Malignant pleural mesothelioma (MPM) tumor cells produce copious amounts of myeloid cell-stimulating factors. The current study examined the prognostic significance of circulating monocytes and tumor-infiltrating macrophages on overall survival in patients with MPM.
The authors retrospectively reviewed 667 patients with MPM who underwent cytoreductive surgery at the Brigham and Women's Hospital in Boston, Massachusetts between 1989 and 2009. Kaplan-Meier and Cox proportional hazards models were used to determine the impact of preoperative circulating monocytes on overall survival. Immunohistochemical staining for CD68 was performed on a tissue microarray of MPM tumors from 52 patients undergoing cytoreductive surgery. The phenotype of circulating monocytes and tumor-infiltrating macrophages in 7 additional patients was determined by flow cytometry.
The median survival for all patients was 13.4 months, and 35% of patients had tumors of nonepithelial histology. For patients with nonepithelial compared with epithelial tumors, survival was significantly worse (9.3 months vs 16.6 months; P < .0001), the number of monocytes was significantly higher (580 ± 20 cells/μL vs 520 ± 10 cells/μL; P = .002), and higher monocyte counts were associated with higher tumor stage. Increasing monocyte counts were correlated with poor survival for all patients with MPM. Within MPM tumors, macrophages comprised 27% ± 9% of the tumor area and demonstrated an immunosuppressive phenotype with high expression of CD163, CD206, and interleukin-4 receptor α. The degree of macrophage infiltration was found to be negatively correlated with survival in patients with nonepithelial (P = .008) but not those with epithelial (P = .7) MPM, independent of disease stage.
Malignant pleural mesothelioma (MPM) is a highly fatal tumor arising from the mesothelial cell lining of the pleura. Although its proclivity for distant metastasis is low, MPM is highly invasive to surrounding tissues and results in failure of adjacent organs. The median survival of patients with MPM is only 4 to 19 months 1-3 and epidemiologic studies suggest that its worldwide incidence continues to rise. 4 MPM is often unresponsive to chemotherapy or radiotherapy, although select patients will benefit from multimodality therapy that incorporates cytoreductive surgery, chemotherapy, and radiation. 3
In recent years, it has become evident that chronic inflammation predisposes individuals to cancer. In the tumor microenvironment, inflammation promotes the proliferation and survival of malignant cells, supports angiogenesis and metastasis, and subverts immune reactions and responses to chemotherapy. 5 Although the mechanisms of carcinogenesis in MPM are incompletely understood, they clearly overlie a background of smoldering inflammation. Occupational exposure to asbestos is the cause of MPM in approximately 80% of individuals with this disease and there is a long latency period between the time of initial exposure and diagnosis, ranging from 20 years to 50 years. 6 In animal models, asbestos causes an influx of mononuclear phagocytes into the tumor that internalize asbestos fibers. In response to asbestos, these phagocytes as well as normal mesothelial cells release tumor necrosis factor (TNF)-α and other proinflammatory cytokines that promote malignant transformation of the mesothelium through NK-κB–dependent mechanisms. 7, 8 In humans, the inflammatory response of MPM tumors is demonstrated by the presence of massive leukocyte infiltrates, comprised mainly of macrophages and CD4 and CD8 T cells. 9, 10
Monocytes originate in the bone marrow from pluripotent stem cells. After several stages of development, they are released into the circulation and differentiate into macrophages upon becoming resident in tissues. A high number of circulating monocytes has been associated with poor overall and cancer-related survival in several human malignancies. 11-16 Normal human mesothelial cells and established human MPM cell lines produce copious amounts of hematopoietic cytokines, including interleukin (IL)-6, IL-8, macrophage inflammatory protein-1, granulocyte-colony stimulating factor, and granulocyte-macrophage-colony stimulating factor. 17, 18 These cytokines provide chemotactic and stimulatory signals to immune cells of the myeloid lineage and recruit monocytes to the tumor mass, where they differentiate into macrophages. Macrophages infiltrate several human cancers and, depending on the type of malignancy, may be positively or negatively associated with survival. 19 In mice, tumor-associated macrophages (TAM) promote tumor progression 20 and both murine and human TAM have been shown to possess immunosuppressive function. 21-23 To our knowledge, the association between monocytes and macrophages and clinical outcome is unknown in patients with MPM. In the current study, we set out to determine the prognostic significance of circulating blood monocytes and tumor-infiltrating macrophages in patients with MPM.
MATERIALS AND METHODS
In accordance with our institution's institutional review board (IRB), we retrospectively reviewed 667 patients with MPM who underwent cytoreductive surgery from 1989 through 2009. Data were obtained from the prospectively maintained International Mesothelioma Program patient data registry and included age, gender, procedure, laterality, pathology, stage of disease according to the Brigham and Women's Hospital (BWH) 3 (Table 1) and TNM staging systems, and overall survival. Preoperative monocyte counts and white blood cell counts (WBC) were determined from the complete blood count and the automated differential drawn within 90 days before surgery.
Table 1. The Brigham and Women's Hospital Staging System for MPM 3
Disease completely resected within the capsule of the parietal pleura without adenopathy: ipsilateral pleura, lung, pericardium, diaphragm, or chest wall disease limited to previous biopsy sites
All of stage I with positive resection margins and/or intrapleural adenopathy
Local extension of disease into the chest wall or mediastinum, heart, or through diaphragm or peritoneum, or with extrapleural lymph node involvement
Distant metastatic disease
Immunostaining was performed on a tissue microarray (TMA) of 58 resectable MPM tumors that was constructed as previously described. 24 Each tumor was represented in duplicate or quadruplicate cores. Six patients were excluded because of damage to their respective cores, resulting in a sample size of 52. Briefly, the TMA slides were soaked in xylene, passed through graded alcohols, and placed in distilled water. Slides were then pretreated with citrate buffer in a steam pressure cooker (Decloaking Chamber; BioCare Medical, Walnut Creek, Calif) as per the manufacturer's instructions followed by washing in distilled water. All further steps were performed at room temperature in a hydrated chamber. Slides were pretreated with peroxidase block (Dako, Carpinteria, Calif) for 5 minutes to quench endogenous peroxidase activity. A mouse antihuman CD68 antibody (clone PG-M1, Catalog #M0876; Dako) or rabbit antihuman CD3 antibody (Catalog #A0452; Dako) was applied at a dilution of 1:200 in Dako diluent for 1 hour. Slides were washed in 50-mM of Tris-Cl (pH 7.4) and detected with the mouse EnVision+ kit (Dako) as per the manufacturer's instructions. After further washing, immunoperoxidase staining was developed using a 3,3′-diaminobenzidine (DAB) chromogen (Dako) and counterstained with hematoxylin.
Slides stained for CD68 and CD3 were scanned at ×200 magnification using an Aperio ScanScope XT workstation (Aperio Technology, Inc, Vista, Calif). Images were visualized and annotated using ImageScope software (version 10.0.35.1800; Aperio Technology). In a blinded fashion, a pathologist (S.R.) identified and annotated areas of tumor as regions of interest (ROI) using standard ImageScope software functions. The ROIs were then analyzed using a standard analysis algorithm to quantitate the percentage of the annotated area that was positive for staining (color deconvolution version 9.0; Aperio Technology). The resulting data were the percentage of the ROI that stained positively for the macrophage marker CD68 or the T-cell identifier CD3. Scores from duplicate or quadruplicate cores for each tumor were averaged.
Under an IRB-approved tissue collection protocol, tumor and matched blood samples were collected on the day of surgery from 7 patients undergoing cytoreductive surgery at the BWH. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation over Ficoll-Paque Plus (GE Healthcare; Pittsburgh, Pa). Tumor tissue was minced into small fragments in cold Hank's balanced salt solution and digested at 37°C for 3 hours in RPMI containing 1 mg/mL of collagenase D (Roche Diagnostics, Indianapolis, Ind) and 100 μg/mL of DNAse (Qiagen, Germantown, Md). After passage through 70-μm and 450-μm filters, the suspension was layered over Ficoll, spun at 1000 g for 20 minutes, and then washed. Immunophenotypic analysis of cells was performed using multicolor flow cytometry on an LSRII flow cytometer (BD Biosciences, San Jose, Calif). Antibodies used for cell analysis included V450-CD45 (clone H130), PE-CD14 (M5E2), APC-CD14 (M5E2), PE-CD163 (GHI/61), APC-CD206 (19.2), APC-cy7-HLA-DR (L243), FITC-CD80 (L307.4), and APC-CD86 (IT2.2), all from BD Biosciences, and APC-IL-4Rα (25463) from R&D Systems (Minneapolis, Minn). Unstained cells and immunoglobulin isotype controls were used to set gates. Flow cytometry data were analyzed with FlowJo software (Tree Star Inc, Ashland, Ore).
Preoperative monocyte count and percentage intratumoral CD68 and CD3 staining were evaluated in histological subtype-specific analyses for epithelial and nonepithelial tumors. Statistical analysis was performed using JMP statistical software (version 8.0; SAS Institute Inc, Cary, NC). Quantitative variables were expressed as the mean ± the standard deviation. Differences between groups of continuous variables were tested using the Wilcoxon rank sum test and the chi-square test was used to compare categorical variables. Actuarial survival was calculated according to the Kaplan-Meier method, and differences in survival were tested for significance using the log-rank test. Cox proportional hazards modeling was used to examine the relation between monocyte count as a continuous variable and overall survival and to estimate 95% confidence intervals. In addition, a Cox proportional hazards model was used to compare the long-term survival based on the number of macrophages using a baseline survival function estimate derived from the Kalbfleisch-Prentice estimator. 25, 26 This derived survival function was subsequently used to create long-term survival plots. P values < .05 were considered statistically significant.
High Preoperative Circulating Monocyte Counts Are Associated With Poor Overall Survival in MPM
The clinical and pathologic characteristics of 667 patients who underwent cytoreductive surgery with either extrapleural pneumonectomy (EPP) or pleurectomy and decortication are shown in Table 2. Approximately 65% (n = 432) of the patients had epithelial histology, and this histology was associated with better overall survival compared with nonepithelial histology (16.6 months vs 9.3 months;, P < .0001) (Table 2) (Fig. 1A). Patients in the nonepithelial group were of slightly older age and were comprised of a slightly greater number of males. There was no difference in the BWH or TNM stage of disease in patients in the epithelial and nonepithelial groups. The number of preoperative circulating monocytes and the total WBC was higher in the nonepithelial histology group. Higher preoperative monocyte counts were found to be negatively correlated with overall survival in all patients with mesothelioma regardless of histology when analyzed using the Kaplan-Meier survival estimates (Figs. 1B-1D) and Cox proportional hazards methods (Table 3).
Patients With Advanced Stage Nonepithelial MPM Have Higher Preoperative Monocyte Counts
Complete pathologic staging data are determinable only for patients who undergo EPP. Staging information was available for 98% of the 481 patients who underwent EPP in the current study. Of this cohort, a higher BWH and TNM stage was found to be correlated with higher monocyte counts for patients in the nonepithelial histology group but not patients in the epithelial histology group (Fig. 2) (data not shown). Despite this correlation, a high monocyte count remained a negative predictor of survival when adjusted for BWH or TNM stage (Table 4) (data not shown).
Table 4. Association Between Monocytes and Survival Unadjusted and Adjusted for BWH Stage
Epithelial and Nonepithelial MPM Are Heavily Infiltrated by Macrophages
To evaluate the degree of macrophage infiltration within mesothelioma tumor tissues, we stained our TMA for CD68, a reliable macrophage-identifying antigen. The characteristics of this group are shown in Table 5. We found, in both epithelial and nonepithelial MPM, that macrophages comprised a major percentage of the cellular infiltrate on tissue sections, accounting for 27% ± 9% of the tumor area. Similarly, using flow cytometry of tumor mononuclear cell suspensions, 28% ± 8% of all immune cells were macrophages. There was no difference with regard to the degree of macrophage infiltration between epithelial and nonepithelial histology (Table 5) (Fig. 3), and the density of infiltrating macrophages was not found to correlate with BWH stage (Fig. 4) or TNM stage (data not shown).
Table 5. Patient Characteristics of the Macrophage Cohorta
(n = 52)
(n = 34)
(n = 18)
Abbreviations: BWH, Brigham and Women's Hospital; +, positive.
Data are represented as the mean ± the standard deviation (%).
55.8 ± 9.1
55.1 ± 9.6
57.1 ± 9.1
Epithelial cell type
Median survival, mo
Infiltrating CD68+ cells, %
26.7 ± 9.7
25.2 ± 9.3
29.7 ± 10.2
Tumor-Infiltrating Macrophages in MPM Exhibit an Immunoregulatory (M2) Phenotype
To determine the phenotype of tumor-infiltrating macrophages of MPM, we performed flow cytometry on mononuclear cell suspensions freshly isolated from tumor and matched blood samples from 7 patients with MPM (Fig. 5). Tumor-infiltrating macrophages displayed a high level of scavenger receptors CD163 and CD206, and also expressed high levels of IL-4 receptor α (IL-4Rα). Although MPM tumor-infiltrating macrophages expressed high levels of the major histocompatibility complex class II molecule human leukocyte antigen complex DR (HLA-DR), only low or modest levels of the costimulatory molecules CD80 and CD86 were present. There was no difference in the mean fluorescence index in any of these markers on tumor-infiltrating macrophages between patients with epithelial (n = 3) and nonepithelial (n = 4) tumors (data not shown).
The Density of Tumor-Infiltrating Macrophages Is Negatively Correlated With Survival in Nonepithelial MPM
Given the association between high monocyte count and poor survival, the high density of tumor-infiltrating macrophages within the tumor, and the immunoregulatory phenotype of MPM tumor-infiltrating macrophages, we examined the impact of the macrophage infiltrate on overall survival. For patients with nonepithelial histology, those with a higher density of tumor-infiltrating macrophages demonstrated significantly worse survival than those with a lower density of tumor-infiltrating macrophages, independent of BWH stage (Fig. 6) (Table 6) or TNM stage (data not shown). For patients with epithelial histology, there was no correlation noted between the macrophage infiltrate and survival. It is interesting to note that a similar finding was noted for CD3-positive (CD3+) tumor-infiltrating T cells; a higher density of CD3+T cells was found to be correlated with worse overall survival in patients with nonepithelial MPM, but not those with epithelial MPM (Table 7).
Table 6. Association Between Tumor-Infiltrating Macrophages and Survival Unadjusted and Adjusted for BWH Stage
In the tumor microenvironment, interaction among tumor cells, immune cells, stromal cells, and the extracellular matrix is vital to tumor progression. 5 The immunologic cellular infiltrates in human tumors, particularly cytotoxic and memory T cells, independently predict cancer-related survival. In fact, these indices are more sensitive than current histological methods for the estimation of survival in patients with colorectal cancer. 27, 28 It has been known for some time that MPM tumors contain an abundance of intratumoral leukocytes, 29 but it is only recently that the composition and phenotype of these cells is being unveiled. Anraku et al demonstrated improved overall survival in patients with MPM tumors that contained a high number of tumor-infiltrating CD8 T cells. 9 In addition, using immunohistochemical stains on 4 patients with MPM, Hegmans et al demonstrated that the MPM leukocyte infiltrate is rich in macrophages. 10 Furthermore, a body of evidence has recently emerged that supports a symbiotic relation between tumor cells and TAM. These data suggest that tumors attract TAM and, reciprocally, TAM sustain the survival of tumors via a variety of mechanisms including promotion of angiogenesis and metastases. In many human cancers including those of the lung, breast, cervix, bladder, ovary, and pancreas, the presence of extensive TAM infiltrates has been shown to correlate with poor prognosis. 19, 30-33 In other tumors, including those of the brain and prostate, there is conflicting evidence regarding the role of macrophages in survival outcomes. 19 To our knowledge, the relation between tumor-infiltrating macrophages and clinical outcomes in patients with MPM is unknown.
Immune dysregulation occurs in patients with cancer. Tumor-derived factors affect immune reactions not only locally within the tumor but also peripherally in the bone marrow. This results in abnormal myelopoiesis and the accumulation of potentially immunosuppressive circulating and tumor-infiltrating myelomonocytic cells. Leukocytosis, for example, has been shown to be a negative predictor of survival for many cancers, including mesothelioma, 34, 35 and may result from unregulated production of tumor-derived hematopoietic cytokines. High levels of macrophage inflammatory protein and monocyte chemotactic protein-1 are found in the pleural effusions of patients with MPM. 10 In addition, strikingly high amounts of the immunosuppressive cytokine transforming growth factor (TGF)-β and the proangiogenic cytokine vascular endothelial growth factor (VEGF) are found in these effusions, 36 both of which induce chemotaxis of monocytes through monocyte expression of the high-affinity TGF-β receptor 37 and the VEGF receptor. 38
Monocytes are central cells of the innate immune system that constitute approximately 10% of the human circulating peripheral leukocyte pool. Their scavenging and bactericidal properties consign them to a pivotal role in innate immune surveillance. Their involvement in tumor progression is incompletely understood but high preoperative blood monocyte counts have been shown to be correlated with worse overall survival in several human tumors including gastric, colorectal, and renal carcinoma; melanoma; and head and neck carcinoma. 12-16 Monocytosis has been shown to predict tumor recurrence in patients with hepatocellular carcinoma 11 and is associated with tumors of greater size and higher grade in soft tissue sarcoma. 39 In the current study, we demonstrated that preoperative monocytosis is associated with worse overall survival in patients with both epithelial and nonepithelial MPM. Although monocyte counts may be influenced by chemotherapy, we did not find a relation between monocyte counts and preoperative chemotherapy in the 9.3% of patients receiving this treatment. Similarly, the preoperative monocyte count retained its predictive value in survival when adjusted for preoperative chemotherapy (data not shown).
One of the most important functions of monocytes is to serve as a systemic reservoir of myeloid precursors for the renewal of tissue macrophages. 40 Macrophages are specialized phagocytic cells that have highly heterogeneous functions depending on their tissue localization and polarization state. Schematically dichotomizing macrophage function, macrophages can be considered to be polarized into M1 (“classically activated”) or M2 (“alternatively activated”) states. In general, M1 macrophages are regarded as soldiers, defending the host from microbial infections and malignancy, producing high amounts of inflammatory cytokines, and activating productive immune responses. M2 macrophages are involved in tissue remodeling, the production of immunoregulatory cytokines such as IL-10, and blunting of immune reactions. The majority of TAM exhibit characteristics of M2 polarization such as expression of M2-associated genes including CD163, the production of anti-inflammatory cytokines, and immunosuppressive function. 41 The tumor microenvironment likely influences the polarization states of macrophages and, in vitro, human MPM cell lines induce human monocyte cell lines to develop functional properties of M2 macrophages. 42
We have found that macrophages within MPM tumors possess a cell surface phenotype in line with an M2, or immunoregulatory, cell type with high expression of the scavenger receptors CD163 and CD206, both of which have been associated with an alternatively activated phenotype of macrophages in humans, 31, 43 and high expression of IL-4Rα. In tumor-bearing mice, IL-4Rα helps to define a population of myeloid-derived suppressor cells (MDSC) that are present in the blood and spleens of tumor-bearing mice and possess immunosuppressive functions. 44 In human tumors, the role of IL-4Rα is not well defined; however, in patients with colon cancer or melanoma, this molecule was recently found to be up-regulated on fractions of circulating mononuclear and polymorphonuclear cells that were suppressors of T-cell function. 45 To our knowledge, this is the first report of high expression of IL-4Rα in human tumor-infiltrating macrophages.
The mechanisms by which circulating and tumor-infiltrating myeloid cells in MPM may contribute to tumor progression were not examined in the current study. In patients with stage IV melanoma, monocyte-derived IL-10 was found to be an independent predictor of survival, 46 and in patients with metastatic renal cell cancer, monocytosis was associated with decreased PBMC cytotoxic activity. 14 In mice, MDSC suppress the functional antitumor activities of CD4 and CD8 T cells via the production of nitric oxide and arginase, and through the induction of regulatory T cells. 47 In patients with renal cell cancer, a circulating pool of arginase-expressing CD11b+CD15+CD14- MDSC-like cells was described that blunted T cell proliferation and interferon-γ production. 48 In the tumor microenvironment, TAM release a wide repertoire of growth factors, cytokines, chemokines, and enzymes that regulate tumor cell proliferation, angiogenesis, invasion, and metastasis. 49 Although the majority of these mechanisms have been elucidated in murine systems, freshly isolated human TAM have been shown to suppress tumor antigen-specific T cell function 22 and produce high levels of IL-10. 23
MPM is a devastating cancer with limited effective treatment options. The nonepithelial histologic variant of MPM is particularly aggressive and patients with this pathology have an even worse prognosis compared with those with the epithelial variant, 3 functionally separating epithelial and nonepithelial MPM into 2 distinct tumor types with different biologic behavior. The results of the current study suggest that the immune response to MPM may differ between patients with epithelial and nonepithelial tumors. Patients with nonepithelial MPM were found to have a higher number of circulating monocytes and although there was no difference with regard to the number of tumor-infiltrating macrophages in these 2 groups, there was a striking difference in their prognostic value, with a high density of tumor-infiltrating macrophages signifying markedly decreased overall survival in the nonepithelial histology group. Although the reason for this is not clear, we surmise that the cytokine milieu and immune cell networks within the tumor microenvironment differ between these 2 pathologic variants of MPM in a way that favors the protumor function of macrophages infiltrating nonepithelial MPM. It is interesting to note that we have also demonstrated a correlation with decreased overall survival and a high CD3 T cell infiltrate in patients with nonepithelial, but not those with epithelial, MPM. The reason for this is uncertain. Because it has been shown that a high CD8 T cell infiltrate is associated with a survival advantage in patients with MPM, 9 we surmise that the association between increased tumor-infiltrating CD3 T cells and poor survival may be related to a T cell subset composition favoring immunoregulatory T cell infiltration.
Monocytes and TAM in patients with MPM most likely represent a continuum of an altered myeloid cell differentiation program induced by tumor-derived factors and potentially support tumor progression and subversion of antitumor immune responses. Targeting this myeloid immune response is a potential therapeutic strategy for this lethal disease. In murine tumor models, for example, pharmacologic skewing of macrophage polarization with cytosine phosphate guanine (CpG) oligodeoxynucleotides and an IL-10 receptor antagonist can switch the M2 macrophage phenotype to an M1 phenotype and sustain antitumor immunity. 50 Furthermore, inhibiting the altered NF-κβ signaling in murine TAM generates cytotoxic antitumor macrophages. 51 Taken together, given the sheer numbers of tumor-infiltrating macrophages already in strategic position within MPM tumors and the altered myeloid immune response noted in these patients, manipulation of myeloid cells in patients with MPM is a rational approach to therapy.
We thank Dr. Christina Wei for her assistance with tissue processing and flow cytometry.
This study was supported by the International Mesothelioma Program of the Brigham and Women's Hospital.