable yield of sufficient RNA for microarray analysis was 60% from bone metastasis biopsies and 80% from bone marrow biopsies. A signature of 133 candidate genes differentially expressed between DTCs and MTCs was identified. Several genes relevant to breast cancer metastasis to bone (osteopontin, CTGF, parathyroid hormone receptor, EGFR) were significantly overexpressed in MTCs as compared to DTCs. Biopsies of bone metastases and bone marrow rarely yield enough tissue for robust molecular biology studies using clinical samples. The findings obtained however are interesting and seem to overlap with the bone metastasis gene expression signature described in murine xenograft models. Larger biopsy specimens or improved RNA extraction techniques may improve analysable yield and feasibility of these techniques.

Imaging the effects of castration on bone turnover and hormone-independent prostate cancer colonization of bone.

INTRODUCTION: Tumor populations may selectively colonize bone that is being actively remodeled. In prostate cancer patients, androgen deprivation directly inhibits tumor growth initially, whilst induced bone loss may facilitate tumor colonization of bone by androgen-insensitive cells. We have tested this hypothesis using a xenograft model of early growth of prostate cancer in bone. METHODS: PC3 cells transfected with Green fluorescent protein (GFP) were injected into castrated and non-castrated athymic mice via intrabial and intracardiac routes. In vivo tumor growth was monitored daily and animals sacrificed 6-9 days following initial GFP-based detection of tumors. Tumor bearing and contra-lateral non-tumor bearing tibias were analyzed extensively by micro-CT and histology/immunohistochemistry for the presence of tumor cells and the effects of tumor and/or castration on bone cells and bone structure evaluated. RESULTS: GFP-positive tumors in bone were visible from 12 days post-injection following intratibial injection, allowing tumors <1 mm diameter to be monitored in live animals. Castration did not affect tumor frequency, tumor volume, or time to initial appearance of tumors injected via intratibial or intracardiac routes. Castration decreased trabecular bone volume in all mice. Significant tumor-induced suppression of numbers of osteoblasts, coupled with increased numbers of activated osteoclasts, was evident in both intact animals and castrated animals. CONCLUSIONS: In vivo GFP imaging allows the detection of early tumor growth at intra-osseous sites. Castration induces bone loss, but PC3-GFP cells are also capable of inducing bone remodeling in intact animals at early time points, independently of pre-existing castration-induced alterations to bone.

Exploring the anti-tumour activity of bisphosphonates in early breast cancer.

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and are firmly established in the management of breast cancer patients with metastatic skeletal disease. There are extensive data that bisphosphonates, particularly nitrogen-containing bisphosphonates such as zoledronic acid, exhibit anti-tumour activity potentially via both indirect and direct mechanisms in vitro. In vivo studies using animal models of breast cancer induced bone disease have shown that bisphosphonates exert anti-tumour effects via inhibiting osteolysis and reducing skeletal tumour burden. Furthermore, pre-clinical studies have demonstrated synergistic anti-tumour effects between chemotherapy agents commonly used in breast cancer treatment and nitrogen-containing bisphosphonates. This, coupled with emerging evidence from pre-clinical in vivo studies suggesting that bisphosphonates may have additional anti-tumour activity outside of the bone microenvironment, could be of significant importance in the clinical management of breast cancer. The evidence in favour of an anti-tumour effect of bisphosphonates in the clinical setting is inconclusive however, with conflicting evidence from several trials. This review focuses on the anti-tumour activity of bisphosphonates in breast cancer, with particular focus on zoledronic acid. The pre-clinical evidence for anti-tumour activity will be reviewed, followed by the synergistic effects with anti-cancer agents. Finally, the clinical relevance and strategies for the evaluation of anti-tumour activity in breast cancer will be discussed. We are currently exploring the potential synergistic anti-tumour effects of the sequential treatment of neoadjuvant chemotherapy followed by zoledronic acid in a randomised phase II study evaluating biological endpoints including apoptosis, proliferation and angiogenesis in patients with breast cancer.

Phenotypic variations of TRAIL sensitivity in cloned populations of prostate cancer cells.

Factors that regulate the induction of apoptosis of tumour cells are potential candidates for therapeutic intervention for the majority of cancers. Studying modifiers of apoptotic responses, such as members of the tumour necrosis factor receptor superfamily, may give clues as to how induction of apoptosis in tumours could be maximized to enhance the benefit of treatment regimes. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumour molecule since its activity is specific for tumour cell populations. TRAIL binds to death receptors, inducing apoptosis in susceptible cells. The mechanisms which determine whether tumour cells are susceptible to TRAIL are unclear, and several mechanisms have been proposed, including expression of osteoprotegerin (OPG), decoy receptors, and factors that affect intracellular signalling of pro-apoptotic molecules, such as c-FLIP. Here we show that experiments to modulate the activity of one of these factors, OPG, by over-expression and also by stable knockdown of OPG expression, alters the TRAIL sensitivity of PC3 prostate cancer cells. However we show that some observed effects, which appear to support the hypothesis that OPG prevents TRAIL-induced apoptosis of tumour cells, may be due to variation of the TRAIL response of sub-clones of tumour cells, even within a cloned population. These results highlight potential limitations of experiments designed to test contribution of factors affecting intrinsic apoptosis susceptibility using cloned tumour cell populations.

Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro.

Breast cancer patients often receive both paclitaxel and zoledronic acid as part of their treatment, and these drugs are reported to have synergistic effects on the induction of apoptosis of breast cancer cells in vitro. We have found that the synergistic interaction is drug sequence dependent, with maximal levels of apoptosis achieved when cells are treated with paclitaxel followed by zoledronic acid, as opposed to the reverse sequence or simultaneous treatment. The synergistic interaction persists at clinically relevant concentrations and incubation periods. We report that the sequential treatment is associated with cell cycle changes and depends on breast cancer cell characteristics, with hormone independence, mutated p53 status and presence of BRCA1 gene being associated with higher levels of apoptosis. Finally, we have found that the synergistic induction of apoptosis is via zoledronic acid-mediated inhibition of the mevalonate pathway.

Expression of receptor activator of nuclear factor kappabeta ligand (RANKL) and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) in breast cancer, and their relations with osteoprotegerin, oestrogen receptor, and clinicopathological variables.

BACKGROUND: Receptor activator of nuclear factor kappabeta ligand (RANKL) has an important role in bone remodelling, and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) can induce apoptosis in cancer cells. Their functions are linked by their interactions with osteoprotegerin (OPG). OBJECTIVE: To investigate the expression of RANKL and TRAIL in a large series of unselected breast cancers and to analyse the relations between these expressions and the expression of OPG, oestrogen receptor, and clinicopathological variables. METHODS: 395 breast cancers were sampled into tissue microarrays and immunohistochemistry undertaken for RANKL and TRAIL. RESULTS: There was strong expression of RANKL in 14% of the cancers and strong expression of TRAIL in 30%. Expression of RANKL had a negative association with expression of oestrogen receptor (p = 0.036). Expression of TRAIL had a negative association with the Nottingham Prognostic Index (p = 0.021). There was a significant negative relation between expression of RANKL and TRAIL (p<0.005). Unsupervised cluster analysis produced a dendrogram that showed a clear division into two groups, and the expression of oestrogen receptor was significantly higher in one of those groups (p = 0.012). CONCLUSIONS: There is apparent loss of expression of RANKL in 86% of breast cancers; those tumours that retain expression tend to be oestrogen receptor negative and of a high histological grade. There is strong expression of TRAIL in 30% of breast cancers and these tend to be of better prognostic type. These results may be important in the processes of metastasis to bone and the apoptotic cell death pathway in cancer.

Expression of osteoprotegerin (OPG), TNF related apoptosis inducing ligand (TRAIL), and receptor activator of nuclear factor kappaB ligand (RANKL) in human breast tumours.

BACKGROUND: Osteoprotegerin (OPG) is involved in the regulation of bone turnover through binding to the receptor activator of nuclear factor kappaB ligand (RANKL), and has also been reported to be a potential survival factor for several different cell types. The survival effects are mediated through inhibition of the activity of tumour necrosis factor related apoptosis inducing ligand (TRAIL). Both breast and prostate cancer cells produce sufficient amounts of OPG to be protected against the effects of TRAIL in vitro. AIMS: To investigate the spatial expression of OPG, RANKL, and TRAIL in non-neoplastic breast tissue and breast cancer, and its relation with oestrogen receptor (ER) expression. METHODS: Forty breast cancers (20 ER+, 20 ER-) and five non-neoplastic breast tissue samples were stained with antibodies against OPG, RANKL, and TRAIL. RESULTS: OPG was not expressed in non-neoplastic breast tissue except when colocalised with altered columnar epithelium. RANKL was expressed at the apical surface of luminal epithelial cells and TRAIL was expressed in myoepithelial cells. All three proteins were expressed in some breast cancers but showed no significant association with tumour type. OPG expression showed a significant positive correlation with ER expression (p = 0.011). CONCLUSIONS: This is the first published study of the spatial expression of OPG, RANKL, and TRAIL in breast tissue and breast cancer. The localisation of each protein was specific and they were not colocalised. This specificity may provide a useful marker of functional differentiation in breast cancer; for example, TRAIL expression as a marker of myoepithelial differentiation.