Interleukin-10 Haplotype May Predict Survival and
Relapse in Resected Non-Small Cell Lung Cancer
Yaw-Cheng Wang
1,2,3., Wen-Wei Sung
1,2., Tzu-Chin Wu
2,3, Lee Wang
4, Wen-Pin Chien
5, Ya-Wen Cheng
1,
Chih-Yi Chen
6, Shwn-Huey Shieh
7, Huei Lee
1,2,8,9*
1 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC, 2 School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC, 3 Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC, 4 Department of Public Health, Chung Shan Medical University, Taichung, Taiwan, ROC, 5 Division of Preclinical Science, Center for Drug Evaluation, Taipei, Taiwan, ROC, 6 Department of Surgery, China Medical University Hospital, Taichung, Taiwan, ROC, 7 Department of Health Services Management, China Medical University and Hospital, Taichung, Taiwan, ROC, 8 Department of Medical Research,
Chung Shan Medical University Hospital, Taichung, Taiwan, ROC, 9 Institute of Medical Sciences, Tzuchi University, Hualien, Taiwan, ROC
Abstract
10 is associated with tumor malignancy via immune escape. We hypothesized that 10 haplotypes categorized by IL-10 promoter polymorphisms at –IL-1082A.G, –819C.T, and –592C.A might influence IL-IL-10 expression and give rise to non-small cell lung cancer (NSCLC) patients with poor outcomes and relapse. We collected adjacent normal tissues from 385 NSCLC patients to determine IL-10 haplotypes by direct sequencing and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 385 tumors, 241 were available to evaluate IL-10 mRNA expression levels by real-time RT-PCR. The influence of IL-10 haplotypes on overall survival (OS) and relapse free survival (RFS) were determined by Kaplan-Meier and multivariate Cox regression analysis. The results showed that IL-10 mRNA levels were significantly higher in tumors with the ATA haplotype than with the ATA haplotype (P = 0.004). Patients with the non-ATA haplotype had shorter OS and RFS periods than did patients with the non-ATA haplotype. This may be associated with the observation that the number of tumor-infiltrating lymphocytes was decreased in the tumors with higher levels of IL-10. Consistently, T cells from the peripheral blood of the patients with non-ATA haplotype were more susceptible to apoptosis and less cytotoxic to tumor cells, compared to those from the patients with ATA haplotype. The results suggest that IL-10 can promote tumor malignancy via promoting T cell apoptosis and tumor cell survival, and IL-10 haplotype evaluated by PCR-RFLP or direct sequencing may be used to predict survival and relapse in resected NSCLC, helping clinicians to make appropriate decisions on treatment of the patients.
Introduction
Interleukin-10 (IL-10), an important immunoinhibitory cyto-kine, is part of a balanced network of cytokines [1–5]. The IL-10 cytokine is produced by several cells including normal and neoplastic B cells, stimulated monocytes, macrophages, and a subset of T cells [1–4]. Many case-control studies have indicated an association of IL-10 promoter polymorphisms (SNPs) with human cancer risks, including risk of lung cancer [6–9]. Of the IL-10 promoter SNPs, ones at 21082A.G, 2819C.T, and 2592G.A have been the focus of recent studies, and the phenotypes of these single nucleotide SNPs have been further confirmed by functional assays in cell models [10,11].
Tumor immune surveillance studies have revealed an associa-tion between IL-10 and the development of human cancers such as large B-cell lymphoma, T-cell non-Hodgkin lymphoma, and colon, prostate, breast, gastric, myeloma, and lung cancers [4,6– 9,12–22]. In lung cancer cases, some reports have indicated that loss of IL-10 in lung tumors may promote tumor progression and
result in poor clinical outcomes in patients; however, an opposite effect has been reported in other studies [23–29]. Interestingly, the absence of IL-10 expression has been associated with poor outcome in stage I non-small cell lung cancer (NSCLC) [24,25], while in late-stage NSCLC, the presence of IL-10-positive macrophages at the tumor margins can be an indicator of poor prognostic outcome [23]. In addition, shorter survival times have been reported in advanced lung cancer patients who had high serum IL-10 levels, when compared with similar patients who had low serum IL-10 levels [26]. A clear role for IL-10 in lung tumorigenesis therefore remains to be identified.
In the present study, we examined normal lung tissues adjacent to surgically resected NSCLC tumors in 385 patients in order to identify IL-10 promoter SNPs at 21082A.G, 2819C.T, and 2592C.A by direct sequencing and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). These three IL-10 promoter SNPs have been reported to produce mainly three haplotypes: GCC, ACC, ATA [30–33]. In the present study, patients were categorized into two haplotypes, ATA and non-ATA
(Fig. 1), which had been used in two previous reports [34,35]. We questioned: 1) whether tumors from non-ATA carriers have higher IL-10 mRNA expression levels than tumors from ATA carriers, 2) whether patients with a non-ATA haplotype or higher IL-10 mRNA levels in lung tumors have greater tumor immune surveillance, and 3) whether IL-10 haplotype or mRNA expression could be used to predict overall survival (OS) and relapse free survival (RFS) in resected NSCLC patients.
Materials and Methods
Patients
This study included 385 patients with NSCLC. All patients were unrelated ethnic Chinese and residents of central Taiwan. Patients had been diagnosed with adenocarcinoma (194; 50.4%) or squamous cell carcinoma (191; 49.6%) and underwent surgical resection at the Division of Thoracic Surgery, Taichung Veterans General Hospital, between 1993 and 2004. Samples were immediately frozen at surgery and kept at 280uC until processed. The study was approved by the Institutional Review Board (Institutional Review Board, Chung Shan Medical University Hospital. CSMUH No: CS11177). Cancer relapse data were obtained by chart review and confirmed by thoracic surgeons. Clinical parameters and OS and RFS data were collected from chart reviews (32 patients had no relapse data) and the Taiwan Cancer Registry, Department of Health, Executive Yuan, ROC.
Genomic DNA extraction, RNA extraction, and
cDNA synthesis
Genomic DNA was extracted by conventional methods. Surgically resected normal tissues adjacent to the lung tumor were prepared by using proteinase K digestion and DNA was extracted using phenol-chloroform, followed by ethanol precipi-tation.
Total RNA was extracted from 241 available lung tumor tissues using TRIzol reagent (Invitrogen). First-strand cDNA synthesis in the presence of random primers was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s instructions.
Table 1. Correlations between IL-10 haplotypes and clinical characteristics in 385 non-small cell lung cancer patients.
IL-10 haplotype
Parameter Case no. ATA Non ATA P value1 Age ,65 163 73 (44.8) 90 (55.2) 0.633 $65 222 94 (42.3) 128 (57.7) Gender Female 117 58 (49.6) 59 (50.4) 0.105 Male 268 109 (40.7) 159 (59.3) Smoking status Nonsmoker 197 95 (48.2) 102 (51.8) 0.049 Smoker 188 72 (38.3) 116 (61.7) Stage I and II 212 94 (44.3) 118 (55.7) 0.673 III 173 73 (42.2) 100 (45.9) T classification 1 and 2 290 121 (41.7) 169 (58.3) 0.253 3 and 4 95 46 (48.4) 49 (51.6) N classification 0 185 92 (49.7) 93 (50.3) 0.016 1 and 2 200 75 (37.5) 125 (62.5) Tumor type SQ 191 85 (44.5) 106 (55.5) 0.658 AD 194 82 (42.3) 112 (57.7) Disease relapse Negative 229 99 (43.2) 130 (56.8) 0.954 Positive 124 54 (43.5) 70 (56.5)
SQ, squamous cell carcinomas; AD, adenocarcinomas. 1Two sided Chi-square test. doi:10.1371/journal.pone.0039525.t001
Figure 1. Allele/haplotype frequencies of IL-10 SNPs. SNPs at positions 2819 and 2592 in the IL-10 promoter were in complete linkage disequilibrium. Three IL-10 haplotypes were observed and the occurrence of each haplotype or haplotype combination is listed in the shaded boxes. doi:10.1371/journal.pone.0039525.g001
Figure 2. IL-10 mRNA expression (mean ± standard error) in tumor tissue by promoter haplotypes.
doi:10.1371/journal.pone.0039525.g002
Table 2. Correlations between tumor infiltrating lymphocyte counts and IL-10 haplotype in 87 non-small cell lung cancer patients.
TIL (count/HPF) IL-10
haplotype Case no. ,25 $25 P value1 All cases ATA 36 18 (50.0) 18 (50.0) 0.236 Non-ATA 51 32 (62.7) 19 (37.3) Early stage ATA 15 7 (46.7) 8 (53.3) 0.988 Non-ATA 28 13 (46.4) 15 (53.0) Late stage ATA 21 11 (52.4) 10 (47.6) 0.032 Non-ATA 23 19 (82.6) 4 (17.4)
1Two sided Chi-square test. doi:10.1371/journal.pone.0039525.t002
PCR-RFLP analysis for IL-10 -592C/A genetic SNP
Genotypes of IL-10 -592C/A were determined by PCR-RFLP as described by Rad et al. [36]. PCR amplification products from 50 samples were randomly selected for direct sequencing to confirm the genotype indicated by PCR-RFLP.Figure 3. Representative immunostaining of CD3+ TILs in lung tumors. (A) Representative of CD3 immunostaining of TILs in lung tumors with low TIL density (,25 CD3+/HPF) (left: 100x; right: 400x); (B) TILs presented in lung tumors show high TIL density ($25 CD3+/HPF) (left: 100x; right: 400x).
