STATE OF THE ART Bone Metastases in Lung Cancer Potential Novel Approaches to Therapy Silvestre Vicent1,2,3*, Naiara Perurena1*, Ramaswamy Govindan4, and Fernando Lecanda1,2,3 1 Division of Oncology, Center for Applied Medical Research, and 2Department of Histology and Pathology, School of Medicine, University of Navarra, Pamplona, Spain; 3IdiSNA, Navarra Institute for Health Research, Pamplona, Spain; and 4Division of Oncology, Washington University School of Medicine, St. Louis, Missouri

Abstract The skeleton is a common site of metastases in lung cancer, an event associated with significant morbidities and poor outcomes. Current antiresorptive therapies provide limited benefit, and novel strategies of prevention and treatment are urgently needed. This review summarizes the latest advances and new perspectives on emerging experimental and clinical approaches to block this deleterious process. Progress propelled by preclinical models has

Contents A Pervasive Clinical Problem Mechanisms Underlying Bone Metastases Genetic Alterations Influencing Bone Metastasis

Lung cancer is the leading cause of cancer-related death globally, accounting for almost 20% of cancer-related fatalities (1, 2). More than 65% of patients present local or disseminated metastatic disease at diagnosis (3, 4). Tumor cells in non–small cell (NSCLC) and small cell lung cancer (SCLC) spread locally to the thoracic cavity and to distant

led to a deeper understanding on the complex interplay of tumor cells in the osseous milieu, unveiling potential new targets for drug development. Improvements in early diagnosis through the use of sophisticated imaging techniques with bone serum biomarkers are also discussed in the context of identifying patients at risk and monitoring disease progression during the course of treatment. Keywords: tyrosine kinase inhibitor; biomarkers; clinical trials

Dissemination of Cancer Cells and Homing to Bone Tissues Potential Novel Therapeutic Opportunities Tumor–Stromal Interactions Tumor Angiogenesis Immune System

organs including the skeleton. Current antiosteolytic therapies delay the deleterious process of bone resorption. However, there is still an urgent need for new agents targeting lung cancer–derived bone metastases. This review summarizes the current state of the art and progress based on preclinical models, which has propelled a deeper

Improved Diagnosis of Skeletal Metastases Increased Imaging Sensitivity of Bone Metastasis Potential Biomarkers of Bone Metastasis Conclusions and Future Perspective

understanding of metastatic progression, unveiling novel vulnerabilities that may open the door to drug development. Strategies blocking tumor-induced angiogenesis, impairing tumor–host interactions, and reversing immunosuppression have provided solid proof of concept supporting further clinical studies.

( Received in original form March 4, 2015; accepted in final form June 25, 2015 ) *S.V. and N.P. contributed equally to this work. Supported by Spanish Ministry of Economy and Competitiveness grants SAF2012-40056 (F.L.) and SAF2013-46423-R (S.V.). S.V. is an investigator from the Ramon ´ y Cajal Program RYC-2011-09042. S.V. was also supported by Marie Curie fellowship PCIG13-GA-2013-618312, a Young Investigator Award from the IASLC, and a research grant by the Fundacion ´ Caja Navarra. N.P. was supported by a predoctoral fellowship (MECD AP2010-2197). This work is the sole responsibility and initiative of the authors. Author Contributions: S.V. and N.P. contributed to the literature search, the writing of several parts of the manuscript, and the preparation of the figures. F.L. and R.G. contributed to the writing and editing of the manuscript. F.L. coordinated the design, the writing, and the editing processes. Correspondence and requests for reprints should be addressed to Fernando Lecanda, Ph.D., Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, 31080 Navarra, Spain. E-mail: [email protected] Am J Respir Crit Care Med Vol 192, Iss 7, pp 799–809, Oct 1, 2015 Copyright © 2015 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201503-0440SO on July 1, 2015 Internet address: www.atsjournals.org

State of the Art

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STATE OF THE ART A Pervasive Clinical Problem Bone metastasis represents one of the most deleterious clinical consequences of lung cancer, associated with dismal prognosis. As many as 30–40% patients with NSCLC develop bone metastases with a median survival rate in these patients of about 6 months, the lowest for all solid tumors metastasizing to bone (5, 6). Five-year survival for these patients with current therapies is less than 5%. Patients with SCLC often present osteolysis, the appearance of which is similar to that encountered in patients with NSCLC. These skeletal lesions involve significant morbidity, metabolic syndromes, pathologic fractures, and spinal cord compression, which entail an overt reduction in quality of life and require costly treatments with limited impact on overall survival. The spine is the preferential site of metastasis (50%) in patients with lung cancer, followed by the ribs (27%), whereas lesions in flat and appendicular bones are infrequent (,6%) (6, 7). This is partially explained by the ease of access to vertebral bodies in the thoracic and lumbar spine through the plexus vertebral system (8, 9) and the high bone marrow flow of some skeletal elements (10). Pain is the most common symptom related to bone metastasis (11, 12). Most of the pain could be attributed to the deleterious effects of osteolysis derived from tumor cells disrupting normal bone homeostasis with the consequent release of pain mediators such as endothelin A (13) and the nerve growth factor produced by the hosting cells (14). Indeed, the observation that decreased osteoclast resorption induced by radiotherapy severely reduces bone pain points to osteolysis as the main contributor to this process (15). In the vast majority of skeletal lesions, also derived from other tumors, osteolysis prevails over bone formation because of the tumor-induced ability to exacerbate osteoclastic activity, an effect derived from the increased number and activation of bone-resorbing osteoclasts. This activity is enhanced by the tumor-secreted factors that alter normal bone homeostasis, tilting the balance between bone formation and resorption. Most frequently tumor-derived factors induce osteoclastic bone resorption, but in rare cases some factors can also 800

promote spurious osteoblastic bone formation by increasing the activity of bone-forming osteoblasts (16). Because during bone remodeling formation is exquisitely coupled to resorption, disruption of this balance in patients with NSCLC often leads to mixed lesions with the presence of an osteosclerotic component, an immature woven bone that is fragile (17). However, although exclusive osteoblastic lesions are seldom predominant in lung cancer, they seem to be fairly common in patients with epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (AD) after therapy with EGFR inhibitors (18–20). The presence of osteolytic lesions concomitantly weakens the mechanical properties of bone tissue leading to the emergence of skeletal-related events (SREs) (21, 22) including malignant hypercalcemia, pathological fractures, and symptomatic compression fractures of the spine that might require palliative vertebroplasty (23). Systemic therapies that block osteoclast activity including bisphosphonates (zoledronic acid) and receptor activator for nuclear factor-kB ligand (RANKL) inhibitors (denosumab) can reduce the incidence of SREs but have a modest impact on overall survival. In a comparative trial, denosumab was slightly superior in delaying the appearance of SREs in NSCLC as compared with zoledronic acid, but showed no differences regarding osteonecrosis of the jaw (24, 25), a serious complication encountered in patients treated long term that has cautioned practitioners against its use (26).

Mechanisms Underlying Bone Metastases Genetic Alterations Influencing Bone Metastasis

In the clinical setting, next-generation sequencing has introduced a comprehensive way to identify somatic alterations, revealing the high degree of heterogeneity among all tumors (27–30). During tumor cell dissemination, tumor clones coevolve in linear fashion or most commonly by a pattern involving ramified evolution, whereby multiple subclones thrive simultaneously, contributing to tumor heterogeneity (31, 32). Understanding the contribution of driver

mutations to the skeletal metastatic phenotype and the clonal evolution could help identify patients at risk for recurrence and distant metastases to specific organs (33). By studying paired samples from primary and corresponding bony metastases, using next-generation sequencing, and identifying clones enriched in osseous metastases, it may be possible to discern the molecular pathways that facilitate osseous colonization. Dissemination of Cancer Cells and Homing to Bone Tissues

Although understanding the molecular mechanisms involved in skeletal colonization is critical for the treatment of established metastases, the majority of patients present circulating tumor cells at diagnosis even without evidence of clinical osseous metastatic disease (34). Interactions between disseminated tumor cells with components of the osseous milieu mediate tumor cell anchorage, survival, micrometastasis, and osseous colonization. A variety of interactions between endothelial and/or stromal cell surface molecules, and/or extracellular matrix proteins and membrane receptors in tumor cells, mediate this process (35). On engagement, critical pathways triggered at the tumor interphase are important in these steps. Several collagen receptors are involved in this process, including the discoidin domain receptor (DDR) family of receptor tyrosine kinases and integrins among others (Figure 1). DDRs are involved in cell adhesion, matrix remodeling, and cell migration and proliferation (36). Of the two members of this small family, DDR1 is expressed in tumor cells of epithelial origin and DDR2 in tumor-associated stroma (37). In addition, DDR2 is mutated in squamous cell cancer (4.4%) (38) and in lung AD (2–5%) (29), whereas DDR1 overexpression is associated with poor prognosis in NSCLC (39, 40). Interestingly, partial responses have been achieved using combinatorial therapies with dasatinib, a multitarget tyrosine kinase inhibitor that also blocks DDR2 (41). DDR1 also participates in collective cell migration, an important step in metastasis (42), and mediates cell survival and adhesion in early stages of bone homing, together with invasiveness by modulating motility and secretion of

American Journal of Respiratory and Critical Care Medicine Volume 192 Number 7 | October 1 2015

STATE OF THE ART

Tumor cell OPG blocks RANK/RANKL

1 VEGFR/PDGFR other TK receptors

Bone

T cell immunosuppressed

5 Anti-PD-1

Stromal cell expressing RANKL

Endothelial cells

2 DDR1, integrins, EPCR

Mononuclear progenitor expressing RANK

Neo-angiogenesis

4 RANKL, cathepsin K, TGF-β, OC function

3 MMPs/ADAMs

Bone matrix-derived growth factors, TGF-β, IGF, miRNAs

6 MMPS, VEGFR/PDGFR, miRNAs, exosomes

Osteoblasts Osteoclast

Figure 1. Mechanisms of osseous tumor cell (TC) colonization as opportunities for therapeutic intervention in preclinical models. (1) TC–endothelial interaction triggers VEGFR/PDGFR signaling in the endothelium that is required for TC homing and colonization. (2) TC–bone matrix protein interactions elicit TC adhesion, survival, and colonization mediated by DDR1 on engagement with collagen type I and by integrin binding to several bone matrix proteins. Expression of EPCR also cooperates with TC survival and osseous colonization. (3) TC–osteoblast/stromal interactions increase the release of MMP/ADAMs and other factors that contribute to bone matrix degradation, osteoblast apoptosis, and TC expansion. (4) “Vicious cycle” of TC-induced osteoclast activity: TC-secreted factors including PTHrP, IL-11, IL-6, IL-8, and tumor necrosis factor-a, among others, stimulate differentiation and fusion of mononuclear precursors. This process is mediated by the RANK–RANKL interaction, the latter expressed in stromal cells. These factors also stimulate osteoclastic activity required for metastatic expansion. Soluble decoy receptor osteoprotegerin or an anti-RANKL antibody (denosumab) blocks this process. Zoledronic acid incorporated in the bone matrix triggers osteoclast apoptosis. Bone resorption entails the secretion of MMP/ADAMs and cathepsins, which releases growth factors from the bone matrix including TGFs and IGFs that further stimulate TC proliferation. (5) TC interaction with T cells through PD-1/PD-L1 promotes immunosuppressive functions. (6) TC colonization and TC-induced angiogenesis are mediated by the secretion of tumor-derived MMP/ADAMs and TC release of proangiogenic factors, including VEGF and exosomes. Exosome cargo contributes to perturb acceptor cells (i.e., endothelial precursors) that lead to altered angiogenesis. ADAM = a disintegrin and metalloproteinase; DDR1 = discoidin domain receptor 1; EPCR = endothelial protein C receptor; IGF = insulin-like growth factor; miRNA = microRNA; MMP = matrix metalloproteinase; OC = osteoclast; OPG = osteoprotegerin; PD-1 = programmed death 1; PD-L1 = programmed death 1 ligand; PDGFR = platelet-derived growth factor receptors; PTHrP = parathyroid hormone–related peptide; RANK = receptor activator for nuclear factor-kB; RANKL = receptor activator for nuclear factor-kB ligand; TGF = transforming growth factor; TK = tyrosine kinase; VEGF = vascular endothelial growth factor; VEGFR = vascular endothelial growth factor receptor.

metalloproteases (MMPs) (43). In animal models, abrogation of DDR1 resulted in marked tumor cell apoptosis and severe reduction in bone metastasis. Because several collagen types bind and activate DDRs by autophosphorylation, triggering intracellular signals (44), DDR1 promotes early events of tumor cell engraftment to the skeleton, 90% of the proteinaceous content of which is constituted by collagen type I (43). Thus, DDR1 blockade, which has shown remarkable effects in preclinical models of metastasis, could be beneficial for increasing the efficacy of conventional radiotherapy in those patients (43). Few factors promoting tumor prosurvival signals conferring endurance to stress in the circulation have been identified. Endothelial protein C receptor triggers prosurvival signals to tumor cells and on binding to its ligand, activated protein C, conferred a significant survival advantage to lung AD cells (45). Endothelial protein C receptor gene State of the Art

silencing or pharmacological blockade of its interaction with activated protein C reduced the infiltration and resulted in impaired osseous prometastatic activity (45) (Figure 1). These findings have the potential to prevent the establishment of metastases during the course of treatment in the clinic. Future studies are required to identify key prosurvival factors that could be targeted to seek a clinical benefit. Integrins are transmembrane receptors composed of a heterodimer of a and b subunits, involved in cell–matrix adhesion and a variety of other cellular functions (46). Integrin-related mechanisms mediate early steps in lung cancer bone metastasis. In preclinical models a5b1-integrin interaction with fibronectin proved essential for lung metastasis development (47). Similarly, host a1-integrin–mediated effects were required for metastasis development in an orthotopic model (48). Although integrin engagement with

other adhesion molecules has been shown to be critical in mediating tumor–stromal interactions, few studies have appropriately addressed this event in lung cancer bone metastasis models, a key step that opens up promising paths for future research.

Potential Novel Therapeutic Opportunities Tumor–Stromal Interactions

In addition to the inherent mutational landscape of the tumor, metastatic spread is heavily directed by the acquisition of selective metastatic traits through subtle changes in gene expression. These are deeply influenced by the local tumor milieu at the primary site and the forceful adaptation to subsequent stringent conditions imposed on cancer cells en route to and at the skeletal sites. Little is known about specific lung cancer–induced pathways involved in 801

STATE OF THE ART bone colonization, which could vary among the various histological subtypes (49, 50). Most of the available information has been derived from animal models or from osseous metastases of other tumor origin. The widely accepted paradigm poses that within the bone microenvironment, the crucial interplay between tumor and neighboring cells elicits a wide spectrum of advantageous mechanisms that greatly contribute to bone colonization (50) (Figure 1). Those mechanisms involved the concomitant activation of novel pathways in tumor cells and its surrounding “normal” stroma. Tumor-released factors such as parathyroid hormone–related peptide (51), cytokines, and other factors induce osteoclast bone resorption, which is critical for bone matrix degradation and the development of osteolytic lesions, key events required for the incipient development of bone micrometastases (16) during early steps of osseous colonization. Because the skeleton is a major reservoir of growth factors including transforming growth factor (TGF)-b and insulin-like growth factors, and a variety of chemotactic factors (52, 53), this process releases active TGF-b and insulin-like growth factors from the bone matrix, which in turn fuels tumor cells, creating a self-maintained “vicious loop” exacerbating osteolytic lesions (54–56) and largely facilitating tumor expansion (Figure 1). Thus, osteoclast blockade has been the focus of research for more than four decades. Current treatments are directed to impair osteoclast activity, survival (57), and/or their differentiation and fusion from mononuclear precursors (58, 59) but show limited efficacy in the clinical setting, partially because tumor-derived factors (proteinases) also promote strong osteoclast-independent bone matrix degradation, allowing metastasis progression. Indeed, the “vicious cycle” of tumor-induced osteoclast activation also entails the secretion of a highly complex panoply of collagenolytic enzymes at the tumor–bone interphase (60). Tumor cells in contact with stromal elements and infiltrating immune cells activate the release of MMP, cathepsin, and ADAM (a disintegrin and metalloproteinase) families of proteases by tumor–stromal cells and other neighboring components (Figure 1). Besides their implication in bone matrix remodeling, ADAMs have extensive roles in 802

signaling through their sheddase activity by processing membrane-bound proteins including cytokines, growth factors, and their receptors (61). In lung cancer, ADAM10 and ADAM17 are involved in the signaling cascade of EGFR, mediating the shedding of its membrane-tethered ligands. Several other ADAMs (62), ADAMs with thrombospondin motifs, and MMPs mediate EGFR transactivation (63). The prognostic marker for lung AD (64, 65), ADAM8 and its novel truncated isoforms showed a dual role contributing to the invasiveness of tumor cells and enhancing osteoclast formation (66) and osteolytic metastasis (67), effects partially mediated by the shedding of secondary targets that participate in osteoclastogenesis (68). In a preclinical model of skeletal metastasis of NSCLC, attenuation of TGF-b by triple gene silencing severely decreased osseous colonization (69). Moreover, a combinatorial regimen of an anti–TGF-b peptide and a global anti-MMP inhibitor had more salient effects than either of these agents alone (60). This global inhibition of TGF-b and MMP/ADAMs might have further benefit in clinically reversing the strong immunosuppression elicited by TGF-b (Figure 1). In patients, the TGF-b kinase inhibitor galunisertib, in combination with nivolumab (see below), is being evaluated in phase 2 trials for advanced refractory NSCLC. Moreover, a safety study of an anti-MMP9 antibody (GS-5745) in combinatorial therapy is also ongoing. Similarly, the contribution of several ADAMs in metastasis indicates the potential benefit of pan-ADAM inhibition in patients. Moreover, the use of global MMP/ADAM inhibitors had shown a marked antimetastatic effect in bone colonization in a preclinical model (60). Yet their use in clinical settings has been hampered in early trials by their lack of specificity, their marked side effects encountered including musculoskeletal pain and inflammation, and sometimes their context-dependent paradoxical tumor-promoting and suppressive effects (70). Tumor Angiogenesis

In the bone microenvironment, established metastatic tumors also implement tumorinduced angiogenesis favoring bone tumor infiltration and colonization. Plateletderived growth factor receptor/vascular

endothelial growth factor (PDGFR/VEGFR) pathways are critical in driving proangiogenesis in endothelial cells, and PDGFRs are crucially involved in pericyte maintenance. This pathway also elicits osteoclast activity and tumor-induced osteolysis (71, 72). A multitarget tyrosine kinase drug, sunitinib, showing potent inhibition of PDGFR/VEGFR pathways, has been approved in the clinical setting for other tumors (73) (Figure 1). The relevance of this pathway (by sunitinib treatment) was manifested in a preclinical model by decreased tumor burden and extended overall survival, despite the presence of marked osteolytic lesions (74). This increase in life span was explained by a potential concomitant role in suppressing tumor-induced cachexia. One of the main mediators associated with cachexia has been identified as the pro-osteoclastogenic parathyroid hormone–related peptide (75), indicating a link between osteolysis and cachexia. A combination of sunitinib with zoledronic acid treatment was able to extend the life span, an effect presumably due to its anticachectic effect, and to maintenance of bone mass. Although clinical studies with this agent in lung cancer have been disappointing, partially due to contrasting subtype-specific effects (76), current clinical phase 2 studies are evaluating drug combinations with other multitarget tyrosine kinase inhibitors, such as cabozantinib, in metastatic NSCLC (Table 1). Further studies to identify combinatorial strategies to inhibit this pathway and identify patients who are likely to respond to antiangiogenic agents are warranted. CXCR4 receptor expressed in tumor cells and its unique ligand CXCL12 (stromal cell–derived factor-1), released in an autocrine or paracrine manner in the tumor microenvironment, also enhance tumor angiogenesis (77), besides their role as chemoattractants, promoting tumor cell survival, growth, and dissemination in lung cancer (78). In preclinical models inhibition of CXCL12 abrogates organspecific metastasis (79). Drugs that block CXCR4/CXCL1, such as AMD3100 and BKT140, attenuate tumor cell development and enhance the effects of chemo- and radiotherapy (80). Given their strong effect on the recirculation of hematopoietic stem cells (81, 82), this issue deserves cautious consideration before designing clinical trials. MicroRNAs (miRNAs), small noncoding RNAs involved in the regulation

American Journal of Respiratory and Critical Care Medicine Volume 192 Number 7 | October 1 2015

STATE OF THE ART Table 1. Ongoing Clinical Trials Relevant to Bone Metastasis or Advanced Disease

Stage Phase 2 Phase 2 Phase 2 Phase 1/1b

Generic Name; Other Name(s) Denosumab Galunisertib; LY2157299 Cabozantinib MGCD516

Phase 2 Phase 2

Lucitanib Famitinib

Phase 2 Phase 1

IMC-3G3; LY3012207 XL999

Phase 2

Dovitinib

Phase 1

Pazopanib

Phase 2

Nintenadib

Phase 1

CC-48; oral azacitidine, Vidaza Phase 2 Radium-223; Xofigo, BrUOG L301 Phase 3 Fractionated radiotherapy Feasibility Ultrasound ExAblate study

Type

Targets

Goal

Humanized moAb RANKL Tyrosine kinase inhibitor TGF-b

Bone metastasis Advanced NSCLC

Tyrosine kinase inhibitor c-Met/VEGFR2 Tyrosine kinase inhibitor MET, AXL, MER, VEGFR, PDGFR, DDR2, Trk and Eph families Tyrosine kinase inhibitor VEGFR, FGFR, PDGFR Tyrosine kinase inhibitor c-Kit, VEGFR2, PDGFR, VEGFR3, Flt-1, and Flt-3 Human anti–PDGFR-a PDGFR-a Tyrosine kinase inhibitor VEGFR, PDGFR, FGFR, Flt-3, and Src Tyrosine kinase inhibitor FGFR, PDGFR, VEGF, c-Kit, Flt-3, CSFR1, Trk, and RET Tyrosine kinase inhibitor VEGFR-1, -2, and -3, PDGFR-a and -b, and c-Kit Tyrosine kinase inhibitor VEGFR, FGFR, and PDGFR

Bone metastasis Advanced tumors Advanced/metastatic Advanced SCLC and NSCLC Advanced/metastatic NSCLC

Regimen

Combined with nivolumab Combined with erlotinib

Alone or in combination with chemotherapy

Mutated or translocated tumors Advanced tumors

Alone or in combination with erlotinib

Recurrent tumors Platinum-sensitive SCLC Antimetabolite of cytidine NSCLC and SCLC

DNA methyltransferase inhibitor Radioisotope

Radiosensitive cells

Bone metastasis

Radiotherapy

Radiosensitive cells

Ultrasound system

Radiofrequency ablation

Bone oligometastatic disease Bone pain relief and local tumor control

Combined with chemotherapy

Definition of abbreviations: AXL = anexelekto gene; CSFR1 = colony-stimulating factor-1 receptor; DDR2 = discoidin domain receptor-2; Eph = ephrin; FGFR = fibroblast growth factor receptor; Flt = fms-related tyrosine kinase 1; MER = MER proto-oncogene; MET = mesenchymal–epithelial transition factor protooncogene; moAb = monoclonal antibody; NSCLC = non–small cell lung cancer; PDGFR-a = platelet-derived growth factor receptor-a; RANKL = receptor activator for nuclear factor-kB ligand; RET = rearranged during transfection proto-oncogene; SCLC = small cell lung cancer; Src = sarcoma proto-oncogene; TGF-b = transforming growth factor-b; Trk = tropomyosin receptor kinase; VEGF = vascular endothelial growth factor; VEGFR = vascular endothelial growth factor receptor. Information is from https://clinicaltrials.gov.

of gene expression, have been implicated in a variety of studies as regulators of metastasis because of their ability to simultaneously regulate hundreds of genes (83, 84). Circulating levels of miRNAs were also detected within microvesicles or exosomes in the extracellular milieu (85). These nanoparticles act as shuttles transferring cellular information (nucleic acids and proteins) to neighboring cells or distant organs (86). In a preclinical model of bone metastasis, released exosomes perturb the surrounding endothelial compartment during tumor-induced angiogenesis, dramatically facilitating tumor expansion at the bone metastatic site (87). In this model, forced expression of an antiangiogenic miR-192 in the cargo of tumor-released exosomes was sufficient to dramatically prevent bone metastatic colonization (87). These findings open new avenues for the design of State of the Art

nanoparticles that could be used to treat metastasis. Immune System

Cellular and innate immunity in the tumor microenvironment play critical roles in the modulation of skeletal bone metastases. Infiltration of immunosuppressive myeloidderived suppressor cells inhibits host antitumor immune responses and promotes tumor growth. In skeletal metastasis, myeloid-derived suppressor cells act as osteoclast progenitors, further increasing tumor-induced osteolysis (88), an effect that has been attributed to signals derived from the tumor and the host milieu (89). The implication of T cells both in the primary tumor and in bone metastasis has also been recognized (90, 91). In normal bone remodeling T cells secrete pro-osteoclastogenic cytokines including RANKL and IL-6 and can

promote osteoclast differentiation (92) (Figure 1). At the primary tumor, CD41 helper T cells (Th cells), important in removing tumor cells, can be functionally divided into Th1, Th2, Th17, and regulatory T (Treg) cells on the basis of their cytokine secretion pattern (93). Th1 cells secrete potent antitumor cytokines such as IFN-g and IL-2, which mediate potent antitumor effects and suppress Th2 cells (characterized by IL-4 and IL-10 secretion), which inhibit cell-mediated antitumor immunity (94). During lung cancer progression, the Th1/Th2 ratio changes and the antitumor Th1 subpopulation decreases relative to the Th2 cell population. This effect is partially mediated by the immunosuppressive cytokines IL-10 and TGF-b, which also favor the development of Treg cells (95). Treg cells exhibit a potent immunosuppressive function in the primary tumor, an effect 803

STATE OF THE ART that contributes to lung cancer progression (96). Paradoxically, in bone metastasis models (breast and prostate), metastasisassociated Treg cells increase in the bone milieu and inhibit bone resorption by exhibiting a wide immunosuppressive profile (97). Thus, in bone metastasis a complex balance between the effects of immunosuppression and bone resorption induced by local cytokines defines global tumor metastatic burden. Advances have shown that binding PD-1 (programmed death 1), a receptor expressed in T and B cells, with its ligands PD-L1 and PD-L2 expressed by tumor cells obliterates T-cell antitumor immune functions (Figure 1). Blockade of the PD-1/PD-L1 axis shifts T-cell populations from Th2 to a tumor-inhibiting Th1/Th17 population (98). Clinical trials using anti–PD-1 in clinical trials (nivolumab) are currently under way for patients with NSCLC, with objective responses in approximately 20% of patients (99) that rise to about 80% in patients with high tumor levels of PD-1 treated with anti–PD-L1 antibodies (100) (Table 1). Regardless of PD-L1 expression levels, nivolumab has shown efficacy in advanced squamous cell cancer (101). Thus, overcoming immunosuppression is a novel therapeutic opportunity showing substantial results in treating advanced tumors. On the basis of the overall response in bone metastasis observed in melanoma (102), one could anticipate a substantial effect in lung cancer bone metastasis, although treatment response in osseous metastases remains to be evaluated. This success story suggests that research to elucidate novel mechanisms of tumor immunosuppression is warranted to develop novel immunotherapeutic drugs. As for other tumors, lung cancer cells activate the classical complement pathway on the direct binding of complement 1q (C1q) to their cell surface (103). Resistance to complement-mediated cytotoxicity is facilitated by the expression of soluble complement regulators (104–106). In a preclinical mouse model of metastasis in breast cancer, the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD81 and CD41 T-cell responses at the metastatic site (107). Moreover, pharmacologic blockade of C5aR or its genetic inhibition in C5aR-deficient mice 804

was sufficient to reduce metastases (107). Interestingly, the complement 3a (C3a) component is secreted by mature osteoclasts and stimulates osteoblastogenesis (108), and together with C5a promotes CD41 T-cell survival (109). In NSCLC, complement 4d (C4d) is deposited in lung primary tumors and is associated with poor prognosis and increased lung cancer risk in asymptomatic individuals (103). Thus, these results suggest the potential role of complement factors in modulating tumor-induced skeletal metastasis. Beyond these findings, the role of the complement system in the initiation and development of skeletal metastasis in NSCLC deserves a thorough investigation to improve current immunotherapy.

Improved Diagnosis of Skeletal Metastases Increased Imaging Sensitivity of Bone Metastasis

The diagnosis of bony metastases is commonly performed by bone scintigraphy screening after injection of technetium99m-labeled methylene diphosphonate (99mTc-MDP), or single-photon emission computed tomography, and is further confirmed by radiography and/or computed tomography (CT) or magnetic resonance imaging (Figure 2). Fluorine-18-labeled fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) scans, which have been shown to have higher specificity and lower rates of false

negative as compared with bone scans (110), have been recognized to be a valuable tool for the evaluation and accurate staging of NSCLC (111). 18F-FDG PET might have better sensitivity and specificity in lytic or mixed lesions but inferior values in sclerotic lesions (112). This could be partially due to the low activity and number of tumor cells inducing bone formation as compared with tumor-induced osteolytic lesions. It also displays poor spatial resolution in comparison with CT or magnetic resonance imaging. In contrast, CT is a convenient tool for skeletal screening and for the initial evaluation of fracture risk with the advantages of its widespread use and low cost (113). However, at initial stages of bone metastasis, these techniques might lack sensitivity, or the radionuclide uptake might be insufficient. In addition, in patients with poor survival, not all these techniques may be appropriate for monitoring skeletal disease progression. Thus the PET–CT combination gives highly informative morphological and functional images with better spatial resolution, higher contrast, and improved tomographic evaluation, making PET–CT the ideal choice in detecting and monitoring bone metastasis (114, 115). However, one challenging problem in the clinic is the distinction between worsening sclerosis and response to disease progression. Scintigraphic/healing flare is a well-recognized problem in patients

Figure 2. Imaging diagnostic strategies of skeletal metastases. (A) Bone positron emission tomography. (B) Computed tomographic scan showing prominent osteosclerotic lesions in the vertebral body (arrow), derived from a rare lung adenocarcinoma. (C) Histological section showing a tumor-induced osteolytic lesion with tartrate acid–resistant purple staining of multinucleated osteoclasts resorbing the bone matrix at the bone (B)–tumor (T) interphase.

American Journal of Respiratory and Critical Care Medicine Volume 192 Number 7 | October 1 2015

STATE OF THE ART Table 2. Bone Turnover Biomarkers of Skeletal Metastases Markers

Sample

Value

Bone alkaline phosphatase Procollagen I carboxy-terminal propeptide Procollagen I amino-terminal propeptide Osteocalcin, bone Gla protein

Serum/plasma Serum/plasma Serum/plasma Serum/plasma

Diagnosis and follow-up Diagnosis Predictor and progression of bone mets ND

Serum/plasma Diagnosis, prognosis, and follow-up Serum/plasma Diagnosis, prognosis, and follow-up

124, 133, 135 125, 133, 135

DPD NTX

Tartrate-resistant acid phosphatase 5b Cross-linked carboxy-terminal telopeptide of type I collagen Deoxypyridinoline Amino-terminal telopeptide of collagen type I

Serum/urine Serum/urine

125 120, 122, 123

CTX

Carboxy-terminal telopeptide of collagen type I

Urine

Formation BAP PICP PINP OC Resorption TRAP5b ICTP

Name

Diagnosis and follow-up Predictor, progression of bone mets, and SRE Extent of osseous metastasis

References

120, 125, 133 134 120, 122, 123

126

Definition of abbreviations: bone mets = bone metastases; ND = not determined; SRE = skeletal-related event.

responding to treatment (116). Similarly, diffuse disease and sclerotic lesions are considered nonmeasurable in CT scans (117), and these lesions perform poorly in FDG PET scans (118). Thus, combinatorial assessment with bone and tumor biomarkers could circumvent this obstacle. Potential Biomarkers of Bone Metastasis

Treatment response is typically assessed in the clinic on the basis of serial radiographs. In the future, a number of biomarkers could potentially aid in the assessment of response to therapy (Table 2). Bone resorption is usually evaluated by the levels of metabolites of collagenolysis, including cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), the C-terminal cross-linked telopeptide of type I collagen, the urine or serum levels of amino-terminal collagen type I telopeptide (NTX) (119), urine deoxypyridinoline and tartrate-resistant acid phosphatase 5b (TRAP5b), a marker of osteoclast activation. Expression of ICTP, procollagen I amino-terminal propeptide (PINP), and NTX is strongly associated with the development and progression of bone metastasis in patients with lung cancer (120–123). Similarly, serum TRAP5b levels constitute a useful tool for the diagnosis and monitoring of bone metastasis in patients with NSCLC (124). Serum ICTP, bone alkaline phosphatase (BAP), and deoxypyridinoline have proven useful in the diagnosis and follow-up of metastatic disease in patients with lung cancer (125). Overall, BAP as bone State of the Art

formation marker and NTX, ICTP, and osteopontin have been found consistently elevated in 11 studies of patients with lung cancer bone metastases (126). These biomarkers could complement other diagnostic technologies and could also be useful for the evaluation of skeletal integrity of patients at risk and extent of metastatic disease. In addition, they could also potentially be used to identify patients most likely to benefit from antiresorptive therapies (127). For instance, elevated NTX levels were highly predictive of osseous SREs in patients treated (122) or untreated (120) with bisphosphonates. miRNAs, which are detectable in body fluids, may become potential biomarkers with prognostic, predictive, and diagnostic value (128, 129). In preclinical models, several miRNAs have been correlated with metastatic progression in lung cancer (130, 131). For instance, circulating levels of miR-326 were the most robust marker closely associated with the extent of metastatic disease, even at early stages of dissemination (132). This miRNA was tightly correlated with PINP, and the extent of tumor burden and osteolysis during bone metastatic progression (132). However, although promising, these results require prospective validation in large cohorts of patients.

Conclusions and Future Perspective The development of animal models closely mimicking the complexity of mutational status could yield valuable new tools to achieve future discoveries. This platform of

preclinical proof-of-concept testing will help identify novel targets of unique relevance favoring tumor cell survival, tumor engagement, and tumor-induced angiogenesis in the stromal hosting milieu. Because all these mechanistic insights are rate-limiting steps in metastatic progression, they provide fertile ground for drug discovery. The introduction of more reliable noninvasive serum biomarkers for patient follow-up, and monitoring of responses to standard therapies, will continue to assist in the management of skeletal metastases. However, these biomarkers require increased sensitivity and robustness before widespread consensus will be reached about their use in standard clinical practice. Serum miRNAs within circulating exosomes hold great promise to meet these expectations, yet clinical evidence requires thorough validation in large well-phenotyped cohorts of patients. Coalescence of basic and translational research will continue to make major impacts in the clinical setting, bringing to light novel opportunities for the management, monitoring, and treatment of skeletal metastases. A rewarding path lies ahead in terms of methods designed to delay or block this process, increasing patient survival and alleviating the inherent societal and economic burden of this devastating disease. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors are grateful to J. M. Lopez-Picazo, ´ M.D., M. Moreno, M.D., and A. Gurpide, ´ M.D., for critical reading of the manuscript.

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