Chemokines are a family of chemoattractant cytokines whose main function is to regulate cell trafficking. To date, studies have identified more than 50 human chemokines and 20 chemokine receptors.1, 2 Chemokines can be classified into 4 subfamilies based on the number and location of the cysteine residues at the N-terminus of the molecule.1 Chemokines are also grouped into 2 main functional subfamilies: inflammatory and homeostatic chemokines. Inflammatory chemokines control the recruitment of leukocytes in inflammation and tissue injury, whereas homeostatic chemokines fulfill housekeeping functions such as navigating leukocytes to and within secondary lymphoid organs as well as in bone marrow and the thymus during hematopoiesis.1, 2 Chemokines bind to specific cell surface transmembrane receptors coupled with heterotrimeric G proteins, whose activation leads to formation of intracellular signaling cascades that prompt migration toward the chemokine source. Directed migration of cells expressing the appropriate chemokine receptors occurs along a chemical ligand gradient known as the chemokine gradient, allowing cells to move toward high local concentrations of chemokines.
Chemokines first emerged as key regulators of leukocyte migration, ensuring the localization of cells to either lymphoid or peripheral tissues depending on their activation state and the inflammatory status of the tissue.1 Appropriate expression of chemokines and their receptors facilitates dendritic cell (DC), B-cell, and T-cell interactions, thereby promoting the development of effective adaptive immune responses.3 Peripheral activation of the innate immune system induces secretion of inflammatory chemokines that attract immature DCs. Upon activation and uptake of antigens, DCs down-regulate receptors for inflammatory chemokines and up-regulate receptors for homeostatic chemokines (eg, CCR7)1, 2 (Table 1). This coordinated switch in chemokine receptor expression allows mature, antigen-loaded DCs to enter regional lymph nodes (LNs), where they activate antigen-specific naive T and B cells.1–3 Naive T cells express CCR7 and CXCR4, the ligands for which (Table 1) are expressed in lymphoid organs and navigate T cells through lymphoid tissue, increasing their chances of encountering DCs carrying the specific antigen. Activated T cells then down-regulate expression of CCR7 and increase expression of other chemokine receptors, the ligands for which are typically expressed in peripheral organs, particularly in inflamed tissues.1–4
However, researchers have found that hematopoietic and nonhematopoietic cells express receptors for various chemokines that are constitutively expressed in distinct tissue microenvironments. The interactions between such receptors and their respective chemokines coordinate the trafficking and organization of cells within different tissues. The chemokine-receptor expression profile in an individual cell is dependent on intrinsic characteristics (eg, lineage, stage of differentiation) and microenvironmental factors, such as chemokine concentration, the presence of inflammatory cytokines, and hypoxia.
Recently, investigators demonstrated that tumor cells also constitutively express chemokines that are inducible in normal tissues as well as functional chemokine receptors.5 Compelling evidence has revealed the multifunctional role of the chemokine network in cancer, including acting as a growth or survival factor, regulating angiogenesis, determining metastatic spread, and controlling leukocyte infiltration into tumors to hinder antitumor immune responses.5
Because of the many critical functions of the chemokine network in cancer progression, manipulation of this network is a potential therapeutic option for cancer. This review focuses on 2 possible chemokine network-related therapeutic interventions: strategies aimed at interfering with chemokine signaling between tumor cells, their microenvironments, and endothelium, leading to the establishment and progression of metastases,6 and approaches for inducing antitumor immune responses.
Chemokines and Cancer Metastases
Recent data suggest that chemokine receptors in cancer cells have a key regulatory role in directing lymphatic and hematogenous spread and influencing the sites of metastatic growth of different tumors.5 Although reports claim that several chemokine receptors are involved in the establishment of metastasis from different tumors, the tissue analysis and animal model data reported thus far for the majority of these receptors are limited. On the other hand, convincing and reproducible evidence of such a role for the chemokine receptors CXCR4 and CCR7 in different tumors is available. CXCR4 is by far the most common chemokine receptor overexpressed in human cancer cells. Apart from leukocytes, in which CXCR4 is ubiquitously present (Table 1), expression of CXCR4 is low or absent in many normal tissues, including breast,6 ovary,7 and colon.8 CXCR4's sole ligand, CXCL12, is constitutively expressed in many tissues, including common sites of metastasis (eg, lung, liver, bone).
In the preclinical setting, CXCR4 activation by CXCL12 induces migration and/or survival of brain,9 colorectal,10 prostate,11 renal,12 and ovarian tumor cells,13 along with melanoma14 and neuroblastoma.15 Elevated CXCR4 expression in primitive tumors is associated with LN metastasis in breast,16–20 head and neck,21 colon,22 and esophageal carcinoma.23 Moreover, CXCR4 expression is associated with intraperitoneal and intrapleural spread of ovarian cancer7 and nonsmall cell lung cancer (NSCLC),24–26 respectively. Researchers found that the majority of metastases from melanoma in their study expressed CXCR4.27 In glioblastoma cells, CXCR4 expression is up-regulated compared with that in normal brain tissue, and tumor cells secrete CXCL12.9 Strong expression of CXR4 has been associated with adverse outcome in osteosarcoma,28 epithelial ovarian cancer,29 NSCLC,24 colon,22, 30 and esophageal carcinoma,23 nasopharyngeal carcinoma,31 and melanoma32 (Table 2).
Table 2. Chemokine Receptor Expression and Clinical Outcome in Different Human Cancers
Predictive for metastases
NA indicates not assessed; predictive, associated with local or distant metastases; NR, not reported; NS, not statistically significant; NO, nonprognostic; HNPC, human nasopharyngeal carcinoma; OS, overall survival; EFS, event-free survival; BM, bone marrow; NSCLC, nonsmall cell lung cancer.
Invasive breast cancer has exhibited higher CXCR4 expression when compared with normal breast tissue and ductal carcinoma in situ. However, different patterns of CXCR4 expression in breast cancer cells may have different prognostic value.33 Cytoplasmic CXCR4 expression is associated with LN-positive tumors, estrogen receptor-negative and HER-2-positive disease, and decreased overall survival rates.18 Conversely, nuclear CXCR4 expression is significantly associated with LN negativity and decreased nuclear grades.16 A recently reported correlation between nuclear and cytoplasmic staining for CXCR4 and histological breast cancer type appears to be particularly relevant.33 Authors reported similar findings in patients with early-stage NSCLC, in whom strong CXCR4 nuclear staining was associated with improved outcome.24
Neoplastic epithelial cells coexist with several distinct stromal cell types that together create the tumor microenvironment. Extensive clinical evidence and experimental models support the contribution of the stromal microenvironment to the development, invasiveness, and metastasis of a variety of tumors. Carcinoma-associated fibroblasts but not normal mammary fibroblasts have high levels of CXCL12 expression.34, 35 Expression of CXCL12 in turn promotes the progression of breast cancer by directly enhancing tumor-cell growth and recruiting endothelial progenitor cells, which are required for tumor angiogenesis. Thus, a high level of expression of CXCL12 in carcinoma-associated fibroblasts apparently can promote breast cancer progression in both a paracrine and endocrine fashion.34
A low oxygen concentration induces a high level of expression of CXCR4 in different cell types, including cancer cells and endothelial cells, which is paralleled by an increase in chemotactic responsiveness to CXCL12.36 Induction of CXCR4 expression by hypoxia depends on both activation of hypoxia-inducible factor (HIF)-1 and transcript stabilization. Moreover, HIF-1 induces CXCL12 expression in ischemic areas in proportion to reduced oxygen tension. Under normal oxygen tension, the von Hippel-Lindau tumor suppressor gene induces degradation of HIF-1. Loss of function of the von Hippel-Lindau tumor suppressor gene and/or hypoxic conditions result in constitutive activation of the HIF-1 pathway,37 leading to inappropriate accumulation of HIF-1 and, as a result, increased expression of CXCR4. An analysis of clear cell renal carcinoma and hemangioblastoma that displayed mutation of the von Hippel-Lindau gene in most cases showed increased expression of both CXCR4 and its ligand through both an autocrine and paracrine signaling pathway.38
Functional CXCR4 is also expressed in nonhematopoietic tissue-committed stem/progenitor cells and breast cancer stem/progenitor cells.39 Researchers have documented the clonogenicity and tumorigenicity of breast cancer stem cells characterized by a CD44+CD24−/low phenotype in NOD/SCID mice.40 CD24, a heavily glycosylated cell-surface protein, negatively affects CXCR4 membrane lipid-raft residence and optimal signaling. CXCR4 appears to be present in membrane rafts in CD24−/low cells, whereas it has been found to be absent in CD24+/+ cells preventing triggering downstream signaling in response to CXCL12 interaction.41
Researchers have investigated another chemokine receptor, CCR7, for its potential role in directing metastasis to regional draining LNs, mimicking its physiological role in mature DCs and naive T cells (Table 1). The CCR7 ligands CCL19 and CCL21 are constitutively expressed in lymphoid tissue. Breast cancer cells have shown chemotaxis and chemoinvasion toward CCL21 gradients,6 suggesting that CCR7/CCL21 interactions play a role in breast cancer metastasis to LNs. Intravenous injection of CCR7-transfected B16 melanoma cells in mice increased the incidence of metastasis to LNs but not to lung; LN metastases could be blocked by administration of an anti-CCL21 monoclonal antibody.42 Finally, studies have positively correlated CCR7 expression in different types of cancer,16, 25, 43–46 with nodal metastasis and poor prognosis (Table 2).
Chemokines and Intracellular Signaling Pathway Interactions
In addition to direct stimulation of tumor-cell survival, proliferation, and spread, chemokines are implicated in cross-talk, with signaling pathways specific to neoplastic cells. Members of the epidermal growth factor (EGF) receptor (EGFR) family (HER-1 and HER-2) can activate different signaling modules leading to tumor proliferation, metastasis, and chemo- and radioresistance.47 It has been observed that overexpression of HER-2 increased the expression of CXCR4, which in turn was required for HER-2-mediated invasion in vitro and lung metastasis in vivo. Studies identified 2 distinct mechanisms of CXR4 overexpression: enhancement of CXCR4 protein synthesis and inhibition of its ligand-induced degradation.48, 49 Conversely, inhibition of HER-2 expression using treatment with trastuzumab or small interfering RNA (siRNA) resulted in a corresponding reduction in CXCR4 expression.
The nuclear factor (NF)-κB family of transcription factors has a fundamental role in cancer development, progression, and treatment resistance.50 Studies have indirectly linked NF-κB with tumor metastasis because of its ability to regulate expression of matrix metalloproteinases, interleukin (IL)-8, vascular endothelial growth factor, and CXCR4.51 Studies have also demonstrated that HER-2 can activate the NF-κB pathway52 and that NF-κB activation results in enhanced CXCR4 expression.53 Furthermore, several reports have shown that ligand-independent transactivation of EGFRs and their downstream signaling pathways is critical for the mitogenic activity of G protein-coupled receptor superfamily members such as CXCR4.54 Researchers have reported transactivation of HER-2 and HER-1 in breast and ovarian cancer cell lines, respectively, after CXCL12/CXCR4 interaction through cytoplasmic tyrosine kinase (c-Src) activation.48, 54
In patients with NSCLC, activation of HER-1 by EGF increases expression of CXCR4 and the migratory capacity of lung cancer cells.26 When NSCLC cells are cultured with EGF under hypoxic conditions, CXCR4 expression is dramatically enhanced.26 Thus, a combination of low oxygen tension and EGFR overexpression in primary NSCLC cells may provide the microenvironmental signals necessary to up-regulate CXCR4/CXCL12 expression and promote metastasis. Conversely, inhibitors of downstream targets activated by EGFR (eg, phosphatidylinositol 3-kinase and mammalian target of rapamycin) may block both activation of HIF-1 and increases in the expression of CXCR4 and prevent chemotaxis of NSCLC cells in response to CXCL12.55 Moreover, intratumoral hypoxia can activate NF-κB, further enhancing CXCR4 expression. Overall, these observations clearly indicate the complexity of the cancer ‘chemokine-chemokine receptor’ network and the sophisticated fine tuning of the CXCL12/CXCR4 axis at both the ligand and receptor level and are likely to help identify or redefine new therapeutic target pathways.
Immunosuppressive Networks in the Tumor Microenvironment: The Role of Chemokines
Convincing evidence indicates that a developing tumor interacts with the host immune system and finally escapes immune destruction.56 This interaction includes hijacking the chemokine network to evade immune recognition. The presence of DCs within human tumors as a consequence of active recruitment by tumor-derived chemokines is well documented.57 Studies suggest that the tumor microenvironment may imbalance their action toward immune paralysis.57, 58 Furthermore, human tumors (eg, ovarian cancer) are often infiltrated by macrophages recruited by tumor-secreted chemokines (eg, CCL2)59 (Table 1). Tumor-associated macrophages are also believed to have primarily protumor biological functions in vivo through the production of several factors that promote tumor angiogenesis and hinder immune reactivity.60 Finally, researchers showed that tumor-derived chemokines can recruit immunosuppressive T-regulatory (Treg) cells.58 Ovarian cancer cells and tumor-associated macrophages were shown to secrete CCL22, which recruited CCR4-positive Treg cells to ovarian tumors and malignant ascites61 (Table 1). Administration of a blocking anti-CCL22 monoclonal antibody reduced Treg-cell migration to tumors, providing compelling evidence that CCL22 is a major chemoattractant for Treg cells in ovarian cancer.61 Importantly, the presence of tumor-infiltrating Treg cells correlated with defective antitumor immune responses and poor patient prognosis.61 At present, the relevance of the CCL22/CCR461 and/or CXCL12/CXCR462 pathways in recruitment of Treg cells to other tumor microenvironments is unclear. Overall, blocking the pathways described above63 (eg, by antibodies to relevant chemokine receptors) may be a strategy for boosting tumor-specific immune responses.
Experimental Evidence for Cancer Chemokine Network-Directed Therapies
The largest body of evidence supporting a role for chemokine and chemokine receptor involvement in metastasis from different tumors centers on the CXCR4/CXCL12 axis. Therefore, researchers thus far have focused therapeutic strategies on this receptor-ligand pair. Molecules already in use with different indications and new specific targeted therapies have shown potential in this respect.
Heparin and Bisphosphonates: New Roles for Old Players
In experimental systems, interference with coagulation can affect tumor biology. A brief course of subcutaneous low molecular weight heparin may prolong survival, at least in certain subgroups of patients with advanced malignancies without venous thromboembolism.64 Chemokines promote specific leukocyte extravasation at sites of inflammation after binding to the heparan sulfate (HSGAG) component of proteoglycans. CXCL12 can be competitively displaced by soluble heparins (including tinzaparin, dalteparin, and nonanticoagulant low molecular weight heparin) in preclinical models in a dose-dependent manner. Researchers have observed complete chemotaxis inhibition of CXCR4-expressing cancer cells induced by heparin.65 Moreover, structural dimerization is essential for in vivo activity of chemokines. Heparin can shift the CXCL12 monomer-dimer equilibrium, promoting the monomeric state.66 These observations add a novel rationale for heparin use in the adjuvant setting.
Osteoclast activation appears to be an important mechanism of bone destruction and bone morbidity from both osteolytic and osteoblastic metastases. Preclinical data suggest that nitrogen-containing bisphosphonates not only block osteoclast-mediated bone resorption but also reduce invasion, proliferation, and survival of metastatic tumor cells in bone.67, 68 In breast and prostate cancer cell lines, zoledronic acid inhibits the chemotactic effect induced by CXCL12, mainly through decreased expression of CXCR4 and reduced cell motility.67 Similarly, researchers showed that minodronate (YM529) decreases CXCR4 expression in both osteoblastic and osteolytic prostate cancer cells in vitro and in vivo, suggesting that its ability to inhibit prostate cancer bone metastases is a result, at least in part, of inhibition of the CXCR4/CXCL12 axis.69
In experimental systems, selective inhibition of CXCR4 suppresses CXCL12-induced migration of cancer cells, invasion, neoangiogenesis and metastases6, 9, 70–77 (Table 3). In immunodeficient mice, studies showed that lung metastases from human breast cancer could be inhibited by a neutralizing antibody against CXCR472 or that their growth could be delayed by inhibiting CXCR4 with an siRNA73 or specific CXCR4 antagonists.70 Similarly, neutralization of CXCR4 significantly impairs the invasive capacity of glioblastoma cells.9
Table 3. Preclinical Evidence of Antitumor Activity Induced by Blocking the CXCL12/CXCR4 Pathway
NA indicates not assessed. VEGF, vascular endothelial growth factor.
Researchers originally developed small molecular CXCR4 antagonists such as AMD3100, T140, and ALX40-4C for treatment of human immunodeficiency virus infection, as CXCR4 functions as a co-receptor for the virus.78 AMD3100 is a bicyclam noncompetitive CXCR4 antagonist that fell short of expectations as an antiviral therapeutic but showed an acceptable toxicity profile.79 Comparative studies of T140, AMD3100, and ALX40-4C found that each of them inhibits CXCR4 via different mechanisms.80 Both AMD3100 and ALX40-4C displayed weak partial agonistic activity on CXCR4 (CXCL12-like).81 Accordingly, administration of AMD3100 results in mobilization of hematopoietic stem cells, mimicking the physiological effect of CXCL12 and thus carrying the risk of hematologic toxic effects if administered in combination with chemotherapy.79 In comparison, T140 has pure antagonistic activity (ie, it cannot induce any residual survival/prometastatic signals upon binding to CXCR4) and suppresses hematopoietic stem cell mobilization in mice.80 Overall, the T140 profile best fulfills the requirement for an anticancer compound. Finally, CTCE-9908 is a new analog of CXCL12 that acts as a competitive inhibitor of CXCR4. Studies showed that it decreased CXCL12 binding to CXCR4 by 50% as determined using radioligand assay, and researchers have successfully tested it in different preclinical models of prostate cancer and osteosarcoma.82, 83 CTCE-9908 has proven to be safe in healthy adults, and investigators recently initiated a phase Ib/II clinical trial of CTCE-9908 for NSCLC and other solid tumors.
Chemokines for Use in Immunotherapy of Cancer
Given the chemoattractive1, 2, 57 effects of chemokines on different leukocyte populations (Table 1), researchers have used intratumoral delivery of chemokines to recruit selected leukocyte populations to tumors as initiators or effectors of antitumor immune responses. Based on the dynamics of an adaptive immune response, one would expect chemoattraction of immature DCs to tumors to initiate a tumor-specific immune response in a physiological manner (Fig. 1A). This strategy would also circumvent the need for preparation of DCs from each patient and expose the whole antigenic repertoire of individual tumors to the immune system. Indeed, injection of CCL2084 and CCL1685 (Table 1) into different subcutaneously established tumors markedly inhibited their growth and prolonged survival, eliciting tumor-specific T-cell immunity. Importantly, intratumoral delivery of CCL16 before excision of a primary tumor prevented metastatic spread and cured 63% of mice as compared with 0% with surgery alone (Table 4).85 Authors have reported substantial antitumor effects of intratumoral administration of chemokines active on other leukocyte subsets, such as CCL19,86 CCL21,87, 88 and CXCL1089, 90 on mature DCs and XCL1 on effector cells (Table 1).91, 92 In these studies, therapeutic efficacy may have depended on the recruitment of Th1, CD8, and natural killer (NK) antitumor effectors (Fig. 1B), whereas the role of DCs after chemokine-mediated recruitment was not clear (Table 4). However, CCL21 differs in receptor specificity in mice and humans, possibly leading to overestimation of antitumor activity in mouse models (Table 1).87 Overall, although researchers reported tumor regression, chemokines used as single agents showed limited efficacy in curing established tumors.85 Efficacy depended largely on the antigenicity of the tumors.84 Thus, investigators have devised different strategies to increase the therapeutic potential of chemokines against tumors. One strategy is modification of the tumor microenvironment to elicit an innate immune response. For example, intratumoral administration of CpG (stretches of bacterial DNA that stimulate innate immunity) coupled with systemic administration of an anti-IL-10 (an immunosuppressive cytokine) monoclonal antibody potentiated the therapeutic efficacy of intratumoral delivery of CCL1693 by switching the function of infiltrating macrophages and DCs from immunosuppression to immunopromotion (Table 4). However, the range of immune cells responding to CpG differs in mice and humans, raising the question of whether activation of innate immunity can be reproduced to the same extent in patients.
Table 4. Preclinical Testing of Chemokine-Based Intratumoral Immunotherapy in Syngeneic Mouse Tumor Models
In several studies, coadministration of immunostimulatory cytokines (IL-2 and IL-12) resulted in augmented antitumor effects when compared with administration of the individual chemokines XCL192, 94 and CXCL1089, 90 or cytokines alone (Table 4). In the report by Narvaiza et al.,89 mice given the combination of chemokines and cytokines also exhibited regression of distant, noninjected tumors, confirming the role of immune effector cells in tumor elimination. Interestingly, injection of CXCL10 and IL-12 into separate tumors in the same animal abrogated much of the antitumor response, demonstrating the importance of colocalization of chemokines with cytokines. This synergistic activity is likely a result of recruitment of T cells to the tumor through CXCL10 expression and their local expansion and activation induced by IL-2 or IL-12, which researchers have referred to as the “attraction-expansion paradigm.” It also offers solutions for reducing potentially dose-limiting toxic effects of systemic cytokine administration.89 Finally, intratumoral administration of chemokines may direct adoptively transferred tumor-specific T cells bearing the relevant chemokine receptor toward tumors.95 Alternatively, T cells can be engineered with receptors for chemokines expressed in tumors.96
In the case of tumors expressing molecularly identified tumor antigens, researchers have successfully used chemokines as vaccine carriers to elicit tumor-specific immunity. Genetic fusion of a model nonimmunogenic tumor antigen with inflammatory chemokines generated antigen-specific, humoral, and cellular protective antitumor immunity in 2 different syngeneic mouse models.97 Generation of immunity depended on chemokine-mediated antigen delivery to chemokine receptors on DCs to induce receptor-mediated uptake and subsequent processing and presentation of the antigen to both CD4+98 and CD8+99 T lymphocytes (Fig. 2A). Investigators have also used chemokines as vaccine adjuvants given their capacity to recruit DCs leading to amplification of antigen presentation (Fig. 2B).100
Conclusions and Perspectives
As described herein, we reviewed the body of evidence strongly suggesting that the ‘chemokine-chemokine receptor’ network plays several key roles in tumor survival, proliferation, metastasis, and immune escape, making it an attractive therapeutic target. Inhibition of the CXCL12/CXCR4 axis has demonstrated significant antineoplastic activity in animal models (Table 4). Because most preclinical studies have failed to demonstrate that CXCR4 inhibition can eliminate established metastases, CXCR4-blocking strategies might be effective, particularly for early-stage cancer. The involvement of chemokines in signaling pathways already targeted by molecular therapies (eg, trastuzumab/HER-2, cetuximab/HER-1) may suggest an association of chemokine interference strategy in order to improve treatment efficacy.
Several reports have highlighted the potential of chemokines in cancer immunotherapy. Intratumoral administration of chemokines relies on the assumption that human tumors express tumor-rejection antigens and that specific T cells amenable to activation exist in the host repertoire.56 A general caveat concerning animal models relates to the use of subcutaneously established tumors, even though the skin is not the most common site of primary or metastatic human cancer. The skin has a complex network of immune cells100 absent in other sites (eg, liver, bone), so it is conceivable to tailor chemokine-based immunotherapy to the target tissue. A second general caveat is that murine tumors, unlike human tumors, often bear retroviral antigens that may be responsible for enhanced immune response.93 Cutaneous melanoma is a tempting target for intratumoral immunotherapy given its accessibility, antigenicity, and lack of effective treatment of recurrent disease. The availability of topical immune adjuvants (eg, imiquimod) used to activate tumor-infiltrating DCs further increases the appeal of such a strategy. Intratumoral chemokine administration to jump-start an immune response before excision of a primary tumor85 is also applicable to patients with common visceral cancers, who undergo invasive diagnostic procedures (eg, colonoscopy) before surgery. Finally, protumor, rather than antitumor, effects after intratumoral expression of the same chemokine (eg, CCL20), have been reported.84, 101
Advances in genomic sequencing will likely expand the number of tumor antigens suitable for patient vaccination. Chemokines have been extensively tested as carriers for induction of specific immunity against the idiotype of syngeneic murine B-cell tumors, in which the results compared favorably with those reported for other vaccine formulations.102 In a clinical perspective, the use of chemokines as vaccine carriers raises the concern that autoimmunity against chemokines may also be elicited,103 with unknown consequences. However, replacing native chemokines with viral chemokines in vaccines would reduce this risk, given the only partial sequence homology between human and viral chemokines.104 With regard to the use of chemokines as adjuvants, little is known of the effects of chemokine administration to humans.105
In conclusion, inhibition of tumor-cell survival and metastatic spread and manipulation of antitumor immune response by interfering with the chemokine-chemokine receptor network have been successful in animal models. Although this does not necessarily predict a positive outcome of clinical trials, testing of these strategies in selected patient populations is warranted.