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Original Research |

Prognostic Impact of Cancer-Associated Stromal Cells in Patients With Stage I Lung AdenocarcinomaStromal Cells Are Important Recurrence Predictors FREE TO VIEW

Masami Ito, MD; Genichiro Ishii, MD; Kanji Nagai, MD; Ryo Maeda, MD; Yasutaka Nakano, MD; Atsushi Ochiai, MD; International Association for the Study of Lung Cancer International Staging Committee
Author and Funding Information

From the Department of Pathology, Research Center for Innovative Oncology (Drs Ito, Ishii, Maeda, and Ochiai), and Department of Thoracic Oncology (Drs Ito, Nagai, and Maeda), National Cancer Center Hospital East, Kashiwa, Chiba; and Division of Respiratory Medicine (Drs Ito and Nakano), Department of Medicine, Shiga University of Medical, Science, Otsu, Shiga, Japan.

Correspondence to: Genichiro Ishii, MD, PhD, Pathology Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, 277-8577, Japan; e-mail address: gishii@east.ncc.go.jp

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

Funding/Support: This work was supported by the Grant-in-Aid for Cancer Research [19-10] from the Ministry of Health, Labour, and Welfare, the Foundation for the Promotion of Cancer Research, 3rd-Term Comprehensive 10-Year Strategy for Cancer Control, Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, and Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (JSPS KAKENHI) [20590417, 215981].


Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.

Funding/Support: This work was supported by the Grant-in-Aid for Cancer Research [19-10] from the Ministry of Health, Labour, and Welfare, the Foundation for the Promotion of Cancer Research, 3rd-Term Comprehensive 10-Year Strategy for Cancer Control, Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, and Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (JSPS KAKENHI) [20590417, 215981].


Chest. 2012;142(1):151-158. doi:10.1378/chest.11-2458
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Background:  The tumor microenvironment, of which cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) are the major cellular components, plays an important role in tumor progression. This study evaluated the significance of podoplanin-positive CAFs and CD204-positive TAMs, which may reflect tumor-promoting CAFs and TAMs, as risk factors for recurrence in patients with stage I lung adenocarcinoma.

Methods:  The expression of podoplanin in CAFs and CD204 in TAMs was analyzed by immunohistochemistry in 304 patients with stage I lung adenocarcinoma who underwent surgical resection between September 1992 and July 2004. The recurrence-free proportion (RFP) was estimated using the Kaplan-Meier method.

Results:  The presence of podoplanin-positive CAFs and the higher number of CD204-positive TAMs were associated with a lower 5-year RFP (P < .001 and P = .001, respectively). Podoplanin-positive CAFs were an independently statistically significant risk factor for recurrence with the highest hazard ratio (3.474, P = .029, by multivariate Cox proportional hazards model). According to subgroup analyses combining podoplanin-positive CAFs and other independent risk factors (visceral pleural invasion and intratumoral vascular invasion), the 5-year RFPs were 95.6%, 92.3%, 80.5%, and 30.3% (P = .294, P = .067, and P < .001) for patients with zero, one, two, or three risk factors, respectively.

Conclusions:  Podoplanin-positive CAFs were the most powerful independent risk factor for recurrence in patients with stage I lung adenocarcinoma. Podoplanin-positive CAFs may be useful for identifying patients with a high risk of recurrence who might benefit from adjuvant chemotherapy.

Figures in this Article

Lung cancer is the leading cause of cancer-related death worldwide.1 The majority of lung cancers are non-small cell lung cancers (NSCLCs), and the most frequent histologic subtype of NSCLC is adenocarcinoma.2 Stage I tumors are now being detected more often due to recent advances in diagnostic techniques.3 However, despite surgical resection, a considerable number of patients develop recurrences. Indeed, although surgical resection is considered the most effective therapy for patients in stage I,4 approximately 40% of patients with stage I disease die within 5 years. Furthermore, while adjuvant chemotherapy after the resection of stage II-IIIA NSCLC is the standard of care, the survival benefit of adjuvant chemotherapy for patients in stage I remains controversial.58 Therefore, it is important to identify risk factors for recurrence in patients in stage I in order to categorize them according to risk, and to assess whether they would benefit from adjuvant chemotherapy.

Cancer tissue is composed not only of cancer cells, but also of stromal cells such as fibroblasts, endothelial cells, and inflammatory cells, which together create the tumor microenvironment. Studies have demonstrated that the biologic behavior of cancers is largely influenced by the tumor microenvironment.913 Although the identification of risk factors for recurrence has focused on cancer cells, findings suggest that tumor progression is not solely determined by the cancer cells themselves, but also by the tumor microenvironment.10,14 The clinical success of an antiangiogenic agent bevacizumab (Avastin), a humanized monoclonal antivascular endothelial growth factor antibody, has provided a new rationale for targeting the tumor microenvironment.15,16

Among tumor microenvironments, fibroblasts and macrophages are the major cellular components, and these fibroblasts and macrophages are termed cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), respectively. Extensive clinical evidence and experimental mouse models have shown that certain types of CAFs and TAMs may have a tumor-promoting phenotype. A previous report demonstrated that CAFs expressing podoplanin, a mucin-like transmembrane glycoprotein that is known as a lymphatic endothelial marker, were capable of enhancing the tumor formation rate, compared with podoplanin-negative CAFs.17 These results suggest that podoplanin may be a marker of CAFs with a tumor-promoting phenotype. In addition, CD204-deficient TAMs inhibit tumor cell proliferation, and tumor growth is delayed in CD204-deficient mice compared with CD204-positive mice, suggesting that CD204 may be a marker of tumor-promoting TAMs.18

Clarification of the role of stromal cells as a prognostic factor may lead to a novel risk classification of patients with lung adenocarcinoma, and more effective treatment strategies. The purpose of this study was to evaluate the significance of podoplanin-positive CAFs and CD204-positive TAMs as risk factors for recurrence in patients with stage I lung adenocarcinoma.

Subjects

A total of 329 consecutive patients with stage I lung adenocarcinoma who underwent surgical resection and whose tumors were formalin-fixed at the National Cancer Center Hospital East (Chiba, Japan) between September 1992 and July 2004 were identified in our division database. Among these patients, 25 were excluded because their surgical specimens were of poor quality. The remaining 304 patients were included in this retrospective study.

Pathologic Evaluation

Ten-percent formalin-fixed and paraffin-embedded surgical specimens were collected from all patients. The samples were cut at 5-mm intervals, and 4-μm-thick sections were stained using the hematoxylin and eosin method. Histologic type was determined according to the World Health Organization classification.19 Disease stages were based on the seventh edition TNM classification.20 Intratumoral vascular invasion (IVI) and visceral pleural invasion (VPI) were analyzed by hematoxylin and eosin and Verhoeff-van-Gieson methods. Lymphatic permeation was evaluated using D2-40.

Immunohistochemistry

To evaluate both podoplanin expression in CAFs and CD204 expression in TAMs in identical samples, immunohistochemical analysis of podoplanin and CD204 was performed on 304 sets of lung adenocarcinoma samples. Four-micrometer-thick sections were deparaffinized with xylene, rehydrated, and antigen-retrieved in a microwave oven for 20 min. Slides were immersed in methanol containing 0.3% hydrogen peroxide for 15 min to inhibit endogenous peroxidase activity. Nonspecific binding was blocked by preincubation with 2% normal swine serum in phosphate-buffered saline for 30 min at room temperature. For podoplanin immunohistochemical staining, individual slides were incubated overnight at 4°C with mouse antipodoplanin antibody (D2-40; Signet Laboratories), at a final dilution of 1:50 in blocking buffer. Tissue sections were also stained with a mouse antihuman CD204 antibody (Scavenger Receptor Class A-E5; Transgenic), at a final dilution of 1:400 in blocking buffer. Slides were subsequently incubated with EnVision (Dako) for 1 h at room temperature. After extensive washing with phosphate-buffered saline, the color reaction was developed for 3 min in 2% 3,3′-diaminobenzidine in 50mM Tris-buffer (pH 7.6) containing 0.3% hydrogen peroxidase. Finally, the sections were counterstained with Meyer hematoxylin, dehydrated, and mounted.

All the immunostained slides and pathologic slides were reviewed by two independent pathologists (M. I. and G. I.), who were unaware of the patients’ clinical data. Spindle cells within tumor stroma were morphologically identified as fibroblasts. A sample was judged to be podoplanin positive if at least 10% of the fibroblasts showed an unequivocal reaction for podoplanin that was equal to that of lymphatic endothelial cells. The four most CD204-positive TAMs-infiltrated areas within a section were selected, and the number of CD204-positive TAMs was counted under a light microscope at a × 400 magnification (0.0625 mm2/field). The average count was recorded as the number of CD204-positive TAMs for each case.

Patient Follow-up

We examined the patients at 3-month intervals for the first 2 years, and typically at 6-month intervals thereafter on an outpatient basis. The follow-up evaluation included a physical examination, chest radiography, and blood examination. Whenever any symptoms or signs of recurrence were detected, further evaluations were performed, including CT scans of the chest and abdomen, brain MRI, and bone scintigraphy. We diagnosed recurrence based on the results of appropriate physical examination and diagnostic imaging findings, and confirmed the diagnosis histologically when clinically feasible. The date of recurrence was defined as the date of histologic proof or, in cases diagnosed based upon clinicoradiologic findings, the date of identification by a physician.

Clinicopathologic Information

All clinical and pathologic patient information was maintained in a regularly updated clinical database. We reviewed the information of each patient, which included age (dichotomized according to an age of 65 years), sex, diameter of the tumor (≤ 3 cm or > 3 cm), VPI (presence or absence), lymphatic permeation (presence or absence), and IVI (presence or absence).

Statistical Analysis

Differences in categorical outcomes were evaluated using the χ2 test. The length of the recurrence-free period was calculated in months from the date of resection to the date of the first recurrence or last follow-up. To calculate the recurrence-free proportion (RFP), patients who died without recurrence or who were known to have no recurrence at the date of last contact were censored. The RFP was calculated using the Kaplan-Meier method, and the differences between groups were analyzed using a log-rank test. Cox proportional hazards multivariate models were used to identify independent predictors. All the P values reported were 2-sided, and the significance level was set at < 0.05. Analyses were performed using the statistical software SPSS 11.0 (Dr. SPSS II for Windows, standard version 11.0; SPSS Inc). This study was conducted as part of a National Cancer Center institutional review board-approved protocol, IRB approval number 2010-095.

Immunohistochemical Staining of Podoplanin-Positive CAFs and CD204-Positive TAMs

A representative case of immunohistochemical staining results for podoplanin and CD204 is shown in Figure 1. The median number of CD204-positive TAMs was 8.0 (per 0.0625 mm2), with a range of 0-88.5. Patients were classified into two groups based on the median number of CD204-positive TAMs in the entire group: a high CD204-positive TAMs group, or a low CD204-positive TAMs group.

Figure Jump LinkFigure 1. Immunohistochemical staining of podoplanin and CD204 in lung adenocarcinomas (original magnification, × 40). The box in the bottom right is a magnified view of each panel (original magnification, × 400). A, A case with podoplanin-positive cancer-associated fibroblasts (CAFs). B, A case without podoplanin-positive CAFs. C, High CD204-positive tumor-associated macrophage (TAM) group. D, Low CD204-positive TAM group.Grahic Jump Location
Associations Between Clinicopathologic Factors and Podoplanin-Positive CAFs or CD204-Positive TAMs

Associations between clinicopathologic variables and the presence of podoplanin-positive CAFs or the number of CD204-positive TAMs are shown in Table 1. Cases with podoplanin-positive CAFs were identified in 105 (35%) samples of adenocarcinoma. Both the presence of podoplanin-positive CAFs and the number of CD204-positive TAMs were associated with male sex, larger tumor size, presence of VPI, presence of lymphatic permeation, and presence of IVI.

Table Graphic Jump Location
Table 1 —Associations Between Clinicopathologic Characteristics and Podoplanin-Positive CAFs or CD204-Positive TAMs

CAF = cancer-associated fibroblast; TAM = tumor-associated macrophage.

a 

χ test.

b 

Indicates significance.

c 

Compared with bronchioloalveolar.

Evaluation of Podoplanin-Positive CAFs and CD204-Positive TAMs as Prognostic Factors in Patients With Stage I Adenocarcinoma

The median follow-up period was 87 months (range 5−181 months). Figures 2A and 2B show the RFP curves of patients with stage I lung adenocarcinoma according to the presence of podoplanin-positive CAFs and the number of CD204-positive TAMs. The 5-year RFP for patients with podoplanin-positive CAFs was significantly lower than that for patients without podoplanin-positive CAFs (71.5% and 94.4%, respectively; P < .001) (Fig 2A). The 5-year RFP for the high CD204-positive TAMs group was also significantly lower than that for the low CD204-positive TAMs group (79.8% and 93.4%, respectively; P = .001) (Fig 2B).

Figure Jump LinkFigure 2. A, Recurrence-free proportion curves of patients with and without podoplanin-positive cancer-associated fibroblasts. B, Recurrence-free proportion curves according to the number of CD204-positive tumor-associated macrophages.Grahic Jump Location
Univariate and Multivariate Analyses of Factors Associated With Prognosis

A univariate analysis identified six significant risk factors for recurrence: tumor size, podoplanin-positive CAFs, CD204-positive TAMs, VPI, lymphatic permeation, and IVI (Table 2). On multivariate analysis with the Cox regression model, the presence of podoplanin-positive CAFs, VPI, and IVI were shown to be statistically significant independent predictors for recurrence (Table 3). Among these factors, podoplanin-positive CAFs were the most powerful risk factor for recurrence with the highest hazard ratio (HR, 3.474).

Table Graphic Jump Location
Table 2 —Univariate Analysis of Risk Factors for Recurrences

See Table 1 for expansion of abbreviations.

a 

Indicates significance.

Table Graphic Jump Location
Table 3 —Multivariate Analysis of Risk Factors for Recurrences

HR = hazard ratio. See Table 1 for expansion of other abbreviations.

a 

Indicates significance.

Subgroup Analysis Combining Podoplanin-Positive CAFs and Other Independent Risk Factors

Subgroup analysis with a combination of podoplanin-positive CAFs and other independent risk factors (VPI and IVI) revealed 5-year RFPs of 95.6%, 92.3%, 80.5%, and 30.3% for patients with zero, one, two, or three risk factors, respectively (Fig 3). The difference in 5-year RFP was statistically significant between the two- and three-risk-factor groups (P < .001), but not between the zero- and one-risk-factor groups (P = .291), or between the one- and two-risk-factor groups (P = .067).

Figure Jump LinkFigure 3. Recurrence-free proportion curves according to the number of independent risk factors.Grahic Jump Location

The present study evaluated the prognostic impact of stromal cells within the tumor microenvironment in a large cohort of patients with stage I lung adenocarcinoma, using a multivariate model that included factors previously reported to influence prognosis.

There is evidence to suggest that focusing on the tumor microenvironment may aid in the management of malignant tumors. Indeed, tumor-promoting CAFs and TAMs may contribute to the proliferation, invasion, and metastasis of cancer cells. In particular, several studies have demonstrated that cocultivation or coinjection of cancer cells with tumor-promoting CAFs and TAMs leads to the enhanced invasiveness or implantation of malignant cells.2124 Furthermore, tumor-promoting CAFs and TAMs release various growth factors, cytokines, and proteinases that create a favorable microenvironment for tumor progression.10,23,25,26

Podoplanin and CD204 may represent useful markers to identify tumor-promoting CAFs and TAMs.17,18,2729 Podoplanin is a glycoprotein which has been used as a specific marker of lymphatic vessels.30,31 In cancer cells, the expression of podoplanin has been reported in several types of carcinomas, suggesting that podoplanin expression is biologically important in tumorigenesis and malignant progression,3236 although opposite results were observed in squamous cell carcinoma of the lung and uterine cervix.37,38 Meanwhile, there is limited information on podoplanin in CAFs.17 CD204 is a prototypic member of a family of transmembrane receptors termed scavenger receptors.39 A previous report indicated that CD204 may be a more useful marker than CD68, a pan-macrophage/monocyte marker, to identify tumor-promoting TAMs in patients with lung adenocarcinoma.29

In the present study, we evaluated the significance of podoplanin-positive CAFs and CD204-positive TAMs as risk factors for recurrence. We showed that both podoplanin-positive CAFs and CD204-positive TAMs were statistically significant predictors for recurrence by univariate analyses. Furthermore, the presence of podoplanin-positive CAFs, which may reflect the presence of a tumor-promoting phenotype of CAFs, was an independently statistically significant predictor for recurrence by multivariate analyses. These results indicate that tumor stromal cells may play a pivotal role in tumor progression.

While the incidence of primary lung adenocarcinoma has increased, stage I tumors are also being detected more often.2,3 Several prognostic factors for NSCLC have been identified, including tumor size, VPI, IVI, and lymphatic permeation.20,4046 Among these factors, VPI was clearly defined in the current edition of the TNM classification. Consequently, stage I NSCLC was defined according to the tumor size and the presence of VPI. Therefore, the inclusion of these established prognostic factors in our analysis strengthens the results of the present study and, in particular, the finding that the presence of podoplanin-positive CAFs were the most powerful independent recurrence predictor (HR, 3.474).

According to the current staging system, patients with stage I NSCLC may have different outcomes. A more precise method to predict prognosis may help clinicians to categorize these patients according to risk, and to identify patients with a high risk who might benefit from adjuvant chemotherapy. In the present study, a combination of the presence of podoplanin-positive CAFs with other independent risk factors enabled the successful identification of patients with poor outcomes. Although surgery alone remains the standard treatment of patients in stage I, a recent study suggests that adjuvant chemotherapy may also be beneficial for certain patients with stage I tumors.47 Our results showed that a combination of 3 independent risk factors, including podoplanin-positive CAFs, allows the statistically significant identification of a high-risk subgroup of patients with stage I adenocarcinoma, and this group may be good candidates for the adjuvant chemotherapy.

Novel therapeutic strategies that target the tumor microenvironment have been highlighted by the introduction of bevacizumab, which has been shown to improve the survival of patients with nonsquamous NSCLC.15,16 Farmer et al48 identified CAF-related gene expression as a marker for resistance to chemotherapy. In addition, Crawford et al49 reported that CAFs are associated with the therapeutic resistance to antivascular endothelial growth factor treatment by secreting mediators that promote CAF-induced angiogenesis independent of tumor cells. Thus, these results suggest that therapy targeted against tumor-promoting CAFs may result in an improved response to lung cancer therapy in patients with podoplanin-positive CAFs. Furthermore, podoplanin in CAFs has been shown using in vivo experiments to be the functional protein that is responsible for the enhancement of tumor formation.17 Therefore, although further studies to clarify the role of podoplanin in CAFs in tumor progression or metastasis are required, it is likely that the podoplanin molecule itself will become a potential therapeutic target for lung adenocarcinoma patients with podoplanin-positive CAFs.

Future studies are required to confirm the prognostic significance of podoplanin-positive CAFs in validation populations, and to clarify whether adjuvant chemotherapy is practically effective for high-risk patients, as identified by independent risk factors including podoplanin-positive CAFs. However, the present study is the first to show that a certain type of tumor stromal cell is a recurrence predictor, independent of the other established prognostic factors in patients with lung cancer. We believe that our data provide a novel rationale for the clinical implications of the role of tumor stromal cells in general.

In conclusion, the presence of podoplanin-positive CAFs was the most powerful and independent recurrence predictor in patients with stage I lung adenocarcinoma. The presence of podoplanin-positive CAFs may be useful for identifying patients with a high risk of recurrence who might benefit from adjuvant chemotherapy.

Author contributions:Dr Ito: contributed to the design and coordination of the study, prepared the manuscript, read and approved the final manuscript, and takes responsibility for the data and its accuracy.

Dr Ishii: contributed to the design and coordination of the study, revised the article for important intellectual content, read and approved the final manuscript, and takes responsibility for the data and its accuracy.

Dr Nagai: contributed to the preparation of the manuscript, and read and approved the final manuscript.

Dr Maeda: contributed to the preparation of the manuscript, and read and approved the final manuscript.

Dr Nakano: contributed to the preparation of the manuscript, and read and approved the final manuscript.

Dr Ochiai: contributed to the design and coordination of the study, revised the article for important intellectual content, read and approved the final manuscript, and takes responsibility for the data and its accuracy.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

Other contributions: All work included in the manuscript was performed at National Cancer Center Hospital East (Kashiwa, Chiba, Japan). The research was approved by the Internal Review Board of the institution. No patient consent was required as the research is a retrospective chart review and no personally identifiable information was included in the manuscript.

CAF

cancer-associated fibroblast

HR

hazard ratio

IVI

intratumoral vascular invasion

NSCLC

non-small cell lung cancer

RFP

recurrence-free proportion

TAM

tumor-associated macrophage

VPI

visceral pleural invasion

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59(4):225-249. [PubMed] [CrossRef]
 
Devesa SS, Bray F, Vizcaino AP, Parkin DM. International lung cancer trends by histologic type: male:female differences diminishing and adenocarcinoma rates rising. Int J Cancer. 2005;117(2):294-299.
 
Seki N, Eguchi K, Kaneko M, et al. The adenocarcinoma-specific stage shift in the Anti-lung Cancer Association project: significance of repeated screening for lung cancer for more than 5 years with low-dose helical computed tomography in a high-risk cohort. Lung Cancer. 2010;67(3):318-324.
 
Scott WJ, Howington J, Feigenberg S, Movsas B, Pisters K American College of Chest Physicians American College of Chest Physicians. Treatment of non-small cell lung cancer stage I and stage II: ACCP evidence-based clinical practice guidelines (2nd edition). Chest. 2007;132(suppl 3):234S-242S.
 
Winton T, Livingston R, Johnson D, et al; National Cancer Institute of Canada Clinical Trials Group National Cancer Institute of Canada Clinical Trials Group National Cancer Institute of the United States Intergroup JBR.10 Trial Investigators National Cancer Institute of the United States Intergroup JBR.10 Trial Investigators. Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer. N Engl J Med. 2005;352(25):2589-2597.
 
Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP, Vansteenkiste J International Adjuvant Lung Cancer Trial Collaborative Group International Adjuvant Lung Cancer Trial Collaborative Group. Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Engl J Med. 2004;350(4):351-360.
 
Douillard JY, Rosell R, De Lena M, et al. Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomised controlled trial [published correction appears inLancet Oncol. 2006;7(10):797]. Lancet Oncol. 2006;7(9):719-727.
 
Pisters KM, Evans WK, Azzoli CG, et al; Cancer Care Ontario Cancer Care Ontario American Society of Clinical Oncology American Society of Clinical Oncology. Cancer Care Ontario and American Society of Clinical Oncology adjuvant chemotherapy and adjuvant radiation therapy for stages I-IIIA resectable non small-cell lung cancer guideline. J Clin Oncol. 2007;25(34):5506-5518.
 
Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 2005;7(3):211-217.
 
Kalluri R. Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer. 2003;3(6):422-433.
 
Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A. PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell. 2005;120(3):303-313.
 
Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology. 2003;37(3):674-685.
 
Cornil I, Theodorescu D, Man S, Herlyn M, Jambrosic J, Kerbel RS. Fibroblast cell interactions with human melanoma cells affect tumor cell growth as a function of tumor progression. Proc Natl Acad Sci U S A. 1991;88(14):6028-6032.
 
Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer. 2001;1(1):46-54.
 
Reck M, von Pawel J, Zatloukal P, et al. Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol. 2009;27(8):1227-1234.
 
Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355(24):2542-2550.
 
Hoshino A, Ishii G, Ito T, et al. Podoplanin-positive fibroblasts enhance lung adenocarcinoma tumor formation: podoplanin in fibroblast functions for tumor progression. Cancer Res. 2011;71(14):4769-4779.
 
Komohara Y, Takemura K, Lei XF, et al. Delayed growth of EL4 lymphoma in SR-A-deficient mice is due to upregulation of nitric oxide and interferon-gamma production by tumor-associated macrophages. Cancer Sci. 2009;100(11):2160-2166.
 
Travis WDBE, Muller-Hermelink HK. World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press; 2004.
 
Goldstraw P, Crowley J, Chansky K, et al; International Association for the Study of Lung Cancer International Staging Committee International Association for the Study of Lung Cancer International Staging Committee Participating Institutions Participating Institutions. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol. 2007;2(8):706-714.
 
Hagemann T, Wilson J, Burke F, et al. Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006;176(8):5023-5032.
 
Hagemann T, Robinson SC, Schulz M, Trümper L, Balkwill FR, Binder C. Enhanced invasiveness of breast cancer cell lines upon co-cultivation with macrophages is due to TNF-alpha dependent up-regulation of matrix metalloproteases. Carcinogenesis. 2004;25(8):1543-1549.
 
Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005;121(3):335-348.
 
Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 1999;59(19):5002-5011.
 
Ono M. Molecular links between tumor angiogenesis and inflammation: inflammatory stimuli of macrophages and cancer cells as targets for therapeutic strategy. Cancer Sci. 2008;99(8):1501-1506.
 
De Wever O, Demetter P, Mareel M, Bracke M. Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer. 2008;123(10):2229-2238.
 
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(11):549-555.
 
Komohara Y, Hirahara J, Horikawa T, et al. AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype. J Histochem Cytochem. 2006;54(7):763-771.
 
Ohtaki Y, Ishii G, Nagai K, et al. Stromal macrophage expressing CD204 is associated with tumor aggressiveness in lung adenocarcinoma. J Thorac Oncol. 2010;5(10):1507-1515.
 
Mäkinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 2001;20(17):4762-4773.
 
Petrova TV, Mäkinen T, Mäkelä TP, et al. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 2002;21(17):4593-4599.
 
Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M. Enhanced expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma. Tumour Biol. 2005;26(4):195-200.
 
Martín-Villar E, Scholl FG, Gamallo C, et al. Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas. Int J Cancer. 2005;113(6):899-910.
 
Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK, Detmar M. Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol. 2005;166(3):913-921.
 
Scholl FG, Gamallo C, Quintanilla M. Ectopic expression of PA2.26 antigen in epidermal keratinocytes leads to destabilization of adherens junctions and malignant progression. Lab Invest. 2000;80(11):1749-1759.
 
Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell. 2006;9(4):261-272.
 
Dumoff KL, Chu CS, Harris EE, et al. Low podoplanin expression in pretreatment biopsy material predicts poor prognosis in advanced-stage squamous cell carcinoma of the uterine cervix treated by primary radiation. Mod Pathol. 2006;19(5):708-716.
 
Ito T, Ishii G, Nagai K, et al. Low podoplanin expression of tumor cells predicts poor prognosis in pathological stage IB squamous cell carcinoma of the lung, tissue microarray analysis of 136 patients using 24 antibodies. Lung Cancer. 2009;63(3):418-424.
 
Platt N, Gordon S. Is the class A macrophage scavenger receptor (SR-A) multifunctional? The mouse’s tale. J Clin Invest. 2001;108(5):649-654.
 
Harpole DH Jr, Herndon JE II, Young WG Jr, Wolfe WG, Sabiston DC Jr. Stage I nonsmall cell lung cancer. A multivariate analysis of treatment methods and patterns of recurrence. Cancer. 1995;76(5):787-796.
 
Ichinose Y, Yano T, Asoh H, Yokoyama H, Yoshino I, Katsuda Y. Prognostic factors obtained by a pathologic examination in completely resected non-small-cell lung cancer. An analysis in each pathologic stage. J Thorac Cardiovasc Surg. 1995;110(3):601-605.
 
Shimizu K, Yoshida J, Nagai K, et al. Visceral pleural invasion classification in non-small cell lung cancer: a proposal on the basis of outcome assessment. J Thorac Cardiovasc Surg. 2004;127(6):1574-1578.
 
Suzuki K, Nagai K, Yoshida J, et al. Conventional clinicopathologic prognostic factors in surgically resected nonsmall cell lung carcinoma. A comparison of prognostic factors for each pathologic TNM stage based on multivariate analyses. Cancer. 1999;86(10):1976-1984.
 
Ogawa J, Tsurumi T, Yamada S, Koide S, Shohtsu A. Blood vessel invasion and expression of sialyl Lewisx and proliferating cell nuclear antigen in stage I non-small cell lung cancer. Relation to postoperative recurrence. Cancer. 1994;73(4):1177-1183.
 
Miyoshi K, Moriyama S, Kunitomo T, Nawa S. Prognostic impact of intratumoral vessel invasion in completely resected pathologic stage I non-small cell lung cancer. J Thorac Cardiovasc Surg. 2009;137(2):429-434.
 
Poleri C, Morero JL, Nieva B, et al. Risk of recurrence in patients with surgically resected stage I non-small cell lung carcinoma: histopathologic and immunohistochemical analysis. Chest. 2003;123(6):1858-1867.
 
Strauss GM, Herndon JE II, Maddaus MA, et al. Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small-cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B, Radiation Therapy Oncology Group, and North Central Cancer Treatment Group Study Groups. J Clin Oncol. 2008;26(31):5043-5051.
 
Farmer P, Bonnefoi H, Anderle P, et al. A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med. 2009;15(1):68-74.
 
Crawford Y, Kasman I, Yu L, et al. PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment. Cancer Cell. 2009;15(1):21-34.
 

Figures

Figure Jump LinkFigure 1. Immunohistochemical staining of podoplanin and CD204 in lung adenocarcinomas (original magnification, × 40). The box in the bottom right is a magnified view of each panel (original magnification, × 400). A, A case with podoplanin-positive cancer-associated fibroblasts (CAFs). B, A case without podoplanin-positive CAFs. C, High CD204-positive tumor-associated macrophage (TAM) group. D, Low CD204-positive TAM group.Grahic Jump Location
Figure Jump LinkFigure 2. A, Recurrence-free proportion curves of patients with and without podoplanin-positive cancer-associated fibroblasts. B, Recurrence-free proportion curves according to the number of CD204-positive tumor-associated macrophages.Grahic Jump Location
Figure Jump LinkFigure 3. Recurrence-free proportion curves according to the number of independent risk factors.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Associations Between Clinicopathologic Characteristics and Podoplanin-Positive CAFs or CD204-Positive TAMs

CAF = cancer-associated fibroblast; TAM = tumor-associated macrophage.

a 

χ test.

b 

Indicates significance.

c 

Compared with bronchioloalveolar.

Table Graphic Jump Location
Table 2 —Univariate Analysis of Risk Factors for Recurrences

See Table 1 for expansion of abbreviations.

a 

Indicates significance.

Table Graphic Jump Location
Table 3 —Multivariate Analysis of Risk Factors for Recurrences

HR = hazard ratio. See Table 1 for expansion of other abbreviations.

a 

Indicates significance.

References

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59(4):225-249. [PubMed] [CrossRef]
 
Devesa SS, Bray F, Vizcaino AP, Parkin DM. International lung cancer trends by histologic type: male:female differences diminishing and adenocarcinoma rates rising. Int J Cancer. 2005;117(2):294-299.
 
Seki N, Eguchi K, Kaneko M, et al. The adenocarcinoma-specific stage shift in the Anti-lung Cancer Association project: significance of repeated screening for lung cancer for more than 5 years with low-dose helical computed tomography in a high-risk cohort. Lung Cancer. 2010;67(3):318-324.
 
Scott WJ, Howington J, Feigenberg S, Movsas B, Pisters K American College of Chest Physicians American College of Chest Physicians. Treatment of non-small cell lung cancer stage I and stage II: ACCP evidence-based clinical practice guidelines (2nd edition). Chest. 2007;132(suppl 3):234S-242S.
 
Winton T, Livingston R, Johnson D, et al; National Cancer Institute of Canada Clinical Trials Group National Cancer Institute of Canada Clinical Trials Group National Cancer Institute of the United States Intergroup JBR.10 Trial Investigators National Cancer Institute of the United States Intergroup JBR.10 Trial Investigators. Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer. N Engl J Med. 2005;352(25):2589-2597.
 
Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP, Vansteenkiste J International Adjuvant Lung Cancer Trial Collaborative Group International Adjuvant Lung Cancer Trial Collaborative Group. Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Engl J Med. 2004;350(4):351-360.
 
Douillard JY, Rosell R, De Lena M, et al. Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomised controlled trial [published correction appears inLancet Oncol. 2006;7(10):797]. Lancet Oncol. 2006;7(9):719-727.
 
Pisters KM, Evans WK, Azzoli CG, et al; Cancer Care Ontario Cancer Care Ontario American Society of Clinical Oncology American Society of Clinical Oncology. Cancer Care Ontario and American Society of Clinical Oncology adjuvant chemotherapy and adjuvant radiation therapy for stages I-IIIA resectable non small-cell lung cancer guideline. J Clin Oncol. 2007;25(34):5506-5518.
 
Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 2005;7(3):211-217.
 
Kalluri R. Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer. 2003;3(6):422-433.
 
Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A. PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell. 2005;120(3):303-313.
 
Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology. 2003;37(3):674-685.
 
Cornil I, Theodorescu D, Man S, Herlyn M, Jambrosic J, Kerbel RS. Fibroblast cell interactions with human melanoma cells affect tumor cell growth as a function of tumor progression. Proc Natl Acad Sci U S A. 1991;88(14):6028-6032.
 
Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer. 2001;1(1):46-54.
 
Reck M, von Pawel J, Zatloukal P, et al. Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol. 2009;27(8):1227-1234.
 
Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355(24):2542-2550.
 
Hoshino A, Ishii G, Ito T, et al. Podoplanin-positive fibroblasts enhance lung adenocarcinoma tumor formation: podoplanin in fibroblast functions for tumor progression. Cancer Res. 2011;71(14):4769-4779.
 
Komohara Y, Takemura K, Lei XF, et al. Delayed growth of EL4 lymphoma in SR-A-deficient mice is due to upregulation of nitric oxide and interferon-gamma production by tumor-associated macrophages. Cancer Sci. 2009;100(11):2160-2166.
 
Travis WDBE, Muller-Hermelink HK. World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press; 2004.
 
Goldstraw P, Crowley J, Chansky K, et al; International Association for the Study of Lung Cancer International Staging Committee International Association for the Study of Lung Cancer International Staging Committee Participating Institutions Participating Institutions. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol. 2007;2(8):706-714.
 
Hagemann T, Wilson J, Burke F, et al. Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006;176(8):5023-5032.
 
Hagemann T, Robinson SC, Schulz M, Trümper L, Balkwill FR, Binder C. Enhanced invasiveness of breast cancer cell lines upon co-cultivation with macrophages is due to TNF-alpha dependent up-regulation of matrix metalloproteases. Carcinogenesis. 2004;25(8):1543-1549.
 
Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005;121(3):335-348.
 
Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 1999;59(19):5002-5011.
 
Ono M. Molecular links between tumor angiogenesis and inflammation: inflammatory stimuli of macrophages and cancer cells as targets for therapeutic strategy. Cancer Sci. 2008;99(8):1501-1506.
 
De Wever O, Demetter P, Mareel M, Bracke M. Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer. 2008;123(10):2229-2238.
 
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(11):549-555.
 
Komohara Y, Hirahara J, Horikawa T, et al. AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype. J Histochem Cytochem. 2006;54(7):763-771.
 
Ohtaki Y, Ishii G, Nagai K, et al. Stromal macrophage expressing CD204 is associated with tumor aggressiveness in lung adenocarcinoma. J Thorac Oncol. 2010;5(10):1507-1515.
 
Mäkinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 2001;20(17):4762-4773.
 
Petrova TV, Mäkinen T, Mäkelä TP, et al. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 2002;21(17):4593-4599.
 
Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M. Enhanced expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma. Tumour Biol. 2005;26(4):195-200.
 
Martín-Villar E, Scholl FG, Gamallo C, et al. Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas. Int J Cancer. 2005;113(6):899-910.
 
Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK, Detmar M. Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol. 2005;166(3):913-921.
 
Scholl FG, Gamallo C, Quintanilla M. Ectopic expression of PA2.26 antigen in epidermal keratinocytes leads to destabilization of adherens junctions and malignant progression. Lab Invest. 2000;80(11):1749-1759.
 
Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell. 2006;9(4):261-272.
 
Dumoff KL, Chu CS, Harris EE, et al. Low podoplanin expression in pretreatment biopsy material predicts poor prognosis in advanced-stage squamous cell carcinoma of the uterine cervix treated by primary radiation. Mod Pathol. 2006;19(5):708-716.
 
Ito T, Ishii G, Nagai K, et al. Low podoplanin expression of tumor cells predicts poor prognosis in pathological stage IB squamous cell carcinoma of the lung, tissue microarray analysis of 136 patients using 24 antibodies. Lung Cancer. 2009;63(3):418-424.
 
Platt N, Gordon S. Is the class A macrophage scavenger receptor (SR-A) multifunctional? The mouse’s tale. J Clin Invest. 2001;108(5):649-654.
 
Harpole DH Jr, Herndon JE II, Young WG Jr, Wolfe WG, Sabiston DC Jr. Stage I nonsmall cell lung cancer. A multivariate analysis of treatment methods and patterns of recurrence. Cancer. 1995;76(5):787-796.
 
Ichinose Y, Yano T, Asoh H, Yokoyama H, Yoshino I, Katsuda Y. Prognostic factors obtained by a pathologic examination in completely resected non-small-cell lung cancer. An analysis in each pathologic stage. J Thorac Cardiovasc Surg. 1995;110(3):601-605.
 
Shimizu K, Yoshida J, Nagai K, et al. Visceral pleural invasion classification in non-small cell lung cancer: a proposal on the basis of outcome assessment. J Thorac Cardiovasc Surg. 2004;127(6):1574-1578.
 
Suzuki K, Nagai K, Yoshida J, et al. Conventional clinicopathologic prognostic factors in surgically resected nonsmall cell lung carcinoma. A comparison of prognostic factors for each pathologic TNM stage based on multivariate analyses. Cancer. 1999;86(10):1976-1984.
 
Ogawa J, Tsurumi T, Yamada S, Koide S, Shohtsu A. Blood vessel invasion and expression of sialyl Lewisx and proliferating cell nuclear antigen in stage I non-small cell lung cancer. Relation to postoperative recurrence. Cancer. 1994;73(4):1177-1183.
 
Miyoshi K, Moriyama S, Kunitomo T, Nawa S. Prognostic impact of intratumoral vessel invasion in completely resected pathologic stage I non-small cell lung cancer. J Thorac Cardiovasc Surg. 2009;137(2):429-434.
 
Poleri C, Morero JL, Nieva B, et al. Risk of recurrence in patients with surgically resected stage I non-small cell lung carcinoma: histopathologic and immunohistochemical analysis. Chest. 2003;123(6):1858-1867.
 
Strauss GM, Herndon JE II, Maddaus MA, et al. Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small-cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B, Radiation Therapy Oncology Group, and North Central Cancer Treatment Group Study Groups. J Clin Oncol. 2008;26(31):5043-5051.
 
Farmer P, Bonnefoi H, Anderle P, et al. A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med. 2009;15(1):68-74.
 
Crawford Y, Kasman I, Yu L, et al. PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment. Cancer Cell. 2009;15(1):21-34.
 
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