Breast cancer as a systemic disease: a view of metastasis

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1 Review Click here for more articles from the symposium doi: /joim Breast cancer as a systemic disease: a view of metastasis A. J. Redig 1 & S. S. McAllister 1,2,3 From the 1 Division of Hematology, Department of Medicine, Brigham and Women s Hospital, Harvard Medical School; 2 Harvard Stem Cell Institute, Boston; and 3 Broad Institute of Harvard and MIT, Cambridge, MA, USA Abstract. Redig AJ, McAllister SS (Brigham and Women s Hospital, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA; Broad Institute of Harvard and MIT, Cambridge, MA, USA). Breast cancer as a systemic disease: a view of metastasis. (Review). J Intern Med 2013; 274: Breast cancer is now the most frequently diagnosed cancer and leading cause of cancer death in women worldwide. Strategies targeting the primary tumour have markedly improved, but systemic treatments to prevent metastasis are less effective; metastatic disease remains the underlying cause of death in the majority of patients with breast cancer who succumb to their disease. The long latency period between initial treatment and eventual recurrence in some patients suggests that a tumour may both alter and respond to the host systemic environment to facilitate and sustain disease progression. Results from studies in animal models suggest that specific subtypes of breast cancer may direct metastasis through recruitment and activation of haematopoietic cells. In this review, we focus on data implicating breast cancer as a systemic disease. Keywords: breast cancer, disseminated tumour cells, metastasis, tumour dormancy, tumour microenvironment, systemic instigation. Introduction Despite advances in the diagnosis and treatment of human malignancy, cancer remains amongst the leading causes of morbidity and mortality worldwide, with 7.5 million deaths attributed to cancer in 2008 ( accessed August 12, Breast cancer is now the most frequently diagnosed cancer and the leading global cause of cancer death in women, accounting for 23% of cancer diagnoses (1.38 million women) and 14% of cancer deaths ( women) each year [1]. Although breast cancer has a markedly higher incidence in developed countries, half of new breast cancer diagnoses and an estimated 60% of breast cancer deaths are now thought to occur in the developing world [1]. Metastatic disease, or the spread of tumour cells throughout the body, is responsible for the vast majority of cancer patient deaths and represents the central clinical challenge of solid tumour oncology. The importance of understanding the mechanisms underlying the metastatic process and the complex interactions between tumour and host during disease progression has been widely recognized and was amongst the subjects discussed at the Nobel Conference on Breast Cancer: Progress and Challenges in Prevention, Risk Prediction, Tumor Biology and Treatment in Stockholm, Sweden, June Our own findings using a breast cancer xenograft model were also presented at this meeting. The aim of this review is to evaluate key clinical and laboratory observations surrounding our emerging understanding about the complexity of metastasis and to consider fundamental questions about the nature of metastatic disease. The clinical challenge of metastasis The decrease in breast cancer-related deaths that has been observed in the developed world since the early 1990s is attributed in part to improved screening, which allows diagnosis at a stage when curative therapy is still an option [2]. It is clear that improving strategies to diagnose and treat breast cancer should remain a high priority in the 21st century. Unfortunately, however, some patients with cancer initially present with distant metastases; these patients are diagnosed with Stage IV disease that is nearly always incurable. Other patients, without detectable metastases at the time of diagnosis, will eventually recur with disease in distant organs. Amongst women diagnosed with breast cancer, only a minority presents with Stage IV disease. Nevertheless, nearly 30% of women initially diagnosed with early-stage disease will ª 2013 The Association for the Publication of the Journal of Internal Medicine 113

2 ultimately develop metastatic lesions, often months or even years later [3]. The goal of tumour staging algorithms is to help determine a patient s recommended therapy based on several features of the underlying malignancy believed to predict the risk of recurrence. Treatment options reflect the underlying staging work-up in two critical ways. First, with an initial diagnosis of Stage IV metastatic disease, multidisciplinary treatment strategies focus on palliation of symptoms, rather than aggressive surgical or medical treatments that are associated with a high degree of morbidity. Second, for patients without detectable metastatic lesions at the time of diagnosis but with features of high-risk disease, the purpose of adjuvant chemotherapy and/or radiation is to destroy the undetectable, occult micrometastases a patient may harbour to prevent future recurrent disease. Standard staging scans or surgical lymph node dissection does not detect individual disseminated tumour cells (DTCs), but the epidemiology of metastatic disease suggests that some tumour cells escape the primary tumour prior to surgical resection. Although strategies for targeting the primary tumour have markedly improved, targeted therapies directed against the elusive micrometastatic population the cells that escaped have been less effective. Despite multidisciplinary treatment algorithms, metastatic disease remains the underlying cause of death in the majority of patients with breast cancer who succumb to their disease [1, 2]. Unfortunately, current clinical strategies fall short both in accurately identifying patients at high risk of recurrence and in treating patients with metastatic disease. A model for metastasis Using our unique breast cancer xenograft model, we have gained insight into the processes by which a malignancy alters and responds to the host environment in order to facilitate and sustain disease progression. It is clear that the manner in which the body responds to certain breast malignancies creates a systemic environment that is amenable to the outgrowth of microscopic disseminated tumours that would otherwise have remained indolent. This breast cancer xenograft model relies on implanting a specific type of aggressively growing human breast tumour into one anatomical site (e.g. subcutaneous flank or orthotopic site) of a nude mouse to elicit a host response, and injecting indolent human breast tumours into distant anatomical sites (e.g. contralateral flank or lung via tail vein injection) to assess the response to the host systemic environment [4 6]. These tumours have no direct physical contact, so the aggressive instigating tumour must induce systemic changes from a distance to drive the otherwise indolent responding tumour to grow. We termed this process systemic instigation, and further mechanistic studies indicated that haematopoietic cells from the bone marrow are key drivers of the process [4 6]. Specifically, bone marrow-derived cells are recruited to the responder tumours and contribute to the development of the tumour microenvironment, thereby allowing the previously indolent tumour cells to grow. Of note, not all breast tumours are able to act as instigators, suggesting that specific features of the primary tumour are required to drive systemic changes in micrometastatic populations. Our model holds particular promise for the study of breast cancer because it represents an in vivo opportunity to dissect the process responsible for incurable metastatic disease, that is, the conversion of indolent micrometastases into overt clinically significant metastases. The indolent responding tumours in this model are analogous to the clinical scenario in which tumour cells disseminate from the primary tumour only to remain dormant in distant tissues for months or even years before continuing to grow as metastatic disease. The instigating tumours are analogous to those primary tumours (or possibly dominant metastatic lesions) that retain the ability to influence the outgrowth of otherwise indolent cancer cells. Some but not all patients with breast cancer will develop recurrent disease, just as some but not all tumours in the xenograft model possess the ability to elicit responder tumour growth. Using this model, the results of mechanistic studies also suggest the possibility of stratifying breast cancers on a functional basis; such classification would directly identify those tumours most likely to drive future metastatic outgrowth. Does traditional classification of breast tumours apply to DTCs? Ongoing research continues to demonstrate the underlying genetic and molecular diversity of breast cancer, suggesting that understanding tumour host interactions may depend upon understanding the unique characteristics of the primary tumour subtype. Traditionally, breast cancer classification relies greatly on the underly- 114 ª 2013 The Association for the Publication of the Journal of Internal Medicine

3 ing histopathological features of the primary tumour, including expression of the oestrogen (ER) and progesterone receptor (PR) and amplification of the HER2/neu oncogene product. Under current clinical guidelines, pathology-based review of tumour tissue and determination of tumour staging provide some information about prognosis as well as suggesting treatment options, notably with the use of anti-oestrogen therapies such as tamoxifen or the anti-her2 agent trastuzumab (Herceptin) in appropriate patient populations [7]. Emerging technology has also been used to develop more detailed molecular profiles of breast cancer subtypes, with some clinical application. Several commercially available methods, including the Oncotype Dx assay (21-gene signature) and the Mammaprint assay (70-gene signature), have been approved for clinical use within the last 10 years to help predict the chance of recurrence and guide therapy in defined patient subsets [8, 9]. More recently, whole-genome profiling of breast cancer has demonstrated that, from a molecular standpoint, breast tumours fall into four general categories: basal-like (which generally corresponds to triple-negative disease), luminal-a (generally ER positive and low grade), luminal-b (also ER positive but high grade) and Her2-positive tumours [10 13]. This evolving paradigm of the molecular subclassification of breast cancer suggests exciting new directions from which to investigate the interactions between primary tumour and systemic environment during the metastatic cascade. In many studies, distinct genes or pathways within a given subtype of breast cancer that appear to play an important prognostic role, including with regard to metastatic behaviour, are now being identified [14, 15]. These approaches have their limitations, and significant gaps in our ability to use either histopathology or molecular profiling to effectively predict recurrence still remain. For instance, it is not yet clear how to combine traditional histopathology, results from limited gene assays (which cannot be used in all patient populations) and whole-genome profiling into a clinically useful algorithm [16]. Findings from molecular profiling studies support clinical observations that breast cancer as a disease entity has several distinct patterns whilst also demonstrating significant heterogeneity amongst patients. In particular, how to effectively utilize molecular classification in the clinical context and whether the same parameters apply to metastatic disease remain unclear. However, novel methodology may eventually change the way in which breast cancer is classified and the way in which traditional biomarkers are evaluated. It was recently demonstrated that whole-genome sequencing of circulating cell-free DNA could be used to identify cancer-associated chromosomal markers in a subset of women with breast cancer [17]. Several studies are assessing the utility of evaluating patient blood samples for circulating tumour cells (CTCs) during risk stratification in the diagnosis of early-stage breast cancer [18] and to predict the likely response in patients undergoing preoperative chemotherapy [19]. There is also some retrospective evidence to suggest that the presence or absence of CTCs in patients with metastatic breast cancer may be of important prognostic significance regardless of molecular subtype [20]. The ability to detect isolated tumour cells outside a primary tumour has led to several attempts to correlate traditional histopathological markers of breast cancer with the presence of either DTCs found in bone marrow or CTCs found in peripheral blood. However, to date, these studies have yielded inconclusive results [8, 9]. In one study of surgical patients, no association was found between the presence of DTCs or CTCs and other markers typically used to assess tumour behaviour, such as tumour size or grade and hormone receptor or lymph node status, or use of neoadjuvant chemotherapy [21]. In a further series of 92 women with early breast cancer, bone marrow aspirates and peripheral blood samples were obtained at the time of surgery, and there was similarly no statistically significant correlation between the presence of CTCs or DTCs and tumour size or grade and lymph node or ER/PR/HER2 status [22]. Nevertheless, some interesting trends were noted when the presence of DTCs was compared to ER, PR and HER2 expression. DTCs were identified in 23% of patients with ER-positive disease but 37% with ERnegative disease; 22% of patients with PR-positive disease but 32% with PR-negative disease; and 0% of patients with HER2-positive disease but 29% with HER2-negative disease [22]. Histopathological subtyping might not provide a basis for predicting the presence or absence of DTCs, but these data suggest that the presence of DTCs varies amongst patients and may reflect underlying functional differences between primary tumours. ª 2013 The Association for the Publication of the Journal of Internal Medicine 115

4 Of particular interest, recent findings challenge previously held assumptions that the histopathological biomarkers of the primary tumour are repeated in metastases. In two recent studies, it was demonstrated that the ER, PR or HER2 status of a biopsy-proven metastatic lesion does not match the pathology of the primary tumour in a substantial fraction of patients (ranging from 15% to 40% of the patient cohort) [23, 24]. Such differences are also seen in the CTC and DTC pools when breast cancer biomarkers are evaluated. In a study focusing on developing a multimarker panel for the characterization of CTCs in patients with metastatic breast cancer, it was incidentally noted that the CTCs were HER2 positive in 27% of the patients with HER2-negative disease [25]. In an earlier study with 254 patients evaluating ERpositive DTCs, the concordance between primary tumour and DTC was only 28% [26]. Furthermore, within the population of DTCs, ER expression was heterogeneous in 26% of patients [26]. The findings of a study comparing the evolution of bone marrow DTCs to that of peripheral blood CTCs also suggest that these two populations may be quite different even within an individual patient. DTCs in the marrow were found with much greater frequency (24% of 431 patients) than CTCs in the peripheral blood (13%) [27]. In addition, DTCs in the bone marrow correlated only with the PR status of the primary tumour, whereas CTCs in the blood correlated with ER status, PR status and node positivity of the primary tumour. A weak (P = 0.05) correlation was noted between the biomarker expressions of DTCs and CTCs from the same patient. However, CTCs in the blood were significantly associated with triple-negative primary tumours and were nearly always triple negative themselves [27]. Consequently, DTC/CTC survival and proliferation into overt metastases may be related to the ability of these cells to regulate critical signalling pathways already known to drive breast cancer cell growth, specifically pathways involving oestrogen, progesterone and HER2. Furthermore, as these studies demonstrate, the histopathology of the primary tumour may not always direct the best treatment options for targeting metastatic disease. From micrometastases to macrometastases Although CTCs can be found with reproducible frequency in the peripheral blood of patients with breast cancer, results from preclinical studies suggest that the majority of CTCs/DTCs will not form a clinically detectable overt metastasis [28]. Indeed, results suggest that <1% of all DTCs are capable of forming an overt tumour [28, 29]. Preclinical findings also indicate that cells from within a heterogeneous primary tumour intravasate into the vasculature, survive in the circulation and extravasate into the parenchyma of a distant tissue. Within this foreign environment, it is thought that DTCs exist in a state of dormancy for unknown and varying periods of time. Some micrometastatic foci will ultimately form overt tumours, ostensibly through their ability to recruit the necessary stroma and vasculature, whilst avoiding detection and elimination by the host immune system [30, 31]. Metastatic inefficiency is thought to explain why some patients present with clinically detectable metastases months or even years after their initial cancer diagnosis and surgery [32 34]. The ultimate determinants of whether or not a given tumour cell or population of cells can give rise to a clinically significant metastasis may thus include not only the biology of the cancer cells themselves but also the relationship between tumour and host. Differences between signalling pathways required for invasion and motility, notably those required for adaptation to a new tissue environment versus those that drive proliferation of an established lesion, may in part explain metastatic inefficiency. Nevertheless, the underlying mechanisms of these distinct processes are not clearly understood. Moreover, there is currently no reliable way to determine which, if any, of the DTCs/CTCs found in an individual patient with cancer will eventually result in overt metastatic disease, making it impossible to translate the technical ability to isolate DTCs/CTCs into a clinically useful algorithm to guide treatment based on a patient s likelihood of recurrence. Determining how to identify which cells pose the greatest threat of recurrent disease as well as elucidating the key molecular mechanism(s) required for their survival and development following dissemination is thus of critical importance in order to target these cells before they lead to clinically devastating outcomes. The presence of DTCs in the bone marrow of women without evidence of metastatic breast cancer has recently been shown to be an independent predictor of decreased recurrence-free survival [35]. The prognostic significance of DTCs also appears to extend to women diagnosed with later stages of 116 ª 2013 The Association for the Publication of the Journal of Internal Medicine

5 breast cancer in whom chemotherapy is incorporated into treatment modalities. It has recently been shown that the presence of DTCs correlates with decreased survival in women receiving neoadjuvant chemotherapy, irrespective of whether DTCs were identified prior to chemotherapy [36]. It is significant that the women in this study were diagnosed with locally advanced disease and represent a subset of patients for whom definitive cure is possible with systemic chemotherapy. The fact that DTCs can be identified and have prognostic significance in breast cancers diagnosed at both early and later stages suggests that the mobilizing events enabling these cells to disseminate represent a pivotal sequence in metastatic progression, and that DTCs may directly or indirectly support the outgrowth of metastases. The relationship between the DTCs in the bone marrow and the CTCs found in peripheral blood adds additional complexity to this systemic view of breast cancer. Are CTCs trafficking directly from the primary tumour or do they represent DTCs from the bone marrow niche? Are metastatic lesions in end organs seeded from the CTC pool or the DTC pool? Which cohort of tumour cells in the periphery represents the population most likely to survive and develop into a metastatic lesion and are thus the best cells to target with chemotherapy or assess for prognostic significance? In some patients, CTCs decrease in number or disappear altogether with chemotherapy [37], but is this sufficient to prevent recurrence? A recent meta-review of 49 different studies collectively enrolling over 6800 patients demonstrated that, like DTCs, CTCs are a statistically significant prognostic marker of disease-free survival in women with early-stage breast cancer as well as progression-free survival in women with metastatic disease [38]. However, despite an overall prognostic role for CTCs, their specific function throughout tumour evolution remains to be determined. In women with locally advanced disease, compared to DTCs, peripheral blood CTCs were found with much lower frequency (~5% of patients before adjuvant chemotherapy compared to 21% of patients with DTCs) [36]. Breast cancer-specific survival was lower for patients with CTCs, but the presence of CTCs was not as prognostically useful as that of DTCs in this population [36]. By contrast, in two studies of women with metastatic disease, the presence of CTCs was found to be an inverse predictor of progression-free survival [39, 40]. Interestingly, surgical data have shown that patients with invasive lobular carcinoma are more likely than those with invasive ductal carcinoma to have detectable DTCs or CTCs at the time of surgery [21]. Together, these findings begin to suggest that the characteristics and function of the CTC population, in particular, may change over time whilst also reflecting both the underlying biology of the primary tumour and the evolution of a systemic response in the host during tumour progression. Functional stratification: a new way to look at breast cancer? As mentioned above, current breast cancer staging is heavily dependent upon the evaluation of pathology specimens. However, recent findings suggest that functional classification of breast tumours may become an important addition to risk prediction and prognosis. Results from preclinical animal models suggest that it might be possible to classify breast cancers on a functional basis, as determined by their ability to promote outgrowth of micrometastatic tumour populations at distant sites. Of note, as presented at the recent Nobel Conference on Breast Cancer, we have the ability to use human tumour cell lines and fresh surgical specimens in our xenograft model to test their ability to promote systemic instigation and/or respond to a pro-tumorigenic host systemic environment [4 6]. The ability to determine whether or not a given tumour has the potential to promote the dissemination of tumour cells from the primary tumour, support the proliferation of otherwise indolent disseminated cells or activate systemic signalling pathways that recruit bone marrow cells to the developing tumour stroma of a metastatic lesion would have significant implications for treatment strategies [41, 42]. The ability to use tumour tissue in functional assays to predict tumour behaviour may enable more accurate identification of patients with a high likelihood of future relapse, thereby allowing for potentially curative treatment during the therapeutic window in which the disease can be controlled. Systemic promotion of angiogenesis The significance of angiogenesis in supporting tumour growth is well established [43], and the role of the angiogenic switch in facilitating outgrowth of dormant metastases continues to be an ª 2013 The Association for the Publication of the Journal of Internal Medicine 117

6 active area of investigation [44 46]. It is noteworthy that the vascular endothelial growth factor (VEGF) inhibitor bevacizumab was initially approved by the US Food and Drug Administration for the treatment of metastatic breast cancer in 2008 [47]. However, approval was revoked in late 2011 after further studies failed to show significant benefit on long-term survival or quality of life [48]. The results of large-scale studies utilizing bevacizumab to treat patients with breast cancer were disappointing given the overwhelming laboratory data pointing to a central role for angiogenesis in metastatic growth. Current translational research efforts are now focusing on studies to define more precisely the patient population in which the benefits of anti-angiogenic therapy are likely to outweigh the risks. Our xenograft model may help to define a specific context in which pro-angiogenic signalling is of critical importance. We recently reported a model of systemic instigation that relies on promotion of angiogenesis, whereby cytokines secreted from certain instigating human luminal breast tumours lead to recruitment of pro-angiogenic platelets and VEGF receptor 2 (VEGFR2)-positive bone marrowderived cells to the responding tumour with subsequent stimulation of angiogenesis [6] (Fig. 1a). Importantly, a variety of pro-angiogenic cytokines were enriched in the platelets from mice bearing the instigating luminal tumours relative to those from cancer-free hosts, but VEGF was not one of these cytokines. Our findings are consistent with other data, for example, from mouse models using human breast cancer xenografts showing that oestrogen signalling helped mobilize and recruit pro-angiogenic myeloid cells from the bone marrow to distant tumour sites [49, 50]. The association between increased angiogenesis in evolving metastases from some subtypes of breast cancers but not others, as well as the need to target pro-angiogenic cytokines in addition to VEGF, may begin to explain the less than remarkable results when bevacizumab was introduced into clinical protocols. Our findings suggest that the patients most likely to benefit from targeted anti-angiogenic therapy may be those with certain luminal breast cancers whose tumours have been primed to utilize angiogenic pathways during metastasis. Hypoxia and the systemic environment The role of hypoxia in driving tumour growth is well established in a wide range of tumour types [51, 52], including breast cancer [53]. Emerging data now suggest that hypoxia within the tumour microenvironment may also serve as a trigger for systemic changes in the macroenvironment that promotes metastasis. In a recent study using a mouse orthoptic implantation model, activation of hypoxia-inducible factors (HIFs) within a tumour led to HIF-dependent recruitment of mesenchymal stem cells to the site of the primary tumour followed by subsequent metastasis of breast cancer cells to lymph nodes and lung [54]. Notably, downstream growth factors activated by HIFs facilitated not only the process of metastasis but also the homing of necessary stromal cells to the primary tumour [54]. In a separate study using human breast cancer orthografts in a mouse model, it was demonstrated that HIF-1 expression specifically promotes metastasis through lymphatic channels by directly transactivating plateletderived growth factor B that serves as a chemotactic agent for lymphatic endothelial cells [55]. This finding raises the intriguing possibility that the specific route of tumour cell metastasis may reflect the milieu of the primary tumour. Molecular changes induced by hypoxia may also be specific to certain subtypes of breast cancer, with resulting implications for design and utilization of targeted therapies. In one study, expression of CD44, cell-surface glycoprotein and a marker associated with stem-like behaviour, was increased under hypoxic conditions specifically in triple-negative breast cancer cell lines [56]. Furthermore, it was recently reported that the protein SHARP1 is a crucial negative regulator of HIFinduced migration in triple-negative breast cancer cells and in primary tumours [57]. In a cohort of 637 breast cancer tumour samples, including 120 specimens from women with triple-negative breast cancer, expression of the hypoxia-associated protein CAIX was significantly associated with triplenegative tumours; 25% of triple-negative tumour specimens expressed CAIX compared with only 7% of the general tumour cohort [58]. In addition, overall survival was decreased in patients expressing high levels of CAIX [58]. In a separate study, expression of the toll-like receptor-9 (TLR9) was induced by hypoxia in breast tumours yet with different functional effect depending upon the specific tumour subtype [59]. In particular, in ERpositive tumours, introduction of sirna targeting TLR9 did not affect hypoxia-induced invasion, but sirna knockdown of TLR9 augmented invasion in triple-negative tumours [59]. Together, these find- 118 ª 2013 The Association for the Publication of the Journal of Internal Medicine

7 (a) Instigating tumour e.g., Luminal breast cancer primary tumour or growing metastatic lesion Pro-angiogenic platelets Circulation Responding tumour e.g., Indolent disseminated tumour cells Normal platelets Aspirin BV Unidentified signal Bone marrow Bone marrow derived cells CD24 + CD24 VEGFR1 VEGFR2 Other BMCs (b) Instigating tumour e.g., Triple negative breast cancer primary tumour or growing metastatic lesion Responding tumour e.g., Indolent disseminated tumour cells Osteopontin and other cytokines Bone marrow GRN + bone marrow derived cells GRN Myofibroblast/CAF Fibroblast GRN GRN + Sca + ckit CD45 + BMCs Fig. 1 (a) Model of systemic instigation by luminal breast cancer. Using our preclinical model, we demonstrated that in hosts with luminal breast cancer, otherwise indolent tumour cells implanted at distant anatomical sites develop into an actively growing tumour. The responding tumour growth is stimulated by pro-angiogenic platelets and bone marrow-derived VEGFR2-positive cells that are recruited to the responding tumour site. As a consequence of this systemic cascade, the responding tumour stroma becomes highly vascularized, and the tumour cells become enriched in the cell-surface marker CD24, a ligand for platelet-specific p-selectin. Of note, this process is distinct from the growth of responding tumours in hosts with triple-negative breast cancer (see Fig. 1b). (b) Model of systemic instigation by triple-negative breast cancer. In hosts with triple-negative breast cancer, otherwise indolent tumour cells implanted at a distant anatomical site develop into an actively growing tumour. Instigating triple-negative breast tumours secrete osteopontin (OPN) and other cytokines into the circulation; this subsequently mobilizes a distinct subpopulation of bone marrow cells characterized by Sca1+/cKit / CD45+ and granulin (GRN) expression. After trafficking to the responding tumour site, these bone marrow-derived cells activate cancer-associated fibroblasts that support tumour growth. ª 2013 The Association for the Publication of the Journal of Internal Medicine 119

8 ings suggest a more nuanced role for hypoxia in modulating tumour metastasis that may depend upon the underlying features of the primary tumour. Systemic promotion of stromal desmoplasia In contrast to luminal instigating tumours that activate pro-angiogenic pathways, we found that some triple-negative instigating tumours establish a tumour-supportive systemic environment that promotes stromal desmoplasia at sites of disseminated responding tumours (Fig. 1b). This systemically induced microenvironment is enriched with reactive myofibroblasts similar to the situation observed in pathology specimens from patients with invasive adenocarcinomas [60, 61]. Under these instigating conditions, responding tumours incorporate pro-tumorigenic host bone marrowderived cells into the tumour microenvironment; however, this was not observed in indolent tumours in control mice [5]. Notably, haematopoietic bone marrow cells from animals bearing these instigating tumours are capable of inducing a desmoplastic reaction when combined with responding tumour cells, yet further experiments demonstrated that these haematopoietic cells do not differentiate into myofibroblasts. Instead, a subset of Sca1+/cKit / Cd45+ bone marrow-derived cells are selectively recruited to responder tumours where they subsequently activate tissue fibroblasts to form the tumour-supportive myofibroblasts that enable tumour growth. These haematopoietic cells act by secreting granulin, a cytokine known to regulate cell growth, fibrosis, inflammation and wound healing [62]. Upon examination of tumour specimens from a cohort of patients with breast cancer, high granulin expression was correlated with triplenegative breast cancer and reduced survival [5]. It is noteworthy that bone marrow cells from mice bearing triple-negative instigating tumours are rendered pro-tumorigenic even prior to their mobilization into the circulation [4]. This process is dependent upon secretion of the protein osteopontin by the primary tumour [4]. Osteopontin has been found at elevated levels in the serum of patients with metastatic breast cancer and is associated with both poor prognosis and increased metastatic burden [63]. Furthermore, the kinetics of this growth process demonstrate that systemic instigation takes time to prime the host environment before responding tumours start to grow, implicating a secreted factor (or factors) in bone marrow-derived cell recruitment. Osteopontin is one such factor, but additional components of the signalling pathways remain to be identified, as osteopontin was necessary, but not sufficient for systemic tumour promotion [4]. Integrating histopathological and functional models of tumour classification Overall, data concerning the functional behaviour of different types of breast cancers indicate that the host systemic response to certain aggressively growing tumours creates a systemic environment that is amenable to the outgrowth of tumour cells that have already disseminated to distant sites but that would otherwise remain indolent in the absence of these systemic signals (Fig. 1b). In our xenograft model, tumours with the ability to promote systemic instigation and responder tumour growth have been restricted to either triple-negative [4, 5] or luminal [6] subtypes. Importantly, the specific histopathological classification of the primary instigating tumour appears to determine the type of stromal microenvironment that develops in the distant responding tumour, as the same population of responding tumour cells was used in the models of both luminal and triplenegative instigators (Fig. 1). The response to the presence of triple-negative instigating tumours results in a desmoplastic stroma enriched in myofibroblasts [5], whilst the response to luminal instigating tumours results in increased angiogenesis within responding tumour stroma [6]. Although we identified a correlation between the type of instigating mechanism and tumour receptor status, it remains unclear whether instigating ability is directly associated with ER/PR/Her2 expression. However, our findings suggest that integration of data from histopathology, molecular profiling and determination of the hallmarks of instigating functional activity may in future improve diagnostic and therapeutic strategies. At present, it is clear that patients diagnosed with metastatic disease have a significantly decreased overall survival rate compared to those with localized disease (Table 1), but to our knowledge, no longitudinal, prospective analysis of relevant tumour classification markers has been conducted in a large cohort of patients with clinical end-points, including progression-free and overall survival. An expanded perspective on biological functions of certain carcinomas could help to explain certain 120 ª 2013 The Association for the Publication of the Journal of Internal Medicine

9 Table 1 A. Relative breast cancer survival by stage at diagnosis [78]. B. Breast cancer survival rates in patients with metastatic disease A Breast cancer stage at diagnosis Five-year relative survival rate (%) Localized 98.6 Regional 83.8 Distant 23.4 Unknown 52.4 B Metastatic breast cancer Median overall by subtype survival (months) Her2 positive (n = 113) [79] 38.0 Her2 negative (n = 62) [80] 12.3 Triple negative (n = 53) [81] 32.8 Data shown here are from selected studies illustrating two significant trends in breast cancer mortality. First, Table 1(a) shows a clear survival advantage for patients diagnosed with localized disease with spread limited to regional nodes. Data are derived from the SEER database of the National Cancer Institute and are representative of survival statistics in the developed world. However, whilst patients with distant metastases do have dramatically decreased overall survival compared to patients with more limited disease, the significance of molecular classification in patients with metastatic disease remains unclear. Table 1(b) shows data from three small cohort studies of patients with metastatic disease enrolled in clinical trials. Variables such as patient age, comorbidities, initial disease presentation and prior therapy have not been adjusted between studies. Furthermore, many clinical trials in patients with metastatic breast cancer do not include overall survival as a study end-point, limiting the comparisons that can be made. As functional classification of breast cancer continues to evolve, future clinical trials in patients with metastatic disease will probably include a more rigorous assessment of molecular profiling in an attempt to link expression patterns of target genes with clinically significant outcome measures. epidemiological trends in clinical oncology for which molecular mechanisms remain elusive. For example, the observed plasticity of disseminated indolent tumours in response to pro-tumorigenic systemic environments suggests that the state in which tumour cells metastasize from a primary tumour, or the state in which they exist during a period of dormancy in a foreign tissue, might not be reflected in the resulting metastatic tumour once it is detected, thus echoing a recent finding that the clinical biomarkers of breast cancer appear to be unstable during disease progression [24]. It is also clear from preclinical experiments using our xenograft model that the degree of responding tumour latency depends upon the identity of the instigating tumour. When responders and triplenegative instigating tumours are injected into opposing flanks simultaneously, responding tumours undergo a considerable period of latency prior to their growth. Yet if the responding tumour is injected after the triple-negative instigating tumour has initiated growth for several weeks, growth of the responding tumour begins almost immediately [4]. By contrast, this reduction in tumour latency is not seen when the instigating tumour is a luminal breast cancer [6]. It is interesting to note that the latency and growth kinetics with which responding tumours formed in hosts bearing different breast cancer subtypes reflected the relative rates of recurrence that are observed in patients with the respective breast cancer subtype. Specifically, patients with triple-negative disease are more likely to experience early relapse than patients with luminal breast cancers [64]. The characteristics of the instigating tumour thus seem to influence not only the tumour microenvironment in the responding tumour but also the type of systemic signalling that is induced, perhaps providing a molecular explanation for the clinical phenomenon in which women diagnosed with similarly staged or treated breast cancer can have widely divergent outcomes with regard to future metastasis. Our studies also expanded an analysis of systemic perturbations and responses to other tumour models and found that colon carcinomas can promote growth of indolent breast tumours (and vice versa), whilst aggressively growing breast tumours can enhance growth of renal cell carcinomas [4, 6]. Although these models require much more investigation, it is well established that patients diagnosed with breast cancer are at increased risk of developing a second malignancy [65]. This trend has been attributed to either morbidity associated with cancer therapies or to the underlying genetic factors that led to the development of the initial malignancy, but it is also plausible that the systemic changes induced by one malignancy may contribute in part to the development of a second. Consequently, the molecular mechanisms that control systemic changes induced by a primary ª 2013 The Association for the Publication of the Journal of Internal Medicine 121

10 tumour and the ability to accurately define and characterize instigating and responding tumours are the subject of intense investigation with the hope that such information will allow accurate identification of those patients who will benefit from specific therapies designed to interrupt these systemic lines of communication. Therapeutic applications Prognosis and diagnosis Consideration of breast cancer as a systemic disease leads to a number of possible prognostic applications. As discussed above, as more becomes known about primary tumour instigating behaviour and the hallmarks that can be used to define tumours as instigators, we might be in a position to determine whether a given primary tumour is likely to support the outgrowth of DTCs. Likewise, as more is learned about disseminated cells that respond to specific systemic cues, we might be in a better position to differentiate life threatening from insignificant DTCs. Such analyses will rely on improved methods for detecting and analysing DTCs and CTCs. As technology continues to develop, it appears that not all methods of assessing CTCs are equally effective. In a prospective trial of 254 patients with breast cancer, blood samples were evaluated using two commercially available methods of detection. Using one assay, the presence of CTCs was a statistically significant prognostic marker for overall survival and progression-free survival, but using the other assay, this association was not noted [66]. It is also important to note that whilst the ability to capture DTCs/ CTCs has markedly improved, it remains technically challenging to analyse these cells once they are isolated; this step will be critical to incorporate CTC/DTC biology into treatment strategies. The use of CTCs/DTCs in predictive algorithms in the nonresearch clinical setting will also require additional clarification of exactly which features in which population of cells should be analysed. Both CTCs and DTCs can have prognostic significance in a range of patients, but a clinical protocol adapted for the use of CTCs obtained via peripheral blood collection would be more broadly applicable than the use of DTCs collected through bone marrow aspiration. Furthermore, it will also be important to determine whether monitoring simply for the presence or absence of CTCs is sufficient, or whether specific molecular analysis of this population is more relevant for choosing therapy. For example, new methodology now allows isolation of single CTCs in the laboratory setting with high-fidelity whole-genome amplification to search for the epidermal growth factor receptor (EGFR) copy number amplification [67]. A gene amplification study revealed enrichment of genes previously associated with metastasis as well as a cohort of genes previously linked to the epithelial mesenchymal transition [68]. Successful in situ hybridization using CTCs has also been reported [39]. Currently, gene expression analysis of primary tumours can help physicians and patients make informed decisions about the benefits of adjuvant chemotherapy. The next step may be expanding tumour profiling to the CTC pool, with a particular emphasis on expression of markers associated with metastasis. The significance of metastatic disease in breast cancer survival has long been recognized, with the presence or absence of metastasis, a critical component of standard staging algorithms. However, current staging only enables assessment of metastasis-positive or metastasis-negative clinical presentation, a binary model that reflects diagnostic capabilities of several decades ago, rather than emerging methods to evaluate DTCs or CTCs. With further technological advances, assessment of tumour cells that have not yet formed an overt metastatic lesion may one day be part of staging algorithms. Finally, beyond assessment of the CTC/DTC pool, it may in future be possible to predict the metastatic potential of a tumour by evaluating either the serum cytokine profile or, possibly, the presence of circulating pro-tumorigenic haematopoietic cells in peripheral blood or bone marrow samples. In animal models, key steps in the process responsible for outgrowth of otherwise dormant micrometastatic lesions include signalling from the primary tumour to the bone marrow compartment, followed by mobilization of pro-tumorigenic haematopoietic cells to the site of metastatic lesions. Further elucidation of critical cytokine mediators (one example of which is osteopontin) or markers of mobilized haematopoietic cells from the bone marrow would allow assessment of these markers during a diagnostic evaluation with potential predictive value for determining metastatic risk. Treatment strategies The potential therapeutic applications arising from consideration of breast cancer as a systemic 122 ª 2013 The Association for the Publication of the Journal of Internal Medicine

11 process are also extensive. First, data demonstrating the evolution of breast cancer biomarkers during the process of metastasis suggest that a more fluid use of drugs targeting the oestrogen receptor or HER2 may lead to clinical benefit. In an albeit small cohort of 10 patients with HER2- positive DTCs detected in bone marrow, the use of trastuzumab was nonetheless able to clear the marrow in most patients (8 of 10) [69]. Notably, the two patients who ultimately presented with metastatic disease were the patients in whom the marrow failed to clear. Amongst patients with metastatic lesions or detectable CTCs/DTCs that do not match the biomarkers of the primary tumour, treating the ER or Her2 status of the metastatic cells may have the potential to improve disease outcomes. Second, in view of the finding that a primary tumour can mobilize pro-tumorigenic bone marrow cells that aid the outgrowth of disseminated tumours, specific therapies targeting these longrange systemic lines of molecular communication may have clinical significance. In a recent study of systemic instigation by luminal carcinomas, we found that the pro-angiogenic role of mobilized platelets was abrogated by aspirin treatment [6]. There is currently debate regarding whether or not aspirin is protective against breast cancer. Several analyses of cohort or case control studies have reported modest decreases in breast cancer risk amongst aspirin users [70, 71], whereas a recent retrospective analysis of over patients demonstrated no significant association with postmenopausal breast cancer [72]. However, these data generally come from vascular biology studies that are neither powered nor designed to specifically address the subtleties of a model of breast cancer in which specific signalling pathways unique to the biology of a primary tumour and/or its metastases exert systemic changes that lead to the mobilization of circulating haematopoietic cells. Preclinical studies suggest that, in the right cohort of patients, aspirin therapy may play a critical role in inhibiting a signalling pathway that drives metastasis. However, our preclinical studies suggest that the secreted proteins osteopontin and granulin may be superior anti-metastasis targets in some patients [4, 5]. As previously discussed, certain triple-negative tumours promote the outgrowth of otherwise indolent disseminated tumours by secreting osteopontin and inducing mobilization of granulinpositive haematopoietic cells from the bone marrow. Interrupting osteopontin signalling or blocking granulin-positive cell mobilization may thus be effective in preventing the growth of otherwise indolent tumours in this setting. It is particularly interesting that elevated plasma levels of osteopontin have been noted in patients with various solid tumours, thus indicating that osteopontin may be a potential cancer biomarker or therapeutic target [73 75]. In a preclinical model of nonsmall-cell lung cancer, anti-osteopontin antibodies were shown to inhibit the growth of further lesions in the lung from the initial primary tumour [76]. Granulin has also been implicated in promoting tumour progression in preclinical models, and in one study, it was shown that inhibition of granulin prevented hepatocellular carcinoma metastasis [77]. Conclusions Models of breast cancer as a systemic disease suggest novel ways to target the process of metastasis, which is responsible for the majority of treatment failures. However, the complexity inherent in the systemic changes induced by a primary tumour also highlights a key consideration in future efforts to develop targeted therapy: selecting the right systemic therapies for the right patients based on functional assessment of their primary disease rather than a static window of tumour pathology. It is clear that the success of targeting the processes that lead to clinical metastasis depends upon appropriate tumour stratification. Future targeting of the metastatic process is likely to involve two critical steps. First, it will be important to identify additional signalling pathways that facilitate metastatic tumour growth to utilize targeted therapies to block progression. Second, it will be equally important to develop efficient and reproducible ways to screen individual patients for the metastasis-promoting pathways used by their specific tumours. Basic experimental and clinical studies have lead to a large knowledge base regarding the underlying biology and clinical presentation of metastatic breast cancer. The next steps will involve integrating information about the systemic nature of breast cancer signalling into ongoing development of individualized therapy. Conflict of interest statement No conflicts of interest to declare. ª 2013 The Association for the Publication of the Journal of Internal Medicine 123