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Cytokine patterns in patients with cancer: a systematic review
Review
Cytokine patterns in patients with cancer: a systematic review Bodo E Lippitz
Active, but dysfunctional, immune responses in patients with cancer have been studied in several tumour types, but owing to the heterogeneity of cancer theories of common reaction mechanisms seem to be obsolete. In this Review of published clinical studies of patients with cancer, expression and interplay of the following cytokines are examined: interleukin 2, interleukin 6, interleukin 8, interleukin 10, interleukin 12, interleukin 18, tumour necrosis factor α (TNFα), transforming growth factor β (TGFβ), interferon-γ, HLA-DR, macrophage migration inhibitory factor (MIF), and C-X-C motif chemokine receptor 4 (CXCR4). Clinical data were analysed in a non-quantitative descriptive manner and interpreted with regard to experimentally established physiological cytokine interactions. The clinical cytokine pattern that emerged suggests that simultaneous immunostimulation and immunosuppression occur in patients with cancer, with increased concentrations of the cytokines MIF, TNFα, interleukin 6, interleukin 8, interleukin 10, interleukin 18, and TGFβ. This specific cytokine pattern seems to have a prognostic effect, since high interleukin 6 or interleukin 10 serum concentrations are associated with negative prognoses in independent cancer types. Although immunostimulatory cytokines are involved in local cancer-associated inflammation, cancer cells seem to be protected from immunological eradication by cytokine-mediated local immunosuppression and a resulting defect of the interleukin 12–interferon-γ–HLA-DR axis. Cytokines produced by tumours might have a pivotal role in this defect. A working hypothesis is that the cancer-specific and histology-independent uniform cytokine cascade is one of the manifestations of the underlying paraneoplastic systemic disease, and this hypothesis links the stage of cancer with both the functional status of the immune system and the patient’s prognosis. Neutralisation of this cytokine pattern could offer novel and so far unexploited treatment approaches for cancer.
The immune system can recognise transformed cells, and both innate and adaptive immune reactions to cancer have long been described. The tumour microenvironment contains macrophages, neutrophils, mast cells, myeloid-derived suppressor cells, dendritic cells (DCs), natural killer (NK) cells, and T and B lymphocytes.1 Tumour-associated antigens and T lymphocytes that are able to recognise tumour-specific antigens have been described.2–5 Tumour-associated macrophages and tumour-infiltrating leucocytes accumulate within neoplastic tissue.6 Activated immune effector cells, such as NK cells, cytotoxic T cells, and macrophages, are present both at the tumour site and in the circulation of patients with cancer, but immune responses against cancer seem to be dysfunctional and tumours progress despite existing immunological activity.5,7 The possibility of local tumour immune escape or even tumour-induced immune suppression has been studied and discussed in detail.7–9 The term immunoediting has been used to describe the immunological selection of resistant tumour variants by elimination of immunosensitive malignant cells.10,11 The resulting avoidance of immune destruction has been defined as a hallmark of cancer.12 An inflammatory microenvironment seems to be a consistent component of malignant tumours, suggesting the presence of a cancer-related immune reaction.1,6 As summarised by Mantovani and colleagues,6 there is increasing evidence that inflammation contributes to the development of cancer and also that cancer seems to directly promote the generation of an inflammatory microenvironment. Trinchieri13 stated in a recent review of the links between inflammation and cancer that “the class of inflammation and immunity that www.thelancet.com/oncology Vol 14 May 2013
Department of Neurosurgery, Karolinska University Hospital Stockholm, Sweden (Prof B E Lippitz MD) Correspondence to: Prof Bodo E Lippitz, Department of Neurosurgery, Karolinska University Hospital Solna, 17176 Stockholm, Sweden [email protected]
is responsible for tumour initiation and early progression is likely also to be the same class that makes the immune system unable to destroy the tumours successfully”. Whether inflammatory conditions increase the local cancer risk or if genetic alterations such as oncogenes cause inflammation and neoplasia is so far unclear,6 but that cancer cells actively interfere with the patient’s immune system has been established. Several recent reviews summarised the cellular interaction between cancer and the immune system in the tumour microenvironment.7–9 However, with respect to the
Pasieka/Science Photo Library
Introduction
Lancet Oncol 2013; 14: e218–28
Interleukin 6 molecule
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See Online for appendix
heterogeneity of cancer, a theory of a common or uniform immune reaction pattern seems so far to be obsolete. Cytokines are intricately involved in all immune reactions. Since cytokines interfere directly or indirectly with each other’s expression, the isolated effect of one cytokine can seem less relevant unless assessed in the context of its position in the hierarchy of a defined cascade. Cytokine interactions comprise sophisticated interdependent positive and negative feedback mechanisms, thus providing homoeostasis and immune control. Any attempt to outline these feedbacks in a model is necessarily simplistic because a more accurate representation of a large feedback system, ideally through a mathematical model, would create a non-linear and practically unpredictable relation. Isolated cytokine reactions have been described in individual tumour types, but so far no systematic model of the cytokinerelated immune reaction in patients with cancer has been developed. In summary, there is increasing evidence from experimental studies that malignant tumours utilise local mechanisms within their microenvironment to prevent activation of immunological effector functions, thereby protecting the tumour from a potential immune attack.8,9 I undertook the present Review of cytokine interactions in patients with cancer with an emphasis on the immunological mechanisms that are associated with clinical disease progression. The approach is intentionally descriptive. Because of this narrow focus, several aspects of cytokine activation and signalling mechanisms were not considered. The format of the present Review is an initial step to detect common patterns in the immune response in patients with cancer, which so far might not be fully represented in experimental systems. The aim of the present Review was to point out reproducible and consistent features among reported clinical data in many
Macrophage migration inhibitory factor expressed in cancer tissue
Lung
Breast
Colorectal
+
+
+
Gastric
and unrelated cancer types. By this translational approach, I attempt to use the context of recent experimental data to interpret the clinical findings. The working hypothesis here is that the emerging cytokine pattern potentially is a manifestation of the underlying systemic paraneoplastic immune response that seems to be a consistent pattern in patients with cancer. Further specification of this system could offer novel therapeutic approaches.7
Transforming growth factor β Transforming growth factor β (TGFβ) has been suggested to be the principal immune-suppressive factor secreted by tumour cells.14 In mice, complete knockout of TGFβ1 results in lethal autoimmunity from a multiorgan inflammatory syndrome.15 TGFβ suppresses interleukin 12 substantially and inhibits interleukin 2 and interleukin-2induced proliferation in T cells.16,17 In CD8+ cytotoxic T lymphocytes and NK cells, TGFβ is a strong antagonist of interferon-γ production. TGFβ has a negative effect on B-cell proliferation and differentiation.18,19 TGFβ is needed for the differentiation of both T-helper-17 (Th17) and induced regulatory T (Treg) cells. The clinical effect of TGFβ in patients has been reviewed in detail.15,20 Malignant cells often secrete large amounts of TGFβ.21 Raised TGFβ1 serum concentrations were independently detected in patients with lung cancer, breast cancer, glioblastoma multiforme, colorectal carcinoma, hepatocellular carcinoma, bladder carcinomas, renalcell carcinoma, and gastric carcinoma (table 1) and were associated with poor prognosis in patients with gastric carcinoma, adenocarcinoma of the lung, and breast cancer. Additionally, TGFβ was associated with metastases in patients with breast cancer, gastric cancer, colorectal cancer, non-small-cell lung cancer (NSCLC), malignant melanoma, and renal-cell cancer (see appendix for further references).
Malignant melanoma
Pancreatic
Malignant glioma
Hepatocellular
+
+
+
+
+
Interleukin 8 produced by tumour cells
+
+
+
+
+
+
Increased serum concentrations of interleukin 6
+
+
+
+
+
+
Decreased expression of interleukin 12
+
+
+
+
+
+
+
+
Decreased interferon-γ production in immune cells
+
Reduced expression of HLA-DR
+
Increased serum concentrations of transforming growth factor-β
+
+
+
+
C-X-C motif chemokine receptor 4 tumour expression
+
+
+
+
+
Increased serum concentrations of interleukin 10
+
+
+
+
+
+
+
+
Renal cell
Head and neck +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Pluses indicate published evidence exists.
Table 1: Published evidence for specific cytokine expression in selected cancer types
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Hence, TGFβ is one of the cytokines produced by tumour cells with immunosuppressive activity and a potent inhibitory effect on several other cytokines. With an essential role in maintenance of homoeostasis, TGFβ suppresses anti-tumour immune responses and creates a local environment of immune tolerance. Increased serum concentrations of the immunosuppressive cytokine TGFβ are a frequent finding in patients with cancer, to the extent that it could potentially represent a general cancer-associated feature.
was associated with metastases in 15 different cancer types independent of their specific histological findings (in NSCLC, gastric cancer, oesophageal cancer, oral squamous-cell cancer, renal-cell cancer metastases, laryngeal and hypopharyngeal squamous-cell cancer, breast cancer, primary squamous cell-carcinoma of the oral cavity, colorectal cancer, cervical adenocarcinoma, hepatocellular cancer, nasopharyngeal cancer, melanoma, and pancreatic cancer; table 2; see appendix for further references).
C-X-C motif chemokine receptor 4
Interleukin 10
Chemokines are molecules that direct cells to specific organs. The chemokine stromal cell-derived factor-1 (SDF-1, also known as CXCL12) binds to C-X-C motif chemokine receptor 4 (CXCR4).22 CXCR4 is expressed in malignant tumour cells whereas its ligand SDF-1 (CXCL12) is expressed in several organs including lung, liver, brain, kidney, skin, and bone marrow, with a probable function during physiological repair mechanisms. Several CXCR4positive cancers seem to metastasise in an organ-specific and CXCL12-dependent manner, and CXCR4 and the ligand CXCL12 seem to be involved in the development of cancer metastasis, with chemokines and particularly CXCR4 possibly being essential components of the metastatic process.22–24 In an experimental model and through genomic analyses on stem cells, CXCR4 upregulation was induced by TGFβ signalling.25,26 Upregulation of CXCR4 by TGFβ, which also occurs in breast-cancer cells,27 would provide a potential link for the described association between TGFβ and metastases. CXCR4 is expressed in various different tumour types (table 1) and has been considered the most widely expressed chemokine receptor in most cancers.23,24 Additionally, expression of CXCR4 in tumour specimens
TGFβ also upregulates the usually immunosuppressive cytokine interleukin 10, whereas interleukin 10 enhances the expression of TGFβ in a positive feedback circuit.28 Interleukin 10 inhibits antigen presentation, MHC class II expression, and the upregulation of costimulatory molecules CD80 and CD86. Interleukin 10 prevents the production of the Th1-associated cytokines interleukin 2 and interferon-γ from antigen-presenting cells (APCs). Physiologically, interleukin 10 significantly suppresses the major inflammatory cytokines interleukin 1, interleukin 6, interleukin 12, and TNFα.29 As a consequence, interleukin 10 was initially classified among the immunosuppressive (Th2) cytokines with a physiological role in the termination of the T-cell-mediated response. Interleukin 10 seems to act primarily on DCs and macrophages and inhibits the differentiation and the antigen-presenting properties of DCs. Interleukin 10 inhibits essential steps in immune detection such as the expression of HLA-DR and costimulatory molecules.28 Control of interleukin 10 expression involves negative feedback mechanisms including interleukin 10 itself. TNFα promotes proinflammatory reactions while upregulating interleukin 10 in macrophages and
Lung
Breast
Colorectal
Gastric
Malignant melanoma
Macrophage migration inhibitory factor expression and negative prognostic effect
+
+
Interleukin 8 is associated with tumour size, depth of infiltration, or increased stage
+
+
Interleukin-6 serum concentration and negative prognostic effect
+
+
Interleukin-18 serum concentration associated with advanced stage
+
+
Increased interleukin-18 serum concentration and negative prognosis
+
High expression of HLA-DR and positive prognosis
+
+
+
C-X-C motif chemokine receptor 4 tumour expression associated with metastases
+
+
+
+
+
Raised interleukin-10 serum concentration associated with a negative prognostic effect
+
+
+
+
Oesophageal Pancreatic
Hepatocellular carcinoma
+
+
+
+
+
+
+
+
+
+
+
+
+ + (+) serum soluble HLA-DR +
Renal cell
+ +
+
Diffuse B-cell lymphoma
+
+
+
+
+
+
(+) downregulated genes
+
+
+
+
+
+
+
+
Pluses indicate published evidence exists.
Table 2: Published evidence for specific cytokine effects in various cancer types
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monocytes as a negative feedback, thereby terminating the inflammatory response. Interleukin 10 suppresses release of TNFα and itself. The cellular sources of interleukin 10 are Th2 cells, a subset of Treg cells, and Th17 cells; however, cytotoxic CD8+ T cells can also produce interleukin 10, as can some subsets of DCs, B cells, granulocytes, eosinophils, mast cells, keratinocytes, and epithelial cells.29,30 Findings from experiments with in-vitro co-cultures showed that colon-cancer cells first stimulated macrophages to produce interleukin 6, which was then followed by interleukin-6-induced production of interleukin 10 by colon-cancer cells.31 Similarly, in vitro, interleukin 6 in association with TGFβ can induce interleukin-10 production in Th17 cells and interleukin 6 can stimulate interleukin-10 production in cancer cells.32–34 Various cancer cells produce interleukin 10; among those are oral squamous-cell carcinoma cells, multiple myeloma cells, melanoma cells, human colon-carcinoma cells, NSCLC cells, renal-cell carcinoma cells, non-Hodgkin lymphoma cells, and chronic lymphocytic leukaemia B lymphocytes. Circulating concentrations of interleukin 10 were raised in 13 different cancer types and were associated with adverse disease stage or with negative prognosis in bone sarcoma, diffuse large B-cell lymphoma, gastric cancer, colon cancer, Hodgkin lymphoma, hepatocellular cancer, melanoma, renal-cell cancer, NSCLC, and pancreatic cancer (table 2; see appendix for further references). In summary, interleukin 10 is highly immunosuppressive and seems to be frequently expressed by tumour cells, resulting in increased circulating serum concentrations of interleukin 10 in most human cancers. The frequent association of raised interleukin-10 concentrations with negative prognosis links the increased systemic interleukin-10 concentrations with the later stages of cancer. Increased serum concentrations of interleukin 10 and the resulting immunosuppression seem to be a common feature of progressive cancer in human beings.
Interleukin 2 Interleukin 10 is also an inhibitor of interleukin-2 production in Th2 cells. Interleukin 2 is a growth factor for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition in adaptive immunity. Interleukin 2 is produced primarily by activated CD4+ T cells and by naive CD8+ T cells and DCs.35 Interleukin 2 stimulates proliferation and differentiation of NK cells.36 Activated cytotoxic T cells need interleukin 2 as a growth factor at late stages of the immune reaction, whereas the initial proliferation of T cells seems to be interleukin-2 independent. Interleukin 2 is also crucial for development and peripheral expansion of Treg cells.35 In the absence of interleukin 2, T-cell receptor engagement results in anergy.36 The soluble interleukin-2 receptor acts as an antagonist of interleukin 2. There is a negative relation between e221
serum concentrations of interferon-γ and production of soluble interleukin-2 receptor. Disease progression or negative prognosis in cancer was associated with reduced interleukin-2 concentrations or an increase in soluble interleukin-2 receptor concentrations (see appendix for further references).
Interaction between interleukin 12, interferon-γ, and HLA-DR The reciprocal activation of APCs and NK cells via interleukin 12 and interferon-γ is one of the central processes in immunodetection and seems to be negatively affected in patients with cancer. APCs, macrophages, and, mainly, DCs bind antigens in the periphery and transport these antigens to the lymph nodes. In the lymph nodes, APCs present MHC-class-IIassociated peptides for recognition by CD4+ lymphocytes. HLA-DR (MHC class II) expression needs stimulation by interferon-γ, which occurs when the innate immune response is activated. The activation of interferon-γ needs stimulation by interleukin 12, which is produced by APCs when presenting antigens to T cells.36 DCs were identified as the main source of interleukin 12 produced early during the innate immune response, which was identified as the functional connection between non-specific innate and antigenspecific adaptive immunity.37 Interleukin 12 enhances the generation and activity of cytotoxic T lymphocytes and has an essential role in cell-mediated immunity. Interleukin 12 promotes maturation and activation of NK cells, the effector cells of the innate immune system. In response to release of interleukin 12 from macrophages and DCs, NK cells produce interferon-γ, which activates macrophages independently from T cells during the early phases of the innate immune response.38 Interleukin 12 and interferon-γ are linked in a positive feedback circuit through phagocytes and DCs. The structure and function of interleukin 12 and the related, but functionally opposing, interleukin 23 have been reviewed in detail.39 Both TGFβ and interleukin 10 suppress interleukin 12 significantly and therefore have an effect on the interaction between interleukin 12, interferon-γ, and HLA-DR and hence on the central circuit in immunodetection.29
Reduced interleukin-12 expression Interleukin 12 is one of the essential proinflammatory cytokines that stimulates Th1 responses; increased expression of interleukin 12 should occur during a physiological immune reaction.37 This step seems to be negatively affected in many patients with cancer: expression of interleukin 12 seems to be decreased in several cancer types, particularly in late stages with larger and more advanced disease, as described in anaplastic astrocytoma, glioblastoma, paediatric soft-tissue sarcomas, renal-cell cancer, head and neck squamouscell carcinoma, malignant melanoma, colorectal cancer, www.thelancet.com/oncology Vol 14 May 2013
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gastric cancer, and hepatocellular carcinoma (table 1; see appendix for further references). Interleukin-12 production by stimulated peripheral blood mononuclear cells decreased significantly in patients with gastric and colorectal cancer with advancing disease. Importantly, interleukin 12 can induce the production of large amounts of interferon-γ from T cells and NK cells. The functional consequence of the described reduction in production of interleukin 12 would be a negative effect on the efficacy of NK cells and secondarily on the activation state of macrophages. Hence, cancerassociated downregulation of interleukin 12 could represent a central dysfunction of APCs, which impair the functionality of effector cells of both innate and adaptive immune systems, thus creating a local environment of immune tolerance.
Interferon-γ Interleukin 12 is essential for the production of interferon-γ, which in turn has an essential role in MHC expression as a central step of immunodetection. Interferon-γ is the principal macrophage-activating cytokine. The major sources of interferon-γ are CD8+ T cells, CD4+ T cells, and NK cells.40 Activated CD8+ T cells produce interferon-γ after antigen stimulation.41 Secretion of interferon-γ by NK cells and APCs is probably important in early host defence, whereas T lymphocytes become the major source of interferon-γ during the adaptive immune response. Interferon-γ inhibits the production of the immunosuppressive factors TGFβ and prostaglandin E2, whereas negative regulators of interferon-γ production include interleukin 4, interleukin 10, and TGFβ.42 TGFβ inhibits interferon-γ production by suppression of a transcription factor (T-bet) that is essential for Th1 differentiation of CD4+ T cells and for interferon-γ production. As a theoretically expected consequence of the aforementioned dysfunction of interleukin 12, decreased interferon-γ production in lymphocytes, NK cells, or peripheral blood mononuclear cells has so far been described in malignant melanoma, gastric cancer, lung cancer, glioblastoma, nasopharyngeal carcinoma, colorectal cancer, and head and neck cancer, and decreased interferon-γ tumour expression in advanced renal-cell carcinoma (table 1). Thus, decreased interferon-γ production potentially represents a common cancerassociated immune dysfunction in human beings (see appendix for further references). Interferon-γ induces expression of the human MHC class II protein, HLA-DR, which is needed for antigen recognition by CD4+ T cells. Interferon-γ also upregulates expression of MHC class I as well as that of genes needed for antigen processing. Hence, reduced concentrations of interferon-γ in patients with cancer is expected to have a negative effect on MHC expression and thereby on tumour immunogenicity. www.thelancet.com/oncology Vol 14 May 2013
HLA-DR expression Reduced expression of MHC class I and class II impairs immunological tumour recognition. Additionally, HLA-DR antigen expression is crucial for activation of naive CD8+ T cells and is hence essential for a successful adaptive immune reaction against cancer cells. Markedly diminished HLA-DR expression on monocytes was reported in six cancer types (glioblastoma, lung cancer, pancreatic carcinoma, colorectal cancer, malignant melanoma, and head and neck cancer; table 1). In 12 different unrelated cancer types, worse prognosis or more advanced disease was associated with reduced HLA-DR expression on tumour tissue or monocytes, or vice versa advanced disease stages coincided with decreased soluble HLA-DR serum concentrations (lung cancer, pancreatic carcinoma, breast cancer, nasopharyngeal carcinoma, colorectal cancer, primary non-Hodgkin gastric lymphoma, laryngeal cancer, hepatocellular carcinoma, malignant melanoma, rectal cancer, head and neck cancer and ovarian serous adenocarcinoma; table 2; see appendix for further references).
Interleukin 18 In addition to interleukin 12, production of interferon-γ is influenced by interleukin 18, which is secreted by APCs and a wide range of cell types including activated monocytes, macrophages, T lymphocytes, and NK cells.43 Interleukin 18 is a costimulatory factor for the induction of interleukin-12-mediated interferon-γ production by T-helper cells, and most peripheral CD4+ T cells express the receptor interleukin-18R alpha. Interferon-γ also induces the expression of the antagonistic interleukin-18 binding protein (interleukin 18bp), which functions as an inhibitor of interleukin-18 activity as a potential direct negative feedback mechanism to control interleukin-18-induced production of interferon-γ.44 In the described feedback circuit, downregulated interleukin 12 and interferon-γ would theoretically result in similarly reduced interleukin-18bp expression. The absence of this negative stimulus could potentially explain an increased concentration of interleukin 18. In fact, high concentrations of interleukin 18 were significantly associated with advanced tumour stages in patients with cancer in seven tumour types (oesophageal squamous-cell carcinoma, breast cancer, hepatocellular carcinoma, lung cancer, renal-cell carcinoma, multiple myeloma, and oral cavity cancer; table 2) and were independently associated with shorter overall survival in seven cancers (see appendix for further references).
TNFα Macrophages and DCs function as APCs. Macrophages respond to inflammatory stimuli by immediate production of TNFα, interleukin 1, and other chemokines. TNFα and interleukin 1 are described as acute-response cytokines. The principal function of TNFα is to stimulate the activation and recruitment of neutrophils and e222
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monocytes to sites of inflammation. TNFα (and also interleukin 1) activates vascular endothelial cells and causes endothelial cells to express adhesion molecules for neutrophils, monocytes, and lymphocytes.45,46 TNFα (and interleukin 1) can elicit tissue-factor production on endothelium and monocytes, which initiates coagulation.47 Additionally, circulating TNFα might have systemic effects on myocyte contractility, resulting in myocardial dysfunction.48 Also, malignant cells constitutively produce small amounts of TNF, causing hyperpermeability of blood vessels.49 This effect promoted pleural effusion in a lung-cancer model.50 Increased serum concentrations of TNFα were described in eight independent cancer types (NSCLC, breast cancer, colorectal cancer, prostate cancer, chronic lymphocytic leukaemia, malignant melanoma, non-Hodgkin lymphoma, and gastric cancer; see appendix for further references). In summary, TNFα is a potent immunostimulatory cytokine with both local effects in the tumour microenvironment and potential systemic effects. In established tumours, TNFα contributes to the maintenance of a proinflammatory environment.
Macrophage migration inhibitory factor Macrophage migration inhibitory factor (MIF) is a rapidly induced immunostimulatory cytokine. MIF is expressed in monocytes, macrophages, T and B lymphocytes, eosinophils, mast cells, basophils, and neutrophils.51 MIF inhibits the migration of macrophages and sustains macrophage viability and hence the inflammatory reaction. Macrophages that do not contain MIF are prone to apoptosis.52 Additionally, findings from an experimental model showed that MIF might be a functional non-cognate ligand for the chemokine receptor CXCR4, and in a non-cancer environment MIF promoted the recruitment of both monocytes and T cells by interacting with CXCR2 and CXCR4.53 MIF potentially promotes tumorigenesis by inhibiting the classic tumour suppressor gene p53.54 p53 can promote cell-cycle arrest and apoptosis in response to DNA damage. MIF stimulates expression of the proinflammatory cytokines TNFα, interferon-γ, interleukin 1β, interleukin 6, and interleukin 8 in a positive feedback circuit.55 Overexpression of MIF has so far been found in hepatocellular cancer, lung cancer, ovarian cancer, breast cancer, oesophageal squamous-cell cancer, bladder cancer, cervical squamous-cell cancer, pancreatic cancer, glioblastomas, prostate cancer, osteosarcoma, colorectal cancer, head and neck cancer, and malignant melanoma (table 1). Significantly increased serum MIF concentrations were confirmed in five different cancer types (see appendix for further references). A negative prognostic effect of high MIF expression was reported in patients with oesophageal squamous-cell cancer, hepatocellular cancer, gastric cancer, breast cancer, glioma, nasopharyngeal cancer, head and neck cancer, and NSCLC (table 2; see appendix for further references). e223
MIF is a proinflammatory cytokine with a crucial role in innate immunity. The uniform overexpression of MIF in clinical cancer is another factor that suggests sustained inflammation occurs in the microenvironment of cancer.
Interleukin 8 Increased serum concentrations of MIF might be associated with higher interleukin-8 concentrations, another factor that is also induced by TNFα (and interleukin 1).56 Interleukin 8 is a member of the CXC chemokine family and has a role as activator and chemoattractant for neutrophils. Interleukin 8 is produced by tumour cells of ten different cancer types (NSCLC, breast cancer, colon cancer, gastric cancer, malignant melanoma, pancreatic cancer, malignant glioma, renal-cell carcinoma, oesophageal adenocarcinoma, and B-cell chronic lymphocytic leukaemia; table 1). Raised serum concentrations of interleukin 8 were associated with tumour size, depth of infiltration, or increasing stage of disease in ten different cancer types (colorectal cancer, hepatocellular cancer, bone sarcomas, gastric cancer, metastatic breast cancer, oesophageal cancer, NSCLC, soft-tissue sarcoma, prostate cancer, and diffuse large B-cell lymphoma; table 2; see appendix for further references).
Interleukin 6 A further effect of TNFα and interleukin 1 is the rapid induction of interleukin-6 gene expression particularly in monocytes and macrophages.57 Interleukin 6 is involved in recruitment of neutrophils and promotes the migration and proliferation of T lymphocytes into the affected tissue.58 Resident fibroblasts produce matrix metalloproteinases after interleukin-6 stimulation and degrade the extracellular matrix. Besides the differentiation of B lymphocytes into immunoglobulin-producing plasma cells, interleukin 6 also promotes T-cell differentiation and activation.59 In experimental conditions, interleukin 6 and TNFα costimulate naive CD8 T cells, resulting in strong cytolytic activity.60 Interleukin 8 and interleukin 6 might be sequentially activated cytokines during inflammation, with initial production of interleukin 8 and other chemoattractants stimulating early neutrophil recruitment in the inflammatory site, followed by interleukin 6 providing monocyte recruitment.61 In inflammation, interleukin 6 has a role in the transition between innate and acquired immune responses.61 Findings show that interleukin 6 provides a key signal in the development of Th17 cells while blocking the differentiation of CD4+ cells into Treg cells.62 Serum concentrations of interleukin 6 were increased in 13 different cancer types (lung cancer, breast cancer, colorectal cancer, gastric cancer, malignant melanoma, pancreatic cancer, hepatocellular carcinoma, renal-cell carcinoma, neuroblastoma, non-Hodgkin lymphoma, nasopharyngeal carcinoma, head and neck squamous-cell carcinoma, and bladder cancer; table 1) and seem to be www.thelancet.com/oncology Vol 14 May 2013
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associated with tumour stage and prognosis. Additionally, there were strong positive associations between serum interleukin-6 concentrations and tumour size, tumour stage, or disease progression in patients with gastric cancer, colorectal cancer, bone sarcoma, breast cancer, hepatocellular cancer, nasopharyngeal cancer, renal-cell cancer, lung cancer, and melanoma (table 2; see appendix for further references).
The Th1–Th2–Th17 paradigm The initial Th1–Th2 paradigm was based on the identification of CD4+ helper-cell subpopulations, termed Th1 and Th2, which produce distinct and opposing patterns of cytokines with immunostimulatory and immunosuppressive functions. After activation, CD4+ T cells of the Th1 lineage secrete interferon-γ, TNFα, interleukin 2, and interleukin 12. Th1-related cytokines are generally regarded as immunostimulatory. Th2-related cytokines inhibit the Th1 responses. Th2 CD4+ T cells express high concentrations of interleukin 4, interleukin 5, interleukin 6, interleukin 10, and interleukin 13, inhibit T-cell-mediated cytotoxicity, and support humoral immune responses via B cells.63 The exclusivity of the initial Th1–Th2 paradigm has recently become unacceptable because further CD4+ T-cell populations have been identified including inducible Treg cells and Th17 cells.64 Th17 cells are inducers of tissue inflammation and Treg cells have immunosuppressive functions, thus preventing the activation of self-reactive cells. In published work, Th17 cells have been widely discussed, but their function is still not entirely understood. Treg cells prevent autoimmunity and limit chronic inflammation by suppressive mechanisms such as those involving inhibitory cytokines, cytolysis, metabolic disruption, and modulation of DC maturation or function. Treg-cellderived cytokines are interleukin 10 and TGFβ (and interleukin 35).65,66 Induced Treg cells develop from T-cell precursors at extrathymic sites. TGFβ is needed for the differentiation of both Th17 and induced Treg cells. Th17 cells and Treg cells are functionally reciprocal.67 Both cell types are found in the cancer environment. In the steady state, TGFβ suppresses inflammatory reactions and maintains self-tolerance through induction of induced Treg cells, but when activation of the innate immune system leads to increased interleukin-6 production, Treg generation is blocked, and Th17 cells are induced, causing a proinflammatory response.67 Hence, the presence of interleukin 6 defines whether the immune response is dominated by proinflammatory Th17 cells or Tregs cells, while interleukin 2 antagonises the differentiation of CD4+ T cells into Th17 cells.62,67,68 Th17 cells are frequently found in cancer tissue and microenvironment in ovarian cancer, oesophageal cancer, breast cancer, hepatocellular carcinoma, gastric cancer, colorectal carcinoma, and oesophageal squamous-cell carcinoma. Additionally, high percentages of Th17 cells in www.thelancet.com/oncology Vol 14 May 2013
tumour-infiltrating leucocytes have been described in ovarian cancer, melanoma, breast cancer, and colon cancer, and interleukin-17 expression of cancer cells was found in NSCLC (see appendix for further references). Although the function of the Th17–Treg cellular dichotomy in cancer still remains to be elucidated, an interesting finding is that the previously described uniform presence of the functionally opposing cytokines TGFβ and interleukin 6 in patients with cancer is a prerequisite for the induction of Th17 cells, which are found in many cancer types.
Interleukin 23 Another crucial factor needed for the expansion of Th17 cells is interleukin 23, which has an essential structural similarity with interleukin 12: both cytokines share a common subunit p40. Interleukin 12 consists of the subunits p40 and p35, while interleukin 23 is comprised of p40 and a p19 subunit.39,69 These subunits are predominantly expressed by activated DCs in vivo.70 Whereas interleukin 12 promotes the differentiation of naive T cells into interferon-γ-producing Th1 cells, interleukin 23 has been linked to Th17 development.71,72 In tumours, where interleukin 23 is mainly produced by tumour-associated macrophages, interleukin 23 and interleukin 12 seem to have antagonistic functions: interleukin 12 promotes infiltration of cytotoxic T cells whereas interleukin 23 induces innate inflammatory infiltration, but reduces the ability of CD8 T cells to infiltrate tumours.73,74 So far, little is known about interleukin 23 in patients with cancer, and hence this cytokine has not been included in the present Review. However, recent studies reported high circulating interleukin-23 serum concentrations in patients with breast cancer, pancreatic cancer, colorectal cancer, and gastric cancer, which would be in line with the experimentally established role of interleukin 23 in the generation of Th17 cells, which are frequently found in several cancer types (see appendix for further references).
Cytokines and prognosis in cancer The association between a high serum concentration of interleukin 6 and a negative prognosis was a consistent finding in 16 different cancer types (diffuse large B-cell lymphoma, renal-cell cancer, pancreatic carcinoma, colorectal cancer, gastric cancer, neuroblastoma, metastatic malignant melanoma, non-Hodgkin lymphoma, nasopharyngeal carcinoma, lung cancer, metastatic breast cancer, bladder cancer, advanced gastric cancer, prostate cancer, neck squamous-cell carcinoma, and oesophageal squamous-cell carcinoma; see appendix for further references). Serum concentrations of interleukin 10 have been associated with negative prognosis in ten cancer types (diffuse large B-cell lymphoma, colorectal cancer, gastric cancer, Hodgkin disease, pancreatic carcinoma, bone sarcoma, hepatocellular carcinoma, metastatic melanoma, renal-cell carcinoma, and NSCLC; table 2). e224
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Positive prognoses were reported when functional concentrations of interleukin 12, interferon-γ, interleukin 2, and HLA-DR were maintained. Although the close relation between high interleukin-6 serum concentrations and negative prognosis represents an activation of the immunostimulatory system, interleukin-10 serum concentrations are representative of the systemic immunoparalysis in advanced cancer or the simultaneously activated Th2 system. Concentrations of both the immunostimulatory cytokine interleukin 6 and the immunoinhibitory cytokine interleukin 10 are strongly associated with prognosis in patients with cancer, independent of the tumour heterogeneity. This obvious contradiction shows the lethal situation of a futile maximum immunostimulation in a simultaneously functionally inhibited state.
Present treatment approaches Even without a comprehensive theory of a uniform immune reaction pattern, several cytokine alterations have been specifically addressed in various experimental treatments. Fresolimumab, a human anti-TGFβ monoclonal antibody, neutralises all active isoforms of TGFβ and was used in a phase 1 study for the treatment of 22 patients with advanced melanoma and renal-cell carcinoma.75 The antisense phosphorothioate oligodeoxynucleotide trabedersen is a specific inhibitor of TGFβ2 biosynthesis and is being assessed in an ongoing phase 1/2 dose-escalation study for patients with advanced pancreatic carcinoma, metastasising melanoma, or metastatic colorectal carcinoma.76 Several other potential anti-TGFβ compounds that are in clinical development in cancer have recently been reviewed.77 Similarly, treatment with highdose interleukin 2 has a proven efficacy in producing durable responses in patients with metastatic renal-cell cancer.78 Cancer immunotherapy by interleukin-12-based cytokine combinations has been summarised in detail: unfortunately the systemic administration of interleukin 12 was associated with substantial toxicity, possibly because of effects mediated by interleukin-12-induced interferon-γ production. TNFα has been suggested to have a role in growth of tumours in experimental models. TNFα antagonists had therapeutic activity in mouse models and were used in phase 1 and 2 clinical cancer trials with some evidence of clinical activity.79–81 A so far clinically unexploited option has been developed experimentally: heparinoids inhibited the interaction between CXCL12 and CXCR4 and reduced the haematogenous metastatic spread in a murine model. The specific neutralisation of cancer-associated simultaneous immunostimulation and immunosuppression could provide new therapeutic approaches (see appendix for further references).
Summary of cytokine interactions in cancer The theoretical evidence that has been established in immunological basic research was used in the present Review to progress from a purely phenomenological e225
clinical description of cytokine interactions in cancer towards an attempted relational and sequential analysis. However, provision of a detailed account of the experimental data that served as a foundation for the interpretation of the clinical findings is beyond the scope of this Review. The result of this translational approach is a necessary simplification of the actual situation, with many cytokine functions and interactions remaining not considered. This systematic review of published clinical data shows a consistent pattern of cytokine reaction in patients with cancer and provides circumstantial evidence for a common paraneoplastic phenomenon in advanced cancer, independent of tumour histology. The functional consequence of this cytokine pattern is tumour-induced immune stimulation with a simultaneous initially local and in later stages generalised environment of immune suppression that protects the cancer cells. Concentrations of specific cytokines were consistently associated with patient prognosis in different unrelated cancer types. The reproducibility of this pattern can be interpreted as a general phenomenon in cancer. Despite tumour heterogeneity, patients with advancedstage cancer seem to experience a simultaneous immunostimulation and immunodepression, with increased concentrations of cytokines MIF, TNFα, interleukin 18, interleukin 8, interleukin 6, TGFβ, and interleukin 10. Experimental data show that the presence of the otherwise antagonistic cytokines interleukin 6 and TGFβ favour the generation of Th17 cells. This initially counterintuitive notion actually seems to match the clinical situation, where both interleukin 6 and TGFβ are present in later stages of cancer and where Th17 cells are frequently found in tumour tissue to an extent that development of tumour-infiltrating Th17 cells was suggested as a general feature in patients with cancer.82 The result would be an inflammatory environment in line with the experimentally established notion of inflammation as a consistent component of malignant tumours.1,6 The clinical effect of interleukin-6 and interleukin-10 serum concentrations shows that the systemic cancerrelated immune reaction has immediate prognostic consequences. The inflammatory process seems to be maintained through stimulatory cytokines and Th17 cells, while the central clinical defect of interleukin 12–interferon-γ–HLA-DR expression prevents the eradication of the cancer cell as origins of the inflammatory reaction. A downregulation of interleukin 12 was frequently and independently reported in various cancer types, whereas a physiological immune reaction would essentially need stimulation of interleukin 12. Because several cytokines are produced by the tumour itself, one can conclude that cancer cells contribute significantly in both Th1 and Th2 stimulation via production of MIF and interleukin 8 on one side and TGFβ and interleukin 10 on the other, resulting in suppression of interleukin 12 (and interleukin 2). The dysfunction of interleukin 12 that could www.thelancet.com/oncology Vol 14 May 2013
Review
represent a central defect in patients with cancer is hardly considered in experimental systems. The downregulation of the interleukin 12–interferon-γ–HLA-DR axis results in dysfunction of APCs and macrophages and in impaired antigen detection and even dysfunction of effector cells of both the innate and adaptive immune system. The net effect of this cytokine pattern is a suppression of innate effector cells through inhibition of NK cells (via interleukin 12) and suppression of the specific immune detection and response through reduction of HLA-DR expression despite recruitment of (ineffective) immune cells through interleukin 6 and interleukin 8. The outlined cytokine reaction pattern would theoretically result in impaired activation of macrophages and their associated functional circuits, while tumourassociated macrophages represent a major and substantial inflammatory stromal tumour component.83–85 The assumed combined cytokine effect is a cancer-induced direct blocking of immunological detection and eradication in the immediate microenvironment of tumours. Present immunotherapies including exogenous cytokine administration, antitumour vaccines or adoptive T-cell transfers might fail, since the necessary final step of tumour detection and in particular immunological tumour destruction seems to be blocked by the tumour itself. TGFβ, interleukin 8, and interleukin 10 are produced in tumours and whether one cytokine initiates the cancer-induced process of immunosuppression is unresolved. TGFβ fulfils all requirements for an initiating substance in the hierarchy of the cytokine reaction because it also contributes to upregulation of interleukin 10. Conversely, both interleukin 10 and TGFβ stimulate each other in a positive feedback. Both cytokines are highly immunosuppressive and act through inhibition of interleukin-12 production from APCs. The net result of the described cytokine pattern is local immunostimulation and inflammation with synchronous functional immunosuppression, attracting immune cells, but protecting the tumour cells locally from detection and destruction. Independent of tumour histological changes, this cytokine cascade links the functional status of the immune system with the extent of the cancer disease and the patient’s prognosis and ultimately is an expression of the paraneoplastic systemic disease in cancer. The interpretation of the clinical results in terms of a uniform reaction pattern in cancer does not take into account the differences in malignant phenotypes and biological behaviours and might seem to contradict the hypothesis of an individual cellular immune signature in cancer. However, the recent theory of immunoediting describes the obvious selection process that originates in the functioning immune system and that ultimately results in tumour cells that are resistant to potential immune attacks. Provided this theory is correct, the malignant cellular selection process must be adapted to the uniform conditions of the immune response, thus www.thelancet.com/oncology Vol 14 May 2013
Search strategy and selection criteria To identify eligible studies, I did systematic searches of PubMed repeatedly over a period of 5 years up to 2012 for all individual cytokines in patients with cancer with the keywords “IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, TNF-alpha, TGF-beta, interferon-γ HLA-DR, MIF or CXCR4” in combination with “serum level”, “patients”, “cancer”, “prognosis” and “metastases”. C-X-C motif chemokine receptor 4 (CXCR4) and HLA-DR are not cytokines, but were reviewed because they are involved in key cytokine pathways. I included only clinical studies. No limits were set on cancer type, date of publication, or duration of follow-up. Studies were classed as eligible for inclusion if they were original research studies that reported on the selected cytokines in patients with cancer. Reviews were excluded. The search identified 307 published clinical studies in patients with cancer. Data on interleukin 1, interleukin 4, interleukin 5, and interleukin 15 were considered so far insufficient or inconclusive and were excluded from the present Review. A thorough review of all papers retrieved was done. When similar facts were reported in successive studies, the initial study was generally included. The data were summarised in a non-quantitative descriptive manner and interpreted with regard to theoretically established physiological cytokine interactions. Because of space restrictions, references for the clinical studies are listed in the appendix.
providing a common biological denominator as a prerequisite for tumour progression. Being part of a large feedback network, which normally functions in homeostasis, cytokines have many, sometimes even contradictory, functions. Thus, the claimed uniform immune reaction pattern is in conflict with the potential diversity of cytokine functions, but could be interpreted as a consistent end stage of a uniform immunological selection process, representing the final result of many cellular modifications and mutations that allowed the cancer to establish. That the described cytokine pattern must have a strong prognostic implication and hence be representative of the later stage of cancer is essential. Despite not considering the variable cause of cancer, the claimed consistent cytokine pattern does not contradict the biological diversity of cancer: although the cytokine expression is a result of a uniform late-stage immunological selection, the process and the time to reach this specific immunological imprint would be defined by the individual cancer biology. The hypothesis that the paraneoplastic immune reaction is cancer induced, uniform, and independent of the cancer type is based on evidence from a plethora of so far uncoordinated clinical studies that report a consistent and common pattern of cytokine reactions in different and unrelated cancer types. The aim of this Review was to point out the consistency and reproducibility of a multitude of clinical data that are in line with theoretically e226
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and experimentally established functional networks of immune response. This translational approach provides both a novel explanation of prognostically essential phenomena as well as new diagnostic and therapeutic perspectives. The conclusion needs to be confirmed by prospective studies in patients at various stages of cancer, but the present evidence seems sufficiently substantial for a valid working hypothesis. Conflicts of interest I declare that I have no conflicts of interest. References 1 Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 140: 883–99. 2 Knuth A, Danowski B, Oettgen HF, Old LJ. T-cell-mediated cytotoxicity against autologous malignant melanoma: analysis with interleukin 2-dependent T-cell cultures. Proc Natl Acad Sci USA 1984; 81: 3511–15. 3 van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991; 254: 1643–47. 4 Sahin U, Tureci O, Pfreundschuh M. Serological identification of human tumor antigens. Curr Opin Immunol 1997; 9: 709–16. 5 Lee PP, Yee C, Savage PA, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med 1999; 5: 677–85. 6 Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature 2008; 454: 436–44. 7 Whiteside TL. Inhibiting the inhibitors: evaluating agents targeting cancer immunosuppression. Expert Opin Biol Ther 2010; 10: 1019–35. 8 Frey AB, Monu N. Effector-phase tolerance: another mechanism of how cancer escapes antitumor immune response. J Leukoc Biol 2006; 79: 652–62. 9 Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005; 5: 263–74. 10 Dunn GP, Koebel CM, Schreiber RD. Interferons, immunity and cancer immunoediting. Nat Rev Immunol 2006; 6: 836–48. 11 Dunn GP, Old LJ, Schreiber RD. The immunobiology of cancer immunosurveillance and immunoediting. Immunity 2004; 21: 137–48. 12 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646–74. 13 Trinchieri G. Cancer and inflammation: an old intuition with rapidly evolving new concepts. Annu Rev Immunol 2012; 30: 677–706. 14 Wojtowicz-Praga S. Reversal of tumor-induced immunosuppression: a new approach to cancer therapy. J Immunother 1997; 20: 165–77. 15 Prud’homme GJ. Pathobiology of transforming growth factor beta in cancer, fibrosis and immunologic disease, and therapeutic considerations. Lab Invest 2007; 87: 1077–91. 16 Kehrl JH, Wakefield LM, Roberts AB, et al. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986; 163: 1037–50. 17 McKarns SC, Schwartz RH, Kaminski NE. Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation. J Immunol 2004; 172: 4275–84. 18 Lee G, Ellingsworth LR, Gillis S, Wall R, Kincade PW. Beta transforming growth factors are potential regulators of B lymphopoiesis. J Exp Med 1987; 166: 1290–99. 19 Lebman DA, Edmiston JS. The role of TGF-beta in growth, differentiation, and maturation of B lymphocytes. Microbes Infect 1999; 1: 1297–304. 20 Kim IY, Kim MM, Kim SJ. Transforming growth factor-beta: biology and clinical relevance. J Biochem Mol Biol 2005; 38: 1–8. 21 Teicher BA. Transforming growth factor-beta and the immune response to malignant disease. Clin Cancer Res 2007; 13: 6247–51. 22 Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 2010; 16: 2927–31.
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25
26
27
28
29 30
31
32
33
34
35 36 37
38 39
40 41
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45
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Zlotnik A. New insights on the role of CXCR4 in cancer metastasis. J Pathol 2008; 215: 211–13. Ben-Baruch A. Organ selectivity in metastasis: regulation by chemokines and their receptors. Clin Exp Metastasis 2008; 25: 345–56. Javelaud D, Mohammad KS, McKenna CR, et al. Stable overexpression of Smad7 in human melanoma cells impairs bone metastasis. Cancer Res 2007; 67: 2317–24. Katoh M. Integrative genomic analyses of CXCR4: transcriptional regulation of CXCR4 based on TGFbeta, Nodal, Activin signaling and POU5F1, FOXA2, FOXC2, FOXH1, SOX17, and GFI1 transcription factors. Int J Oncol 2010; 36: 415–20. Zhao XP, Huang YY, Huang Y, et al. Transforming growth factor-beta1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells. Acta Pharmacol Sin 2010; 31: 347–54. Mocellin S, Marincola FM, Young HA. Interleukin-10 and the immune response against cancer: a counterpoint. J Leukoc Biol 2005; 78: 1043–51. Mosser DM, Zhang X. Interleukin-10: new perspectives on an old cytokine. Immunol Rev 2008; 226: 205–18. Ryan JJ, Kashyap M, Bailey D, et al. Mast cell homeostasis: a fundamental aspect of allergic disease. Crit Rev Immunol 2007; 27: 15–32. Herbeuval JP, Lelievre E, Lambert C, Dy M, Genin C. Recruitment of STAT3 for production of IL-10 by colon carcinoma cells induced by macrophage-derived IL-6. J Immunol 2004; 172: 4630–36. Jin JO, Han X, Yu Q. Interleukin-6 induces the generation of IL-10-producing Tr1 cells and suppresses autoimmune tissue inflammation. J Autoimmun 2013; 40: 28–44. Suzuki S, Mita S, Kamohara H, Sakamoto K, Ishiko T, Ogawa M. IL-6 and IFN-gamma regulation of IL-10 production by human colon carcinoma cells. Int J Oncol 2001; 18: 581–86. Kovacs E. Interleukin-6 leads to interleukin-10 production in several human multiple myeloma cell lines. Does interleukin-10 enhance the proliferation of these cells? Leuk Res 2010; 34: 912–16. Nelson BH. IL-2, regulatory T cells, and tolerance. J Immunol 2004; 172: 3983–88. Abbas KA. Cellular and molecular immunology, fifth edition. Philadelphia, PA: Saunders Elsevier, 2005. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13: 251–76. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003; 3: 133–46. Trinchieri G, Pflanz S, Kastelein RA. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 2003; 19: 641–44. Beatty GL, Paterson Y. Regulation of tumor growth by IFN-gamma in cancer immunotherapy. Immunol Res 2001; 24: 201–10. Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol 2000; 18: 275–308. Schindler H, Lutz MB, Rollinghoff M, Bogdan C. The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J Immunol 2001; 166: 3075–82. Fukao T, Matsuda S, Koyasu S. Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells. J Immunol 2000; 164: 64–71. Vidal-Vanaclocha F, Mendoza L, Telleria N, et al. Clinical and experimental approaches to the pathophysiology of interleukin-18 in cancer progression. Cancer Metastasis Rev 2006; 25: 417–34. Mantovani A, Dejana E. Cytokines as communication signals between leukocytes and endothelial cells. Immunol Today 1989; 10: 370–75. Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harb Perspect Biol 2012; 4: pii:a006049. Esmon CT. Possible involvement of cytokines in diffuse intravascular coagulation and thrombosis. Baillieres Best Pract Res Clin Haematol 1999; 12: 343–59. Zanotti-Cavazzoni SL, Hollenberg SM. Cardiac dysfunction in severe sepsis and septic shock. Curr Opin Crit Care 2009; 15: 392–97.
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55 56
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Balkwill F. Tumour necrosis factor and cancer. Nat Rev Cancer 2009; 9: 361–71. Stathopoulos GT, Kollintza A, Moschos C, et al. Tumor necrosis factor-alpha promotes malignant pleural effusion. Cancer Res 2007; 67: 9825–34. Calandra T. Macrophage migration inhibitory factor and host innate immune responses to microbes. Scand J Infect Dis 2003; 35: 573–76. Mitchell RA, Liao H, Chesney J, et al. Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response. Proc Natl Acad Sci USA 2002; 99: 345–50. Bernhagen J, Krohn R, Lue H, et al. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med 2007; 13: 587–96. Conroy H, Mawhinney L, Donnelly SC. Inflammation and cancer: macrophage migration inhibitory factor (MIF)—the potential missing link. Q JM 2010; 103: 831–36. Calandra T, Roger T. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol 2003; 3: 791–800. Ren Y, Tsui HT, Poon RT, et al. Macrophage migration inhibitory factor: roles in regulating tumor cell migration and expression of angiogenic factors in hepatocellular carcinoma. Int J Cancer 2003; 107: 22–29. Schuett H, Luchtefeld M, Grothusen C, Grote K, Schieffer B. How much is too much? Interleukin-6 and its signalling in atherosclerosis. Thromb Haemost 2009; 102: 215–22. Romano M, Sironi M, Toniatti C, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 1997; 6: 315–25. Fujihashi K, Kono Y, Kiyono H. Effects of IL6 on B cells in mucosal immune response and inflammation. Res Immunol 1992; 143: 744–49. Sepulveda H, Cerwenka A, Morgan T, Dutton RW. CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation. J Immunol 1999; 163: 1133–42. Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol 2003; 24: 25–29. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441: 235–38. DeNardo DG, Andreu P, Coussens LM. Interactions between lymphocytes and myeloid cells regulate pro- versus anti-tumor immunity. Cancer Metastasis Rev 2010; 29: 309–16. Alexander J, Brombacher F. T helper1/t helper2 cells and resistance/susceptibility to leishmania infection: is this paradigm still relevant? Front Immunol 2012; 3: 80. Vignali DA, Collison LW, Workman CJ. How regulatory T cells work. Nat Rev Immunol 2008; 8: 523–32. Facciabene A, Motz GT, Coukos G. T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res 2012; 72: 2162–71. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol 2009; 27: 485–517.
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77 78
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80
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Laurence A, Tato CM, Davidson TS, et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 2007; 26: 371–81. Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 2000; 13: 715–25. Croxford AL, Mair F, Becher B. IL-23: one cytokine in control of autoimmunity. Eur J Immunol 2012; 42: 2263–73. O’Garra A, Hosken N, Macatonia S, Wenner CA, Murphy K. The role of macrophage- and dendritic cell-derived IL12 in Th1 phenotype development. Res Immunol 1995; 146: 466–72. Mangan PR, Harrington LE, O’Quinn DB, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006; 441: 231–34. Kortylewski M, Xin H, Kujawski M, et al. Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell 2009; 15: 114–23. Langowski JL, Zhang X, Wu L, et al. IL-23 promotes tumour incidence and growth. Nature 2006; 442: 461–65. Lonning S, Mannick J, McPherson JM. Antibody targeting of TGF-β in cancer patients. Curr Pharm Biotechnol 2011; 12: 2176–89. Jaschinski F, Rothhammer T, Jachimczak P, Seitz C, Schneider A, Schlingensiepen KH. The antisense oligonucleotide trabedersen (AP 12009) for the targeted inhibition of TGF-beta2. Curr Pharm Biotechnol 2011; 12: 2203–13. Hawinkels LJ, Ten Dijke P. Exploring anti-TGF-beta therapies in cancer and fibrosis. Growth Factors 2011; 29: 140–52. McDermott DF, Regan MM, Clark JI, et al. Randomized phase III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients with metastatic renal cell carcinoma. J Clin Oncol 2005; 23: 133–41. Madhusudan S, Foster M, Muthuramalingam SR, et al. A phase II study of etanercept (Enbrel), a tumor necrosis factor alpha inhibitor in patients with metastatic breast cancer. Clin Cancer Res 2004; 10: 6528–34. Harrison ML, Obermueller E, Maisey NR, et al. Tumor necrosis factor alpha as a new target for renal cell carcinoma: two sequential phase II trials of infliximab at standard and high dose. J Clin Oncol 2007; 25: 4542–49. Balkwill F. Tumour necrosis factor and cancer. Nat Rev Cancer 2009; 9: 361–71 Su X, Ye J, Hsueh EC, Zhang Y, Hoft DF, Peng G. Tumor microenvironments direct the recruitment and expansion of human Th17 cells. J Immunol 2010; 184: 1630–41. al-Sarireh B, Eremin O. Tumour-associated macrophages (TAMS): disordered function, immune suppression and progressive tumour growth. J R Coll Surg Edinb 2000; 45: 1–16. Mantovani A, Schioppa T, Porta C, Allavena P, Sica A. Role of tumor-associated macrophages in tumor progression and invasion. Cancer Metastasis Rev 2006; 25: 315–22. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002; 23: 549–55.
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Cytokine patterns in patients with cancer: a systematic review Bodo E Lippitz
Active, but dysfunctional, immune responses in patients with cancer have been studied in several tumour types, but owing to the heterogeneity of cancer theories of common reaction mechanisms seem to be obsolete. In this Review of published clinical studies of patients with cancer, expression and interplay of the following cytokines are examined: interleukin 2, interleukin 6, interleukin 8, interleukin 10, interleukin 12, interleukin 18, tumour necrosis factor α (TNFα), transforming growth factor β (TGFβ), interferon-γ, HLA-DR, macrophage migration inhibitory factor (MIF), and C-X-C motif chemokine receptor 4 (CXCR4). Clinical data were analysed in a non-quantitative descriptive manner and interpreted with regard to experimentally established physiological cytokine interactions. The clinical cytokine pattern that emerged suggests that simultaneous immunostimulation and immunosuppression occur in patients with cancer, with increased concentrations of the cytokines MIF, TNFα, interleukin 6, interleukin 8, interleukin 10, interleukin 18, and TGFβ. This specific cytokine pattern seems to have a prognostic effect, since high interleukin 6 or interleukin 10 serum concentrations are associated with negative prognoses in independent cancer types. Although immunostimulatory cytokines are involved in local cancer-associated inflammation, cancer cells seem to be protected from immunological eradication by cytokine-mediated local immunosuppression and a resulting defect of the interleukin 12–interferon-γ–HLA-DR axis. Cytokines produced by tumours might have a pivotal role in this defect. A working hypothesis is that the cancer-specific and histology-independent uniform cytokine cascade is one of the manifestations of the underlying paraneoplastic systemic disease, and this hypothesis links the stage of cancer with both the functional status of the immune system and the patient’s prognosis. Neutralisation of this cytokine pattern could offer novel and so far unexploited treatment approaches for cancer.
The immune system can recognise transformed cells, and both innate and adaptive immune reactions to cancer have long been described. The tumour microenvironment contains macrophages, neutrophils, mast cells, myeloid-derived suppressor cells, dendritic cells (DCs), natural killer (NK) cells, and T and B lymphocytes.1 Tumour-associated antigens and T lymphocytes that are able to recognise tumour-specific antigens have been described.2–5 Tumour-associated macrophages and tumour-infiltrating leucocytes accumulate within neoplastic tissue.6 Activated immune effector cells, such as NK cells, cytotoxic T cells, and macrophages, are present both at the tumour site and in the circulation of patients with cancer, but immune responses against cancer seem to be dysfunctional and tumours progress despite existing immunological activity.5,7 The possibility of local tumour immune escape or even tumour-induced immune suppression has been studied and discussed in detail.7–9 The term immunoediting has been used to describe the immunological selection of resistant tumour variants by elimination of immunosensitive malignant cells.10,11 The resulting avoidance of immune destruction has been defined as a hallmark of cancer.12 An inflammatory microenvironment seems to be a consistent component of malignant tumours, suggesting the presence of a cancer-related immune reaction.1,6 As summarised by Mantovani and colleagues,6 there is increasing evidence that inflammation contributes to the development of cancer and also that cancer seems to directly promote the generation of an inflammatory microenvironment. Trinchieri13 stated in a recent review of the links between inflammation and cancer that “the class of inflammation and immunity that www.thelancet.com/oncology Vol 14 May 2013
Department of Neurosurgery, Karolinska University Hospital Stockholm, Sweden (Prof B E Lippitz MD) Correspondence to: Prof Bodo E Lippitz, Department of Neurosurgery, Karolinska University Hospital Solna, 17176 Stockholm, Sweden [email protected]
is responsible for tumour initiation and early progression is likely also to be the same class that makes the immune system unable to destroy the tumours successfully”. Whether inflammatory conditions increase the local cancer risk or if genetic alterations such as oncogenes cause inflammation and neoplasia is so far unclear,6 but that cancer cells actively interfere with the patient’s immune system has been established. Several recent reviews summarised the cellular interaction between cancer and the immune system in the tumour microenvironment.7–9 However, with respect to the
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heterogeneity of cancer, a theory of a common or uniform immune reaction pattern seems so far to be obsolete. Cytokines are intricately involved in all immune reactions. Since cytokines interfere directly or indirectly with each other’s expression, the isolated effect of one cytokine can seem less relevant unless assessed in the context of its position in the hierarchy of a defined cascade. Cytokine interactions comprise sophisticated interdependent positive and negative feedback mechanisms, thus providing homoeostasis and immune control. Any attempt to outline these feedbacks in a model is necessarily simplistic because a more accurate representation of a large feedback system, ideally through a mathematical model, would create a non-linear and practically unpredictable relation. Isolated cytokine reactions have been described in individual tumour types, but so far no systematic model of the cytokinerelated immune reaction in patients with cancer has been developed. In summary, there is increasing evidence from experimental studies that malignant tumours utilise local mechanisms within their microenvironment to prevent activation of immunological effector functions, thereby protecting the tumour from a potential immune attack.8,9 I undertook the present Review of cytokine interactions in patients with cancer with an emphasis on the immunological mechanisms that are associated with clinical disease progression. The approach is intentionally descriptive. Because of this narrow focus, several aspects of cytokine activation and signalling mechanisms were not considered. The format of the present Review is an initial step to detect common patterns in the immune response in patients with cancer, which so far might not be fully represented in experimental systems. The aim of the present Review was to point out reproducible and consistent features among reported clinical data in many
Macrophage migration inhibitory factor expressed in cancer tissue
Lung
Breast
Colorectal
+
+
+
Gastric
and unrelated cancer types. By this translational approach, I attempt to use the context of recent experimental data to interpret the clinical findings. The working hypothesis here is that the emerging cytokine pattern potentially is a manifestation of the underlying systemic paraneoplastic immune response that seems to be a consistent pattern in patients with cancer. Further specification of this system could offer novel therapeutic approaches.7
Transforming growth factor β Transforming growth factor β (TGFβ) has been suggested to be the principal immune-suppressive factor secreted by tumour cells.14 In mice, complete knockout of TGFβ1 results in lethal autoimmunity from a multiorgan inflammatory syndrome.15 TGFβ suppresses interleukin 12 substantially and inhibits interleukin 2 and interleukin-2induced proliferation in T cells.16,17 In CD8+ cytotoxic T lymphocytes and NK cells, TGFβ is a strong antagonist of interferon-γ production. TGFβ has a negative effect on B-cell proliferation and differentiation.18,19 TGFβ is needed for the differentiation of both T-helper-17 (Th17) and induced regulatory T (Treg) cells. The clinical effect of TGFβ in patients has been reviewed in detail.15,20 Malignant cells often secrete large amounts of TGFβ.21 Raised TGFβ1 serum concentrations were independently detected in patients with lung cancer, breast cancer, glioblastoma multiforme, colorectal carcinoma, hepatocellular carcinoma, bladder carcinomas, renalcell carcinoma, and gastric carcinoma (table 1) and were associated with poor prognosis in patients with gastric carcinoma, adenocarcinoma of the lung, and breast cancer. Additionally, TGFβ was associated with metastases in patients with breast cancer, gastric cancer, colorectal cancer, non-small-cell lung cancer (NSCLC), malignant melanoma, and renal-cell cancer (see appendix for further references).
Malignant melanoma
Pancreatic
Malignant glioma
Hepatocellular
+
+
+
+
+
Interleukin 8 produced by tumour cells
+
+
+
+
+
+
Increased serum concentrations of interleukin 6
+
+
+
+
+
+
Decreased expression of interleukin 12
+
+
+
+
+
+
+
+
Decreased interferon-γ production in immune cells
+
Reduced expression of HLA-DR
+
Increased serum concentrations of transforming growth factor-β
+
+
+
+
C-X-C motif chemokine receptor 4 tumour expression
+
+
+
+
+
Increased serum concentrations of interleukin 10
+
+
+
+
+
+
+
+
Renal cell
Head and neck +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Pluses indicate published evidence exists.
Table 1: Published evidence for specific cytokine expression in selected cancer types
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Hence, TGFβ is one of the cytokines produced by tumour cells with immunosuppressive activity and a potent inhibitory effect on several other cytokines. With an essential role in maintenance of homoeostasis, TGFβ suppresses anti-tumour immune responses and creates a local environment of immune tolerance. Increased serum concentrations of the immunosuppressive cytokine TGFβ are a frequent finding in patients with cancer, to the extent that it could potentially represent a general cancer-associated feature.
was associated with metastases in 15 different cancer types independent of their specific histological findings (in NSCLC, gastric cancer, oesophageal cancer, oral squamous-cell cancer, renal-cell cancer metastases, laryngeal and hypopharyngeal squamous-cell cancer, breast cancer, primary squamous cell-carcinoma of the oral cavity, colorectal cancer, cervical adenocarcinoma, hepatocellular cancer, nasopharyngeal cancer, melanoma, and pancreatic cancer; table 2; see appendix for further references).
C-X-C motif chemokine receptor 4
Interleukin 10
Chemokines are molecules that direct cells to specific organs. The chemokine stromal cell-derived factor-1 (SDF-1, also known as CXCL12) binds to C-X-C motif chemokine receptor 4 (CXCR4).22 CXCR4 is expressed in malignant tumour cells whereas its ligand SDF-1 (CXCL12) is expressed in several organs including lung, liver, brain, kidney, skin, and bone marrow, with a probable function during physiological repair mechanisms. Several CXCR4positive cancers seem to metastasise in an organ-specific and CXCL12-dependent manner, and CXCR4 and the ligand CXCL12 seem to be involved in the development of cancer metastasis, with chemokines and particularly CXCR4 possibly being essential components of the metastatic process.22–24 In an experimental model and through genomic analyses on stem cells, CXCR4 upregulation was induced by TGFβ signalling.25,26 Upregulation of CXCR4 by TGFβ, which also occurs in breast-cancer cells,27 would provide a potential link for the described association between TGFβ and metastases. CXCR4 is expressed in various different tumour types (table 1) and has been considered the most widely expressed chemokine receptor in most cancers.23,24 Additionally, expression of CXCR4 in tumour specimens
TGFβ also upregulates the usually immunosuppressive cytokine interleukin 10, whereas interleukin 10 enhances the expression of TGFβ in a positive feedback circuit.28 Interleukin 10 inhibits antigen presentation, MHC class II expression, and the upregulation of costimulatory molecules CD80 and CD86. Interleukin 10 prevents the production of the Th1-associated cytokines interleukin 2 and interferon-γ from antigen-presenting cells (APCs). Physiologically, interleukin 10 significantly suppresses the major inflammatory cytokines interleukin 1, interleukin 6, interleukin 12, and TNFα.29 As a consequence, interleukin 10 was initially classified among the immunosuppressive (Th2) cytokines with a physiological role in the termination of the T-cell-mediated response. Interleukin 10 seems to act primarily on DCs and macrophages and inhibits the differentiation and the antigen-presenting properties of DCs. Interleukin 10 inhibits essential steps in immune detection such as the expression of HLA-DR and costimulatory molecules.28 Control of interleukin 10 expression involves negative feedback mechanisms including interleukin 10 itself. TNFα promotes proinflammatory reactions while upregulating interleukin 10 in macrophages and
Lung
Breast
Colorectal
Gastric
Malignant melanoma
Macrophage migration inhibitory factor expression and negative prognostic effect
+
+
Interleukin 8 is associated with tumour size, depth of infiltration, or increased stage
+
+
Interleukin-6 serum concentration and negative prognostic effect
+
+
Interleukin-18 serum concentration associated with advanced stage
+
+
Increased interleukin-18 serum concentration and negative prognosis
+
High expression of HLA-DR and positive prognosis
+
+
+
C-X-C motif chemokine receptor 4 tumour expression associated with metastases
+
+
+
+
+
Raised interleukin-10 serum concentration associated with a negative prognostic effect
+
+
+
+
Oesophageal Pancreatic
Hepatocellular carcinoma
+
+
+
+
+
+
+
+
+
+
+
+
+ + (+) serum soluble HLA-DR +
Renal cell
+ +
+
Diffuse B-cell lymphoma
+
+
+
+
+
+
(+) downregulated genes
+
+
+
+
+
+
+
+
Pluses indicate published evidence exists.
Table 2: Published evidence for specific cytokine effects in various cancer types
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monocytes as a negative feedback, thereby terminating the inflammatory response. Interleukin 10 suppresses release of TNFα and itself. The cellular sources of interleukin 10 are Th2 cells, a subset of Treg cells, and Th17 cells; however, cytotoxic CD8+ T cells can also produce interleukin 10, as can some subsets of DCs, B cells, granulocytes, eosinophils, mast cells, keratinocytes, and epithelial cells.29,30 Findings from experiments with in-vitro co-cultures showed that colon-cancer cells first stimulated macrophages to produce interleukin 6, which was then followed by interleukin-6-induced production of interleukin 10 by colon-cancer cells.31 Similarly, in vitro, interleukin 6 in association with TGFβ can induce interleukin-10 production in Th17 cells and interleukin 6 can stimulate interleukin-10 production in cancer cells.32–34 Various cancer cells produce interleukin 10; among those are oral squamous-cell carcinoma cells, multiple myeloma cells, melanoma cells, human colon-carcinoma cells, NSCLC cells, renal-cell carcinoma cells, non-Hodgkin lymphoma cells, and chronic lymphocytic leukaemia B lymphocytes. Circulating concentrations of interleukin 10 were raised in 13 different cancer types and were associated with adverse disease stage or with negative prognosis in bone sarcoma, diffuse large B-cell lymphoma, gastric cancer, colon cancer, Hodgkin lymphoma, hepatocellular cancer, melanoma, renal-cell cancer, NSCLC, and pancreatic cancer (table 2; see appendix for further references). In summary, interleukin 10 is highly immunosuppressive and seems to be frequently expressed by tumour cells, resulting in increased circulating serum concentrations of interleukin 10 in most human cancers. The frequent association of raised interleukin-10 concentrations with negative prognosis links the increased systemic interleukin-10 concentrations with the later stages of cancer. Increased serum concentrations of interleukin 10 and the resulting immunosuppression seem to be a common feature of progressive cancer in human beings.
Interleukin 2 Interleukin 10 is also an inhibitor of interleukin-2 production in Th2 cells. Interleukin 2 is a growth factor for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition in adaptive immunity. Interleukin 2 is produced primarily by activated CD4+ T cells and by naive CD8+ T cells and DCs.35 Interleukin 2 stimulates proliferation and differentiation of NK cells.36 Activated cytotoxic T cells need interleukin 2 as a growth factor at late stages of the immune reaction, whereas the initial proliferation of T cells seems to be interleukin-2 independent. Interleukin 2 is also crucial for development and peripheral expansion of Treg cells.35 In the absence of interleukin 2, T-cell receptor engagement results in anergy.36 The soluble interleukin-2 receptor acts as an antagonist of interleukin 2. There is a negative relation between e221
serum concentrations of interferon-γ and production of soluble interleukin-2 receptor. Disease progression or negative prognosis in cancer was associated with reduced interleukin-2 concentrations or an increase in soluble interleukin-2 receptor concentrations (see appendix for further references).
Interaction between interleukin 12, interferon-γ, and HLA-DR The reciprocal activation of APCs and NK cells via interleukin 12 and interferon-γ is one of the central processes in immunodetection and seems to be negatively affected in patients with cancer. APCs, macrophages, and, mainly, DCs bind antigens in the periphery and transport these antigens to the lymph nodes. In the lymph nodes, APCs present MHC-class-IIassociated peptides for recognition by CD4+ lymphocytes. HLA-DR (MHC class II) expression needs stimulation by interferon-γ, which occurs when the innate immune response is activated. The activation of interferon-γ needs stimulation by interleukin 12, which is produced by APCs when presenting antigens to T cells.36 DCs were identified as the main source of interleukin 12 produced early during the innate immune response, which was identified as the functional connection between non-specific innate and antigenspecific adaptive immunity.37 Interleukin 12 enhances the generation and activity of cytotoxic T lymphocytes and has an essential role in cell-mediated immunity. Interleukin 12 promotes maturation and activation of NK cells, the effector cells of the innate immune system. In response to release of interleukin 12 from macrophages and DCs, NK cells produce interferon-γ, which activates macrophages independently from T cells during the early phases of the innate immune response.38 Interleukin 12 and interferon-γ are linked in a positive feedback circuit through phagocytes and DCs. The structure and function of interleukin 12 and the related, but functionally opposing, interleukin 23 have been reviewed in detail.39 Both TGFβ and interleukin 10 suppress interleukin 12 significantly and therefore have an effect on the interaction between interleukin 12, interferon-γ, and HLA-DR and hence on the central circuit in immunodetection.29
Reduced interleukin-12 expression Interleukin 12 is one of the essential proinflammatory cytokines that stimulates Th1 responses; increased expression of interleukin 12 should occur during a physiological immune reaction.37 This step seems to be negatively affected in many patients with cancer: expression of interleukin 12 seems to be decreased in several cancer types, particularly in late stages with larger and more advanced disease, as described in anaplastic astrocytoma, glioblastoma, paediatric soft-tissue sarcomas, renal-cell cancer, head and neck squamouscell carcinoma, malignant melanoma, colorectal cancer, www.thelancet.com/oncology Vol 14 May 2013
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gastric cancer, and hepatocellular carcinoma (table 1; see appendix for further references). Interleukin-12 production by stimulated peripheral blood mononuclear cells decreased significantly in patients with gastric and colorectal cancer with advancing disease. Importantly, interleukin 12 can induce the production of large amounts of interferon-γ from T cells and NK cells. The functional consequence of the described reduction in production of interleukin 12 would be a negative effect on the efficacy of NK cells and secondarily on the activation state of macrophages. Hence, cancerassociated downregulation of interleukin 12 could represent a central dysfunction of APCs, which impair the functionality of effector cells of both innate and adaptive immune systems, thus creating a local environment of immune tolerance.
Interferon-γ Interleukin 12 is essential for the production of interferon-γ, which in turn has an essential role in MHC expression as a central step of immunodetection. Interferon-γ is the principal macrophage-activating cytokine. The major sources of interferon-γ are CD8+ T cells, CD4+ T cells, and NK cells.40 Activated CD8+ T cells produce interferon-γ after antigen stimulation.41 Secretion of interferon-γ by NK cells and APCs is probably important in early host defence, whereas T lymphocytes become the major source of interferon-γ during the adaptive immune response. Interferon-γ inhibits the production of the immunosuppressive factors TGFβ and prostaglandin E2, whereas negative regulators of interferon-γ production include interleukin 4, interleukin 10, and TGFβ.42 TGFβ inhibits interferon-γ production by suppression of a transcription factor (T-bet) that is essential for Th1 differentiation of CD4+ T cells and for interferon-γ production. As a theoretically expected consequence of the aforementioned dysfunction of interleukin 12, decreased interferon-γ production in lymphocytes, NK cells, or peripheral blood mononuclear cells has so far been described in malignant melanoma, gastric cancer, lung cancer, glioblastoma, nasopharyngeal carcinoma, colorectal cancer, and head and neck cancer, and decreased interferon-γ tumour expression in advanced renal-cell carcinoma (table 1). Thus, decreased interferon-γ production potentially represents a common cancerassociated immune dysfunction in human beings (see appendix for further references). Interferon-γ induces expression of the human MHC class II protein, HLA-DR, which is needed for antigen recognition by CD4+ T cells. Interferon-γ also upregulates expression of MHC class I as well as that of genes needed for antigen processing. Hence, reduced concentrations of interferon-γ in patients with cancer is expected to have a negative effect on MHC expression and thereby on tumour immunogenicity. www.thelancet.com/oncology Vol 14 May 2013
HLA-DR expression Reduced expression of MHC class I and class II impairs immunological tumour recognition. Additionally, HLA-DR antigen expression is crucial for activation of naive CD8+ T cells and is hence essential for a successful adaptive immune reaction against cancer cells. Markedly diminished HLA-DR expression on monocytes was reported in six cancer types (glioblastoma, lung cancer, pancreatic carcinoma, colorectal cancer, malignant melanoma, and head and neck cancer; table 1). In 12 different unrelated cancer types, worse prognosis or more advanced disease was associated with reduced HLA-DR expression on tumour tissue or monocytes, or vice versa advanced disease stages coincided with decreased soluble HLA-DR serum concentrations (lung cancer, pancreatic carcinoma, breast cancer, nasopharyngeal carcinoma, colorectal cancer, primary non-Hodgkin gastric lymphoma, laryngeal cancer, hepatocellular carcinoma, malignant melanoma, rectal cancer, head and neck cancer and ovarian serous adenocarcinoma; table 2; see appendix for further references).
Interleukin 18 In addition to interleukin 12, production of interferon-γ is influenced by interleukin 18, which is secreted by APCs and a wide range of cell types including activated monocytes, macrophages, T lymphocytes, and NK cells.43 Interleukin 18 is a costimulatory factor for the induction of interleukin-12-mediated interferon-γ production by T-helper cells, and most peripheral CD4+ T cells express the receptor interleukin-18R alpha. Interferon-γ also induces the expression of the antagonistic interleukin-18 binding protein (interleukin 18bp), which functions as an inhibitor of interleukin-18 activity as a potential direct negative feedback mechanism to control interleukin-18-induced production of interferon-γ.44 In the described feedback circuit, downregulated interleukin 12 and interferon-γ would theoretically result in similarly reduced interleukin-18bp expression. The absence of this negative stimulus could potentially explain an increased concentration of interleukin 18. In fact, high concentrations of interleukin 18 were significantly associated with advanced tumour stages in patients with cancer in seven tumour types (oesophageal squamous-cell carcinoma, breast cancer, hepatocellular carcinoma, lung cancer, renal-cell carcinoma, multiple myeloma, and oral cavity cancer; table 2) and were independently associated with shorter overall survival in seven cancers (see appendix for further references).
TNFα Macrophages and DCs function as APCs. Macrophages respond to inflammatory stimuli by immediate production of TNFα, interleukin 1, and other chemokines. TNFα and interleukin 1 are described as acute-response cytokines. The principal function of TNFα is to stimulate the activation and recruitment of neutrophils and e222
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monocytes to sites of inflammation. TNFα (and also interleukin 1) activates vascular endothelial cells and causes endothelial cells to express adhesion molecules for neutrophils, monocytes, and lymphocytes.45,46 TNFα (and interleukin 1) can elicit tissue-factor production on endothelium and monocytes, which initiates coagulation.47 Additionally, circulating TNFα might have systemic effects on myocyte contractility, resulting in myocardial dysfunction.48 Also, malignant cells constitutively produce small amounts of TNF, causing hyperpermeability of blood vessels.49 This effect promoted pleural effusion in a lung-cancer model.50 Increased serum concentrations of TNFα were described in eight independent cancer types (NSCLC, breast cancer, colorectal cancer, prostate cancer, chronic lymphocytic leukaemia, malignant melanoma, non-Hodgkin lymphoma, and gastric cancer; see appendix for further references). In summary, TNFα is a potent immunostimulatory cytokine with both local effects in the tumour microenvironment and potential systemic effects. In established tumours, TNFα contributes to the maintenance of a proinflammatory environment.
Macrophage migration inhibitory factor Macrophage migration inhibitory factor (MIF) is a rapidly induced immunostimulatory cytokine. MIF is expressed in monocytes, macrophages, T and B lymphocytes, eosinophils, mast cells, basophils, and neutrophils.51 MIF inhibits the migration of macrophages and sustains macrophage viability and hence the inflammatory reaction. Macrophages that do not contain MIF are prone to apoptosis.52 Additionally, findings from an experimental model showed that MIF might be a functional non-cognate ligand for the chemokine receptor CXCR4, and in a non-cancer environment MIF promoted the recruitment of both monocytes and T cells by interacting with CXCR2 and CXCR4.53 MIF potentially promotes tumorigenesis by inhibiting the classic tumour suppressor gene p53.54 p53 can promote cell-cycle arrest and apoptosis in response to DNA damage. MIF stimulates expression of the proinflammatory cytokines TNFα, interferon-γ, interleukin 1β, interleukin 6, and interleukin 8 in a positive feedback circuit.55 Overexpression of MIF has so far been found in hepatocellular cancer, lung cancer, ovarian cancer, breast cancer, oesophageal squamous-cell cancer, bladder cancer, cervical squamous-cell cancer, pancreatic cancer, glioblastomas, prostate cancer, osteosarcoma, colorectal cancer, head and neck cancer, and malignant melanoma (table 1). Significantly increased serum MIF concentrations were confirmed in five different cancer types (see appendix for further references). A negative prognostic effect of high MIF expression was reported in patients with oesophageal squamous-cell cancer, hepatocellular cancer, gastric cancer, breast cancer, glioma, nasopharyngeal cancer, head and neck cancer, and NSCLC (table 2; see appendix for further references). e223
MIF is a proinflammatory cytokine with a crucial role in innate immunity. The uniform overexpression of MIF in clinical cancer is another factor that suggests sustained inflammation occurs in the microenvironment of cancer.
Interleukin 8 Increased serum concentrations of MIF might be associated with higher interleukin-8 concentrations, another factor that is also induced by TNFα (and interleukin 1).56 Interleukin 8 is a member of the CXC chemokine family and has a role as activator and chemoattractant for neutrophils. Interleukin 8 is produced by tumour cells of ten different cancer types (NSCLC, breast cancer, colon cancer, gastric cancer, malignant melanoma, pancreatic cancer, malignant glioma, renal-cell carcinoma, oesophageal adenocarcinoma, and B-cell chronic lymphocytic leukaemia; table 1). Raised serum concentrations of interleukin 8 were associated with tumour size, depth of infiltration, or increasing stage of disease in ten different cancer types (colorectal cancer, hepatocellular cancer, bone sarcomas, gastric cancer, metastatic breast cancer, oesophageal cancer, NSCLC, soft-tissue sarcoma, prostate cancer, and diffuse large B-cell lymphoma; table 2; see appendix for further references).
Interleukin 6 A further effect of TNFα and interleukin 1 is the rapid induction of interleukin-6 gene expression particularly in monocytes and macrophages.57 Interleukin 6 is involved in recruitment of neutrophils and promotes the migration and proliferation of T lymphocytes into the affected tissue.58 Resident fibroblasts produce matrix metalloproteinases after interleukin-6 stimulation and degrade the extracellular matrix. Besides the differentiation of B lymphocytes into immunoglobulin-producing plasma cells, interleukin 6 also promotes T-cell differentiation and activation.59 In experimental conditions, interleukin 6 and TNFα costimulate naive CD8 T cells, resulting in strong cytolytic activity.60 Interleukin 8 and interleukin 6 might be sequentially activated cytokines during inflammation, with initial production of interleukin 8 and other chemoattractants stimulating early neutrophil recruitment in the inflammatory site, followed by interleukin 6 providing monocyte recruitment.61 In inflammation, interleukin 6 has a role in the transition between innate and acquired immune responses.61 Findings show that interleukin 6 provides a key signal in the development of Th17 cells while blocking the differentiation of CD4+ cells into Treg cells.62 Serum concentrations of interleukin 6 were increased in 13 different cancer types (lung cancer, breast cancer, colorectal cancer, gastric cancer, malignant melanoma, pancreatic cancer, hepatocellular carcinoma, renal-cell carcinoma, neuroblastoma, non-Hodgkin lymphoma, nasopharyngeal carcinoma, head and neck squamous-cell carcinoma, and bladder cancer; table 1) and seem to be www.thelancet.com/oncology Vol 14 May 2013
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associated with tumour stage and prognosis. Additionally, there were strong positive associations between serum interleukin-6 concentrations and tumour size, tumour stage, or disease progression in patients with gastric cancer, colorectal cancer, bone sarcoma, breast cancer, hepatocellular cancer, nasopharyngeal cancer, renal-cell cancer, lung cancer, and melanoma (table 2; see appendix for further references).
The Th1–Th2–Th17 paradigm The initial Th1–Th2 paradigm was based on the identification of CD4+ helper-cell subpopulations, termed Th1 and Th2, which produce distinct and opposing patterns of cytokines with immunostimulatory and immunosuppressive functions. After activation, CD4+ T cells of the Th1 lineage secrete interferon-γ, TNFα, interleukin 2, and interleukin 12. Th1-related cytokines are generally regarded as immunostimulatory. Th2-related cytokines inhibit the Th1 responses. Th2 CD4+ T cells express high concentrations of interleukin 4, interleukin 5, interleukin 6, interleukin 10, and interleukin 13, inhibit T-cell-mediated cytotoxicity, and support humoral immune responses via B cells.63 The exclusivity of the initial Th1–Th2 paradigm has recently become unacceptable because further CD4+ T-cell populations have been identified including inducible Treg cells and Th17 cells.64 Th17 cells are inducers of tissue inflammation and Treg cells have immunosuppressive functions, thus preventing the activation of self-reactive cells. In published work, Th17 cells have been widely discussed, but their function is still not entirely understood. Treg cells prevent autoimmunity and limit chronic inflammation by suppressive mechanisms such as those involving inhibitory cytokines, cytolysis, metabolic disruption, and modulation of DC maturation or function. Treg-cellderived cytokines are interleukin 10 and TGFβ (and interleukin 35).65,66 Induced Treg cells develop from T-cell precursors at extrathymic sites. TGFβ is needed for the differentiation of both Th17 and induced Treg cells. Th17 cells and Treg cells are functionally reciprocal.67 Both cell types are found in the cancer environment. In the steady state, TGFβ suppresses inflammatory reactions and maintains self-tolerance through induction of induced Treg cells, but when activation of the innate immune system leads to increased interleukin-6 production, Treg generation is blocked, and Th17 cells are induced, causing a proinflammatory response.67 Hence, the presence of interleukin 6 defines whether the immune response is dominated by proinflammatory Th17 cells or Tregs cells, while interleukin 2 antagonises the differentiation of CD4+ T cells into Th17 cells.62,67,68 Th17 cells are frequently found in cancer tissue and microenvironment in ovarian cancer, oesophageal cancer, breast cancer, hepatocellular carcinoma, gastric cancer, colorectal carcinoma, and oesophageal squamous-cell carcinoma. Additionally, high percentages of Th17 cells in www.thelancet.com/oncology Vol 14 May 2013
tumour-infiltrating leucocytes have been described in ovarian cancer, melanoma, breast cancer, and colon cancer, and interleukin-17 expression of cancer cells was found in NSCLC (see appendix for further references). Although the function of the Th17–Treg cellular dichotomy in cancer still remains to be elucidated, an interesting finding is that the previously described uniform presence of the functionally opposing cytokines TGFβ and interleukin 6 in patients with cancer is a prerequisite for the induction of Th17 cells, which are found in many cancer types.
Interleukin 23 Another crucial factor needed for the expansion of Th17 cells is interleukin 23, which has an essential structural similarity with interleukin 12: both cytokines share a common subunit p40. Interleukin 12 consists of the subunits p40 and p35, while interleukin 23 is comprised of p40 and a p19 subunit.39,69 These subunits are predominantly expressed by activated DCs in vivo.70 Whereas interleukin 12 promotes the differentiation of naive T cells into interferon-γ-producing Th1 cells, interleukin 23 has been linked to Th17 development.71,72 In tumours, where interleukin 23 is mainly produced by tumour-associated macrophages, interleukin 23 and interleukin 12 seem to have antagonistic functions: interleukin 12 promotes infiltration of cytotoxic T cells whereas interleukin 23 induces innate inflammatory infiltration, but reduces the ability of CD8 T cells to infiltrate tumours.73,74 So far, little is known about interleukin 23 in patients with cancer, and hence this cytokine has not been included in the present Review. However, recent studies reported high circulating interleukin-23 serum concentrations in patients with breast cancer, pancreatic cancer, colorectal cancer, and gastric cancer, which would be in line with the experimentally established role of interleukin 23 in the generation of Th17 cells, which are frequently found in several cancer types (see appendix for further references).
Cytokines and prognosis in cancer The association between a high serum concentration of interleukin 6 and a negative prognosis was a consistent finding in 16 different cancer types (diffuse large B-cell lymphoma, renal-cell cancer, pancreatic carcinoma, colorectal cancer, gastric cancer, neuroblastoma, metastatic malignant melanoma, non-Hodgkin lymphoma, nasopharyngeal carcinoma, lung cancer, metastatic breast cancer, bladder cancer, advanced gastric cancer, prostate cancer, neck squamous-cell carcinoma, and oesophageal squamous-cell carcinoma; see appendix for further references). Serum concentrations of interleukin 10 have been associated with negative prognosis in ten cancer types (diffuse large B-cell lymphoma, colorectal cancer, gastric cancer, Hodgkin disease, pancreatic carcinoma, bone sarcoma, hepatocellular carcinoma, metastatic melanoma, renal-cell carcinoma, and NSCLC; table 2). e224
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Positive prognoses were reported when functional concentrations of interleukin 12, interferon-γ, interleukin 2, and HLA-DR were maintained. Although the close relation between high interleukin-6 serum concentrations and negative prognosis represents an activation of the immunostimulatory system, interleukin-10 serum concentrations are representative of the systemic immunoparalysis in advanced cancer or the simultaneously activated Th2 system. Concentrations of both the immunostimulatory cytokine interleukin 6 and the immunoinhibitory cytokine interleukin 10 are strongly associated with prognosis in patients with cancer, independent of the tumour heterogeneity. This obvious contradiction shows the lethal situation of a futile maximum immunostimulation in a simultaneously functionally inhibited state.
Present treatment approaches Even without a comprehensive theory of a uniform immune reaction pattern, several cytokine alterations have been specifically addressed in various experimental treatments. Fresolimumab, a human anti-TGFβ monoclonal antibody, neutralises all active isoforms of TGFβ and was used in a phase 1 study for the treatment of 22 patients with advanced melanoma and renal-cell carcinoma.75 The antisense phosphorothioate oligodeoxynucleotide trabedersen is a specific inhibitor of TGFβ2 biosynthesis and is being assessed in an ongoing phase 1/2 dose-escalation study for patients with advanced pancreatic carcinoma, metastasising melanoma, or metastatic colorectal carcinoma.76 Several other potential anti-TGFβ compounds that are in clinical development in cancer have recently been reviewed.77 Similarly, treatment with highdose interleukin 2 has a proven efficacy in producing durable responses in patients with metastatic renal-cell cancer.78 Cancer immunotherapy by interleukin-12-based cytokine combinations has been summarised in detail: unfortunately the systemic administration of interleukin 12 was associated with substantial toxicity, possibly because of effects mediated by interleukin-12-induced interferon-γ production. TNFα has been suggested to have a role in growth of tumours in experimental models. TNFα antagonists had therapeutic activity in mouse models and were used in phase 1 and 2 clinical cancer trials with some evidence of clinical activity.79–81 A so far clinically unexploited option has been developed experimentally: heparinoids inhibited the interaction between CXCL12 and CXCR4 and reduced the haematogenous metastatic spread in a murine model. The specific neutralisation of cancer-associated simultaneous immunostimulation and immunosuppression could provide new therapeutic approaches (see appendix for further references).
Summary of cytokine interactions in cancer The theoretical evidence that has been established in immunological basic research was used in the present Review to progress from a purely phenomenological e225
clinical description of cytokine interactions in cancer towards an attempted relational and sequential analysis. However, provision of a detailed account of the experimental data that served as a foundation for the interpretation of the clinical findings is beyond the scope of this Review. The result of this translational approach is a necessary simplification of the actual situation, with many cytokine functions and interactions remaining not considered. This systematic review of published clinical data shows a consistent pattern of cytokine reaction in patients with cancer and provides circumstantial evidence for a common paraneoplastic phenomenon in advanced cancer, independent of tumour histology. The functional consequence of this cytokine pattern is tumour-induced immune stimulation with a simultaneous initially local and in later stages generalised environment of immune suppression that protects the cancer cells. Concentrations of specific cytokines were consistently associated with patient prognosis in different unrelated cancer types. The reproducibility of this pattern can be interpreted as a general phenomenon in cancer. Despite tumour heterogeneity, patients with advancedstage cancer seem to experience a simultaneous immunostimulation and immunodepression, with increased concentrations of cytokines MIF, TNFα, interleukin 18, interleukin 8, interleukin 6, TGFβ, and interleukin 10. Experimental data show that the presence of the otherwise antagonistic cytokines interleukin 6 and TGFβ favour the generation of Th17 cells. This initially counterintuitive notion actually seems to match the clinical situation, where both interleukin 6 and TGFβ are present in later stages of cancer and where Th17 cells are frequently found in tumour tissue to an extent that development of tumour-infiltrating Th17 cells was suggested as a general feature in patients with cancer.82 The result would be an inflammatory environment in line with the experimentally established notion of inflammation as a consistent component of malignant tumours.1,6 The clinical effect of interleukin-6 and interleukin-10 serum concentrations shows that the systemic cancerrelated immune reaction has immediate prognostic consequences. The inflammatory process seems to be maintained through stimulatory cytokines and Th17 cells, while the central clinical defect of interleukin 12–interferon-γ–HLA-DR expression prevents the eradication of the cancer cell as origins of the inflammatory reaction. A downregulation of interleukin 12 was frequently and independently reported in various cancer types, whereas a physiological immune reaction would essentially need stimulation of interleukin 12. Because several cytokines are produced by the tumour itself, one can conclude that cancer cells contribute significantly in both Th1 and Th2 stimulation via production of MIF and interleukin 8 on one side and TGFβ and interleukin 10 on the other, resulting in suppression of interleukin 12 (and interleukin 2). The dysfunction of interleukin 12 that could www.thelancet.com/oncology Vol 14 May 2013
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represent a central defect in patients with cancer is hardly considered in experimental systems. The downregulation of the interleukin 12–interferon-γ–HLA-DR axis results in dysfunction of APCs and macrophages and in impaired antigen detection and even dysfunction of effector cells of both the innate and adaptive immune system. The net effect of this cytokine pattern is a suppression of innate effector cells through inhibition of NK cells (via interleukin 12) and suppression of the specific immune detection and response through reduction of HLA-DR expression despite recruitment of (ineffective) immune cells through interleukin 6 and interleukin 8. The outlined cytokine reaction pattern would theoretically result in impaired activation of macrophages and their associated functional circuits, while tumourassociated macrophages represent a major and substantial inflammatory stromal tumour component.83–85 The assumed combined cytokine effect is a cancer-induced direct blocking of immunological detection and eradication in the immediate microenvironment of tumours. Present immunotherapies including exogenous cytokine administration, antitumour vaccines or adoptive T-cell transfers might fail, since the necessary final step of tumour detection and in particular immunological tumour destruction seems to be blocked by the tumour itself. TGFβ, interleukin 8, and interleukin 10 are produced in tumours and whether one cytokine initiates the cancer-induced process of immunosuppression is unresolved. TGFβ fulfils all requirements for an initiating substance in the hierarchy of the cytokine reaction because it also contributes to upregulation of interleukin 10. Conversely, both interleukin 10 and TGFβ stimulate each other in a positive feedback. Both cytokines are highly immunosuppressive and act through inhibition of interleukin-12 production from APCs. The net result of the described cytokine pattern is local immunostimulation and inflammation with synchronous functional immunosuppression, attracting immune cells, but protecting the tumour cells locally from detection and destruction. Independent of tumour histological changes, this cytokine cascade links the functional status of the immune system with the extent of the cancer disease and the patient’s prognosis and ultimately is an expression of the paraneoplastic systemic disease in cancer. The interpretation of the clinical results in terms of a uniform reaction pattern in cancer does not take into account the differences in malignant phenotypes and biological behaviours and might seem to contradict the hypothesis of an individual cellular immune signature in cancer. However, the recent theory of immunoediting describes the obvious selection process that originates in the functioning immune system and that ultimately results in tumour cells that are resistant to potential immune attacks. Provided this theory is correct, the malignant cellular selection process must be adapted to the uniform conditions of the immune response, thus www.thelancet.com/oncology Vol 14 May 2013
Search strategy and selection criteria To identify eligible studies, I did systematic searches of PubMed repeatedly over a period of 5 years up to 2012 for all individual cytokines in patients with cancer with the keywords “IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, TNF-alpha, TGF-beta, interferon-γ HLA-DR, MIF or CXCR4” in combination with “serum level”, “patients”, “cancer”, “prognosis” and “metastases”. C-X-C motif chemokine receptor 4 (CXCR4) and HLA-DR are not cytokines, but were reviewed because they are involved in key cytokine pathways. I included only clinical studies. No limits were set on cancer type, date of publication, or duration of follow-up. Studies were classed as eligible for inclusion if they were original research studies that reported on the selected cytokines in patients with cancer. Reviews were excluded. The search identified 307 published clinical studies in patients with cancer. Data on interleukin 1, interleukin 4, interleukin 5, and interleukin 15 were considered so far insufficient or inconclusive and were excluded from the present Review. A thorough review of all papers retrieved was done. When similar facts were reported in successive studies, the initial study was generally included. The data were summarised in a non-quantitative descriptive manner and interpreted with regard to theoretically established physiological cytokine interactions. Because of space restrictions, references for the clinical studies are listed in the appendix.
providing a common biological denominator as a prerequisite for tumour progression. Being part of a large feedback network, which normally functions in homeostasis, cytokines have many, sometimes even contradictory, functions. Thus, the claimed uniform immune reaction pattern is in conflict with the potential diversity of cytokine functions, but could be interpreted as a consistent end stage of a uniform immunological selection process, representing the final result of many cellular modifications and mutations that allowed the cancer to establish. That the described cytokine pattern must have a strong prognostic implication and hence be representative of the later stage of cancer is essential. Despite not considering the variable cause of cancer, the claimed consistent cytokine pattern does not contradict the biological diversity of cancer: although the cytokine expression is a result of a uniform late-stage immunological selection, the process and the time to reach this specific immunological imprint would be defined by the individual cancer biology. The hypothesis that the paraneoplastic immune reaction is cancer induced, uniform, and independent of the cancer type is based on evidence from a plethora of so far uncoordinated clinical studies that report a consistent and common pattern of cytokine reactions in different and unrelated cancer types. The aim of this Review was to point out the consistency and reproducibility of a multitude of clinical data that are in line with theoretically e226
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and experimentally established functional networks of immune response. This translational approach provides both a novel explanation of prognostically essential phenomena as well as new diagnostic and therapeutic perspectives. The conclusion needs to be confirmed by prospective studies in patients at various stages of cancer, but the present evidence seems sufficiently substantial for a valid working hypothesis. Conflicts of interest I declare that I have no conflicts of interest. References 1 Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 140: 883–99. 2 Knuth A, Danowski B, Oettgen HF, Old LJ. T-cell-mediated cytotoxicity against autologous malignant melanoma: analysis with interleukin 2-dependent T-cell cultures. Proc Natl Acad Sci USA 1984; 81: 3511–15. 3 van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991; 254: 1643–47. 4 Sahin U, Tureci O, Pfreundschuh M. Serological identification of human tumor antigens. Curr Opin Immunol 1997; 9: 709–16. 5 Lee PP, Yee C, Savage PA, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med 1999; 5: 677–85. 6 Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature 2008; 454: 436–44. 7 Whiteside TL. Inhibiting the inhibitors: evaluating agents targeting cancer immunosuppression. Expert Opin Biol Ther 2010; 10: 1019–35. 8 Frey AB, Monu N. Effector-phase tolerance: another mechanism of how cancer escapes antitumor immune response. J Leukoc Biol 2006; 79: 652–62. 9 Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005; 5: 263–74. 10 Dunn GP, Koebel CM, Schreiber RD. Interferons, immunity and cancer immunoediting. Nat Rev Immunol 2006; 6: 836–48. 11 Dunn GP, Old LJ, Schreiber RD. The immunobiology of cancer immunosurveillance and immunoediting. Immunity 2004; 21: 137–48. 12 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646–74. 13 Trinchieri G. Cancer and inflammation: an old intuition with rapidly evolving new concepts. Annu Rev Immunol 2012; 30: 677–706. 14 Wojtowicz-Praga S. Reversal of tumor-induced immunosuppression: a new approach to cancer therapy. J Immunother 1997; 20: 165–77. 15 Prud’homme GJ. Pathobiology of transforming growth factor beta in cancer, fibrosis and immunologic disease, and therapeutic considerations. Lab Invest 2007; 87: 1077–91. 16 Kehrl JH, Wakefield LM, Roberts AB, et al. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986; 163: 1037–50. 17 McKarns SC, Schwartz RH, Kaminski NE. Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation. J Immunol 2004; 172: 4275–84. 18 Lee G, Ellingsworth LR, Gillis S, Wall R, Kincade PW. Beta transforming growth factors are potential regulators of B lymphopoiesis. J Exp Med 1987; 166: 1290–99. 19 Lebman DA, Edmiston JS. The role of TGF-beta in growth, differentiation, and maturation of B lymphocytes. Microbes Infect 1999; 1: 1297–304. 20 Kim IY, Kim MM, Kim SJ. Transforming growth factor-beta: biology and clinical relevance. J Biochem Mol Biol 2005; 38: 1–8. 21 Teicher BA. Transforming growth factor-beta and the immune response to malignant disease. Clin Cancer Res 2007; 13: 6247–51. 22 Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 2010; 16: 2927–31.
e227
23 24
25
26
27
28
29 30
31
32
33
34
35 36 37
38 39
40 41
42
43
44
45
46 47
48
Zlotnik A. New insights on the role of CXCR4 in cancer metastasis. J Pathol 2008; 215: 211–13. Ben-Baruch A. Organ selectivity in metastasis: regulation by chemokines and their receptors. Clin Exp Metastasis 2008; 25: 345–56. Javelaud D, Mohammad KS, McKenna CR, et al. Stable overexpression of Smad7 in human melanoma cells impairs bone metastasis. Cancer Res 2007; 67: 2317–24. Katoh M. Integrative genomic analyses of CXCR4: transcriptional regulation of CXCR4 based on TGFbeta, Nodal, Activin signaling and POU5F1, FOXA2, FOXC2, FOXH1, SOX17, and GFI1 transcription factors. Int J Oncol 2010; 36: 415–20. Zhao XP, Huang YY, Huang Y, et al. Transforming growth factor-beta1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells. Acta Pharmacol Sin 2010; 31: 347–54. Mocellin S, Marincola FM, Young HA. Interleukin-10 and the immune response against cancer: a counterpoint. J Leukoc Biol 2005; 78: 1043–51. Mosser DM, Zhang X. Interleukin-10: new perspectives on an old cytokine. Immunol Rev 2008; 226: 205–18. Ryan JJ, Kashyap M, Bailey D, et al. Mast cell homeostasis: a fundamental aspect of allergic disease. Crit Rev Immunol 2007; 27: 15–32. Herbeuval JP, Lelievre E, Lambert C, Dy M, Genin C. Recruitment of STAT3 for production of IL-10 by colon carcinoma cells induced by macrophage-derived IL-6. J Immunol 2004; 172: 4630–36. Jin JO, Han X, Yu Q. Interleukin-6 induces the generation of IL-10-producing Tr1 cells and suppresses autoimmune tissue inflammation. J Autoimmun 2013; 40: 28–44. Suzuki S, Mita S, Kamohara H, Sakamoto K, Ishiko T, Ogawa M. IL-6 and IFN-gamma regulation of IL-10 production by human colon carcinoma cells. Int J Oncol 2001; 18: 581–86. Kovacs E. Interleukin-6 leads to interleukin-10 production in several human multiple myeloma cell lines. Does interleukin-10 enhance the proliferation of these cells? Leuk Res 2010; 34: 912–16. Nelson BH. IL-2, regulatory T cells, and tolerance. J Immunol 2004; 172: 3983–88. Abbas KA. Cellular and molecular immunology, fifth edition. Philadelphia, PA: Saunders Elsevier, 2005. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13: 251–76. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003; 3: 133–46. Trinchieri G, Pflanz S, Kastelein RA. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 2003; 19: 641–44. Beatty GL, Paterson Y. Regulation of tumor growth by IFN-gamma in cancer immunotherapy. Immunol Res 2001; 24: 201–10. Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol 2000; 18: 275–308. Schindler H, Lutz MB, Rollinghoff M, Bogdan C. The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J Immunol 2001; 166: 3075–82. Fukao T, Matsuda S, Koyasu S. Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells. J Immunol 2000; 164: 64–71. Vidal-Vanaclocha F, Mendoza L, Telleria N, et al. Clinical and experimental approaches to the pathophysiology of interleukin-18 in cancer progression. Cancer Metastasis Rev 2006; 25: 417–34. Mantovani A, Dejana E. Cytokines as communication signals between leukocytes and endothelial cells. Immunol Today 1989; 10: 370–75. Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harb Perspect Biol 2012; 4: pii:a006049. Esmon CT. Possible involvement of cytokines in diffuse intravascular coagulation and thrombosis. Baillieres Best Pract Res Clin Haematol 1999; 12: 343–59. Zanotti-Cavazzoni SL, Hollenberg SM. Cardiac dysfunction in severe sepsis and septic shock. Curr Opin Crit Care 2009; 15: 392–97.
www.thelancet.com/oncology Vol 14 May 2013
Review
49 50
51
52
53
54
55 56
57
58
59
60
61
62
63
64
65 66
67
Balkwill F. Tumour necrosis factor and cancer. Nat Rev Cancer 2009; 9: 361–71. Stathopoulos GT, Kollintza A, Moschos C, et al. Tumor necrosis factor-alpha promotes malignant pleural effusion. Cancer Res 2007; 67: 9825–34. Calandra T. Macrophage migration inhibitory factor and host innate immune responses to microbes. Scand J Infect Dis 2003; 35: 573–76. Mitchell RA, Liao H, Chesney J, et al. Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response. Proc Natl Acad Sci USA 2002; 99: 345–50. Bernhagen J, Krohn R, Lue H, et al. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med 2007; 13: 587–96. Conroy H, Mawhinney L, Donnelly SC. Inflammation and cancer: macrophage migration inhibitory factor (MIF)—the potential missing link. Q JM 2010; 103: 831–36. Calandra T, Roger T. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol 2003; 3: 791–800. Ren Y, Tsui HT, Poon RT, et al. Macrophage migration inhibitory factor: roles in regulating tumor cell migration and expression of angiogenic factors in hepatocellular carcinoma. Int J Cancer 2003; 107: 22–29. Schuett H, Luchtefeld M, Grothusen C, Grote K, Schieffer B. How much is too much? Interleukin-6 and its signalling in atherosclerosis. Thromb Haemost 2009; 102: 215–22. Romano M, Sironi M, Toniatti C, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 1997; 6: 315–25. Fujihashi K, Kono Y, Kiyono H. Effects of IL6 on B cells in mucosal immune response and inflammation. Res Immunol 1992; 143: 744–49. Sepulveda H, Cerwenka A, Morgan T, Dutton RW. CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation. J Immunol 1999; 163: 1133–42. Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol 2003; 24: 25–29. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441: 235–38. DeNardo DG, Andreu P, Coussens LM. Interactions between lymphocytes and myeloid cells regulate pro- versus anti-tumor immunity. Cancer Metastasis Rev 2010; 29: 309–16. Alexander J, Brombacher F. T helper1/t helper2 cells and resistance/susceptibility to leishmania infection: is this paradigm still relevant? Front Immunol 2012; 3: 80. Vignali DA, Collison LW, Workman CJ. How regulatory T cells work. Nat Rev Immunol 2008; 8: 523–32. Facciabene A, Motz GT, Coukos G. T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res 2012; 72: 2162–71. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol 2009; 27: 485–517.
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69
70 71
72
73
74 75 76
77 78
79
80
81 82
83
84
85
Laurence A, Tato CM, Davidson TS, et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 2007; 26: 371–81. Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 2000; 13: 715–25. Croxford AL, Mair F, Becher B. IL-23: one cytokine in control of autoimmunity. Eur J Immunol 2012; 42: 2263–73. O’Garra A, Hosken N, Macatonia S, Wenner CA, Murphy K. The role of macrophage- and dendritic cell-derived IL12 in Th1 phenotype development. Res Immunol 1995; 146: 466–72. Mangan PR, Harrington LE, O’Quinn DB, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006; 441: 231–34. Kortylewski M, Xin H, Kujawski M, et al. Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell 2009; 15: 114–23. Langowski JL, Zhang X, Wu L, et al. IL-23 promotes tumour incidence and growth. Nature 2006; 442: 461–65. Lonning S, Mannick J, McPherson JM. Antibody targeting of TGF-β in cancer patients. Curr Pharm Biotechnol 2011; 12: 2176–89. Jaschinski F, Rothhammer T, Jachimczak P, Seitz C, Schneider A, Schlingensiepen KH. The antisense oligonucleotide trabedersen (AP 12009) for the targeted inhibition of TGF-beta2. Curr Pharm Biotechnol 2011; 12: 2203–13. Hawinkels LJ, Ten Dijke P. Exploring anti-TGF-beta therapies in cancer and fibrosis. Growth Factors 2011; 29: 140–52. McDermott DF, Regan MM, Clark JI, et al. Randomized phase III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients with metastatic renal cell carcinoma. J Clin Oncol 2005; 23: 133–41. Madhusudan S, Foster M, Muthuramalingam SR, et al. A phase II study of etanercept (Enbrel), a tumor necrosis factor alpha inhibitor in patients with metastatic breast cancer. Clin Cancer Res 2004; 10: 6528–34. Harrison ML, Obermueller E, Maisey NR, et al. Tumor necrosis factor alpha as a new target for renal cell carcinoma: two sequential phase II trials of infliximab at standard and high dose. J Clin Oncol 2007; 25: 4542–49. Balkwill F. Tumour necrosis factor and cancer. Nat Rev Cancer 2009; 9: 361–71 Su X, Ye J, Hsueh EC, Zhang Y, Hoft DF, Peng G. Tumor microenvironments direct the recruitment and expansion of human Th17 cells. J Immunol 2010; 184: 1630–41. al-Sarireh B, Eremin O. Tumour-associated macrophages (TAMS): disordered function, immune suppression and progressive tumour growth. J R Coll Surg Edinb 2000; 45: 1–16. Mantovani A, Schioppa T, Porta C, Allavena P, Sica A. Role of tumor-associated macrophages in tumor progression and invasion. Cancer Metastasis Rev 2006; 25: 315–22. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002; 23: 549–55.
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