Variations in HSPA1B at 6P21.3 Are Associated with Lung Cancer Risk and Prognosis in Chinese Populations

Published OnlineFirst October 28, 2011; DOI: 10.1158/0008-5472.CAN-11-1409

Cancer Prevention and Epidemiology Research

Variations in HSPA1B at 6p21.3 Are Associated with Lung Cancer Risk and Prognosis in Chinese Populations

Huan Guo1, Qifei Deng1, Chen Wu4, Lingmin Hu5, Sheng Wei1, Ping Xu3, Dan Kuang1, Li Liu2, Zhibin Hu5, Xiaoping Miao1, Hongbing Shen5, Dongxin Lin4, and Tangchun Wu1

Abstract The heat shock protein Hsp70 is crucial for regulating cellular homeostasis in stressed cells. Although the tumorigenic potential and prognostic applications of Hsp70 have been widely investigated, it remains unclear whether genetic variations of the human isoforms HSPA1L, HSPA1A, and HSPA1B are associated with cancer risk and prognosis. In this study, we genotyped six tagSNPs in these genes in 1,152 paired patients with lung cancer and controls, and then validated the results in additional cohorts of 1,781 patients with lung cancer and 1,038 controls. In addition, we evaluated the associations of these tagSNPs with survival in 330 patients with advanced non–small cell lung cancer (NSCLC) with additional validation in another 331 patients with advanced NSCLC. Functions of the risk variants identified were investigated using cell-based reporter assays. We found that the HSPA1B rs6457452T allele was associated with increased lung cancer risk compared with the rs6457452C allele in both data sets and also pooled analysis (adjusted OR ¼ 1.41; P ¼ 2.8 10 5). The HSPA1B rs2763979TT genotype conferred poor survival outcomes for patients with advanced NSCLC in two independent cohorts and pooled analysis [adjusted hazard ratio (HR) ¼ 1.80, 1.61, and 1.66; P ¼ 0.013, 0.036, and 0.002, respectively]. Lastly, we also found that the rs2763979T and rs6457452T alleles were each sufficient to reduce expression of transcriptional reporter constructs, when compared with the rs2763979C and rs6457452C alleles, respectively. Taken together, our findings define that functional HSPA1B variants are associated with lung cancer risk and survival. These Hsp70 genetic variants may offer useful biomarkers to predict lung cancer risk and prognosis. Cancer Res; 71(24); 1–11. 2011 AACR.

Introduction researchers have still not identified specific biomarkers for lung cancer risk assessment and prognosis prediction. Lung cancer is a leading cause of cancer-related deaths in Published genome-wide association studies (GWAS) in Cau- the world (1). Although tobacco smoking accounts for more casians have mapped a lung cancer susceptibility locus to than 80% of all cases (2), only around 15% of all heavy smokers chromosome 6p21.3, a gene-rich region of the major histocom- develop lung cancer, indicating that genetic factors may also patibility complex (MHC; refs. 3–5). The reported significant play a crucial role in susceptibility to the disease. To date, variants (rs3117582, rs3131379, and rs4324798) at 6p21.3 do not prognosis for patients with non–small cell lung carcinoma exist in Asians according to the HapMap project, and whether (NSCLC) remains poor, with an overall 5-year survival rate of single nucleotide polymorphisms (SNP) in this region are only 10% to 15% (1). Despite intense efforts in the past decades, related to lung cancer risk in Chinese populations is unknown. The 3 Hsp70 genes (HSPA1L, HSPA1A, and HSPA1B), encod- ing 3 highly homologous Hsp70 proteins (Hsp70-1, Hsp70-2, Authors' Affiliations: 1Institute of Occupational Medicine and Ministry of Education Key Lab for Environment and Health, School of Public Health, and Hsp70-hom, respectively), are located parallel to each Tongji Medical College, 2Cancer Center, Union Hospital, Huazhong Uni- other within the MHC class-III region at 6p21.3, only approx- 3 versity of Science and Technology; Department of Oncology, Wuhan Iron imately 50-kb away from rs3131379 (3). HSPs, and Hsp70 in and Steel (Group) Corporation Staff-Worker Hospital, Wuhan; 4Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese particular, are the major stress-inducible proteins that func- Academy of Medical Sciences and Peking Union Medical College, Beijing; tion as key components in regulating cellular homeostasis, and 5Department of Epidemiology and Biostatistics, Cancer Center of Nanjing Medical University, Nanjing, China probably through their molecular chaperone activities to inhibit accumulation of protein aggregates (6, 7) or through Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). their cytoprotective properties to protect against DNA damage (8), thus promoting cell survival under stressful stimuli (9, 10). Corresponding Author: Tangchun Wu, Institute of Occupational Medi- cine, School of Public Health, Tongji Medical College, HUST, 13 Hangkong Accumulating evidence indicates their potential associations Rd, Wuhan 430030, Hubei, China. Phone: 86-27-83692347; Fax: 86-27- with tumorigenesis and clinical outcomes of patients with 83692560; E-mail: [email protected] cancer (11, 12). A case–control study nested in a large-scale doi: 10.1158/0008-5472.CAN-11-1409 Japanese cohort indicated that serum Hsp70 level might be an 2011 American Association for Cancer Research. important biomarker for lung cancer risk (13). Because of its

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peptide chaperone capacity and its ability to bind to profes- cancer can be affected by many factors, including genetic sional antigen-presenting cells (APC), the immunologic prop- background, treatments, and socioeconomic status. To min- erties of Hsp70 indicate implications of its potential clinical imize the bias due to patient selection, inconsistency of applications as personalized vaccines for human cancers (14). therapies, and patient economic status among different hos- Because of the vital role of Hsp70 in cancer development and pitals, we limited our survival analysis to the 330 patients with survival progress and the results from GWAS in Caucasian advanced NSCLC (stage III or IV) from Wuhan Steel Group/ populations that identified risk alleles at 6p21.3 near HSPA11L– Corporation Staff-Worker Hospital. HSPA1A–HSPA1B (3), we hypothesized that genetic variations Among these, 255 patients with advanced NSCLC received in HSPA1L–HSPA1A–HSPA1B may modulate Hsp70 expression chemotherapy, 84 (32.9%) patients received a gemcitabine/ levels and/or protein structures and, thus, influence lung cisplatin regimen, 99 (38.8%) received a vinorelbine/cisplatin cancer susceptibility and prognosis in Chinese populations. regimen, 22 (8.6%) received a docetaxel/cisplatin regimen, 8 (3.1%) received an etoposide/cisplatin regimen, and the Materials and Methods remaining 42 (16.5%) patients received chemotherapy that was a combination of the above regimens. Case–control study to identify risk variants In addition, 353 patients with advanced NSCLC from the In this study, we carried out 3 independent case–control Cancer Hospital of Jiangsu Province and the First Affiliated analyses. The discovery set included 1,152 patients with lung Hospital of Nanjing Medical University were used as the cancer (recruited from Union Hospital Cancer Center, Wuhan validation set (17). Among these 353 patients, only 331 patients Steel Group/Corporation Staff-Worker Hospital, and Wuhan with sufficient DNA data were evaluated. In this data set, 300 Zhongnan Hospital from January 2003 to February 2009) and patients received chemotherapy. Among these patients, 142 1,152 age- (5 years) and sex frequency–matched controls (47.3%) received a gemcitabine/cisplatin regimen, 14 (4.7%) (randomly selected from 4,073 individuals participating in a received a vinorelbine/cisplatin regimen, 36 (12.0%) received a community screening program in the same regions during the docetaxel/cisplatin regimen, 13 (4.3%) received an etoposide/ same period; ref. 15). Data from patients with lung cancer and cisplatin regimen, and 95 (31.7%) received chemotherapy that controls from northern China were used to validate the iden- was a combination of the above regimens. The TNM stage tified risk variants (validation set) and it included an additional classification was evaluated by medical oncologists according 1,781 patients with lung cancer (recruited at the Cancer to the Staging Manual of the AJCC/UICC (2003; ref. 18). Patients Hospital, Chinese Academy of Medical Sciences, between July were followed up by telephone calls every 3 months. Survival 2000 and November 2008) as well as 1,038 age- (5 years) and duration was calculated from the date when patients first sex-matched controls (cancer-free individuals selected from a received confirmed diagnoses to the date of the last follow- community nutritional survey of 6,450 individuals in the same up or death. Dates of death were obtained from inpatient and region during the same period; ref. 16). In addition, 500 newly outpatient records or from the patients' families through enrolled patients with lung cancer (from Union Hospital telephonic follow-up. In the discovery set, the mean and Cancer Center and Wuhan Steel Group/Corporation Staff- median follow-up periods were 17.5 and 12.2 months, respec- Worker Hospital) and 500 age- (5 years) and sex frequency– tively, whereas the follow-up period ranged from 1.0 to 84.0 matched controls were chosen for further fine-mapping anal- months. In the validation set, the mean and median follow-up ysis. In addition, these 500 control subjects were randomly periods were 19.6 and 16.0 months, respectively, whereas the selected from the 4,073 individuals used for selecting controls follow-up period ranged from 1.0 to 70.2 months. in the discovery set, and they were independent of the 1,152 All subjects in this study were unrelated ethnic Han Chinese, controls used in the discovery set. and general information about all the subjects was collected through personal interviews. Subjects who had smoked an Follow-up study to identify prognostic variants average of less than 1 cigarette per day and for less than 1 year For the survival analysis, we followed up 450 patients with in their lifetimes were defined as nonsmokers; those who primary lung cancer of Wuhan Steel Group/Corporation Staff- stopped smoking more than 1 year previously were considered Worker Hospital as the discovery set. These patients were former smokers; and those who were still smoking in the admitted to the Department of Oncology of Wuhan Iron and previous year were defined as current smokers. This study Steel Group/Corporation Staff-Worker Hospital and had suf- was approved by the Institutional Review Board of Tongji ficient follow-up data available (15). Patients enrolled in this Medical College, the Chinese Academy of Medical Sciences hospital lived in the same region and had a similar socioeco- Cancer Institute, and Nanjing Medical University. nomic status. Of the 450 patients, 4 (0.1%) patients did not have TNM stage classification of tumors. Among the remaining 446 TagSNPs selection patients with completed follow-up/clinical information, 28 Haplotype-tagging SNPs (tagSNP) of the HSPA1L– (6.3%) were patients with small cell lung cancer (SCLC) and HSPA1A–HSPA1B gene cluster on 6p21.3 (Chr6: 31,885,375 418 (93.7%) were patients with NSCLC. There were 88 (21.1%) to 31,906,010) in Chinese populations were selected on the patients with early NSCLC (stage I or II) and 330 (78.9%) basis of Han Chinese Beijing (CHB) data in the HapMap patients with advanced NSCLC (stage III or IV). Eight of project [HapMap data Rel24/phaseII Nov08, on NCBI B36 the patients with advanced NSCLC were lost to follow-up. As assembly, dbSNP b126, minor allele frequency (MAF 0.05), we know, the survival outcomes of patients with NSCLC lung Hardy–Weinberg equilibrium (HWE) P value 0.05, and call

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rate 90%] with Haploview 4.0 software on a block-by-block reproducibility was 100%. All primers and probes used in analysis (r2 threshold, 0.8). Linkage disequilibrium (LD) this study are listed in Supplementary Table S1. blocks were determined according to the method described by Gabriel and colleagues with default settings (19). By Luciferase reporter assay this method, we selected 5 tagSNPs, rs2075800 (HSPA1L_þ Referring to the transcription initiation site of the HSPA1B 2763C>T), rs2227956 (HSPA1L_þ2437A>G), rs1043618 gene as "þ1," we constructed 6 reporter plasmids based on the (HSPA1A_þ190G>C), rs2763979 (HSPA1B_912C>T), and pGL3-Basic vector (Promega), including 4 fragments from rs6457452 (HSPA1B_þ39C>T). In addition, the rs1008438 1031 to þ216 according to their haplotypes (rs2763979C/ (HSPA1A_110A/C) identified by our resequencing results rs6457452C, rs2763979C/rs6457452T, rs2763979T/rs6457452C, was also selected (Supplementary Fig. S1; ref. 20). and rs2763979T/rs6457452T, respectively) and 2 core regula- The risk polymorphisms [rs3117582 (chr6: 31728499) and tion regions from 456 to þ216 containing either the rs3131379 (chr6: 31829012)] reported by GWAS in Caucasians rs6457452C or the rs6457452T allele (Fig. 2A). These were all did not exist in Asians. Because rs3117582 was located in the inserted into KpnI/HindIII enzyme sites of the pGL3-Basic. block from chr6: 31722081 to 31733520, we further carried out a Primer pairs designed for plasmid constructs and site-specific fine-mapping analysis from this block to the HSPA1L–HSPA1A– mutagenesis are listed in Supplementary Table S1. The direc- HSPA1B cluster. The same HapMap-CHB database and selection and sequence authenticity of the 6 constructs were tion criteria described above were used to select 29 tagSNPs at validated by restriction analysis and DNA sequencing. 6p21.3 (from 31722081 to 31906010; Supplementary Fig. S2). The human bronchial epithelial 16HBE cells were kindly provided by Dr. Yiguo Jiang of the Guangzhou Medical College, Genotyping China, whereas the human lung carcinoma A549 cells and Six tagSNPs located in the HSPA1L–HSPA1A–HSPA1B hepatoma HepG2 Cells were purchased from the China Center cluster were genotyped by TaqMan methods, and the of Type Culture Collection in Wuhan, China. The 2 104 additional 24 tagSNPs at 6p21.3 were genotyped by the 16HBE, HepG2, and A549 cells were seeded into 96-well plates MassArray system (Sequenom). However, genotyping of and co-transfected with 100 ng reporter plasmids and 1 ng rs6905572 failed because no appropriate primers could be pRL-SV40 (Promega) using Lipofectamine-2000 (Invitrogen). designed for this SNP. In the 331 patients with advanced The relative luciferase activity (RLA) was determined 24 hours NSCLC in the validation set, 6 samples (1.8%) failed in after transfection as previously described (15, 20). genotyping of rs2763979 and 8 samples (2.4%) failed in genotyping of rs6457452 due to their lower DNA quantity. Statistical analysis As a result, 325 and 323 patients with advanced NSCLC in the A 2-phase screening was used to search for tagSNPs in the validation set were successfully genotyped for HSPA1B gene HSPA1L–HSPA1A–HSPA1B cluster associated with lung cancer rs2763979 and rs6457452, respectively. For all tested sam- risk and survival (Fig. 1). Distributions of numerical data were ples, a 5% random sample was reciprocally tested, and the examined by the 1-sample Komogorov–Smirnov normality

HSPA1L–HSPA1A–HSPA1B

Case-only Case-control study survival analysis 6 tagSNPs

HSPA1L: rs2075800, rs2227956 HSPA1A: rs1008438, rs1043618 HSPA1B: rs2763979, rs6457452 1,152 patients Discovery set 330 advanced 1,152 controls NSCLC patients Risk SNP Risk SNPs HSPA1B HSPA1B Figure 1. Patients and study rs6457452 rs2763979, rs6457452 strategy. Validate Validate

Validation set 1,781 patients 331 advanced 1,038 controls NSCLC patients

Risk SNP Risk SNP HSPA1B: rs6457452 HSPA1B: rs2763979

Fine-mapping analysis: Genotyped 29 tagSNPs Risk SNP within surrounding 185-kb Function analyses region at 6p21.3 500 patients 500 controls Luciferase reporter assay

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A –1,031 –456 +1 +216 HSPA1B gene Promoter 5′-UTR coding region

pGL3-basic –912C +39C pGL3 constructs p-C-C –912C +39T p-C-T –912T +39C Figure 2. Luciferase reporter assays p-T-C for the HSPA1B rs2763979C>T –912T +39T p-T-T and rs6457452C>T +39C polymorphisms. A, schematic p-rs6457452C +39T representation of HSPA1B gene p-rs6457452T and 6 reporter gene constructs. B, Luciferase expression of these B constructs. The 6 reporter 30.0 constructs and empty pGL3-Basic P < 0.001 16HBE were transfected into 16HBE, P = 0.002 A549 A549, and HepG2 cells. All P = 0.002 25.0 HepG2 constructs were co-transfected with pRL-SV40 to standardize the P < 0.001 P < 0.001 transfection efﬁciency. Fold P = 0.001 P = 0.001 increase of luciferase activity was measured by deﬁning the activity of 20.0 P = 0.006 P = 0.003 the empty pGL3-Basic vector as 1. Mean SD of relative luciferase activity (RLA) values were from 6 15.0 independent transfection experiments, each done in triplicate. Arbitary units 10.0

5.0

0.0 p-C-C p-C-T p-T-C p-T-T p-rs6457452C p-rs6457452T

test. We used c2 or the Student t tests to examine the HWE of were compared using one-way ANOVA, Student–Newman– SNPs, and the distributions of selected characteristics and Keul test, and Student t test. Two-sided P < 0.05 was genotypes between patients and controls. Multivariate logis- considered significant and the SPSS v.12.0 software (SPSS tic regression models were used to estimate the associations Inc.) was used for all analyses. between SNPs and lung cancer risk after adjustment for age, sex, and pack-years smoked. The interactions of age, sex, and Results smoking habits with SNP genotypes in the pooled case– control data were tested by using the Wald test in the Associations between tagSNPs of the HSPA1L–HSPA1A– multivariate logistic regression models. The associations HSPA1B cluster and lung cancer risk between overall survival time and demographic and clinical Distributions of selected characteristics for subjects in the characteristics were estimated using the Kaplan–Meier discovery and validation sets and fine-mapping analyses are method and log-rank test. The effect modifications by these listed in Table 1. As shown in Table 2, in the discovery set, characteristics and the effects of SNPs on death risk in compared with the HSPA1B rs6457452CC genotype, subjects patients with advanced NSCLC were assessed by using the with the rs6457452CT or combined rs6457452CT þ TT geno- Wald test in the multivariate Cox proportional hazards types had a 1.39-fold increased risk of lung cancer (P ¼ 0.013 regression models after adjusting for the confounders. The and 0.010, respectively). There was a dose–response effect of proportional hazards assumption was examined by testing the increasing number of the rs6457452T alleles on increased interactions between the genotypes and time (all P-values > lung cancer risk (Ptrend ¼ 0.026). However, no associations were 0.05). The statistical software packages R and QVALUE were found between rs2075800, rs2227956, rs1008438, rs1043618, used to calculate false discovery rate. Differences of RLA and rs2763979 and lung cancer risk (Table 2).

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Table 1. Distribution of selected characteristics among patients with lung cancer and controls in 3 panels of Chinese populations

Discovery set Validation set Fine-mapping

Lung cancer Lung cancer Lung cancer patients Controls patients Controls patients Controls (N ¼ 1,152) (N ¼ 1,152) (N ¼ 1,781) (N ¼ 1,038) (N ¼ 500) (N ¼ 500) Variable N (%) N (%) N (%) N (%) N (%) N (%)

Age, y 60 562 (48.8) 598 (51.9) 974 (54.7) 578 (55.7) 269 (53.8) 269 (53.8) >60 590 (51.2) 554 (48.1) 807 (45.3) 460 (44.3) 231 (46.2) 231 (46.2) Sex Male 924 (80.2) 951 (82.6) 1,288 (72.3) 721 (69.5) 386 (77.2) 386 (77.2) Female 228 (19.8) 201 (17.4) 493 (27.7) 317 (30.5) 114 (22.8) 114 (22.8) Smoking status Never 340 (29.5) 525 (45.6) 711 (39.9) 644 (62.0) 164 (32.8) 242 (48.4) Former smokers 343 (29.8) 129 (11.2) 399 (22.4) 100 (9.6) 161 (32.2) 60 (12.0) Current smokers 469 (40.7) 498 (43.2) 671 (37.7) 294 (28.3) 175 (35.0) 198 (39.6) Pack-years smoked 0 340 (29.5) 525 (45.6) 711 (39.9) 644 (62.0) 164 (32.8) 242 (48.4) 26 210 (18.2) 314 (27.3) 359 (20.2) 207 (19.9) 90 (18.0) 134 (26.8) >26 602 (52.3) 313 (27.2) 711 (39.9) 187 (18.0) 246 (49.2) 124 (24.8) Histologic type Adenocarcinoma 370 (32.1) 866 (48.6) 181 (36.2) Squamous cell carcinoma 344 (29.9) 678 (38.1) 145 (29.0) Small cell carcinoma 47 (4.1) 112 (6.3) 35 (7.0) Othersa 391 (33.9) 125 (7.0) 139 (27.8)

aOthers include large cell, bronchioalveolar, mixed cell, undifferentiated and pathologic not otherwise speciﬁed carcinomas.

In the validation set, logistic regression analysis indicated HSPA1L–HSPA1A–HSPA1B tagSNPs and survival of that compared with the HSPA1B rs6457452CC genotype, advanced NSCLC patients subjects with rs6457452CT or rs6457452CT þ TT genotypes The demographic and clinical characteristics of patients had a significantly increased risk of lung cancer (adjusted OR with lung cancer in the survival discovery and validation sets ¼ 1.34 and 1.32; P ¼ 0.009 and 0.012, respectively). Pooled are presented in Table 3. In the discovery set, in which the mean analysis of the discovery and validation sets showed that the age was 63.25 10.07 years, 257 (77.9%) patients died of lung adjusted ORs were 1.41 for the rs6457452CT genotype (P ¼ cancer, 89 (27.0%) received surgical operations, 255 (77.3%) 3.0 10 5), and 1.41 for the rs6457452CT þ TT genotype received chemotherapies, and 152 (46.1%) received radiothera- (P ¼ 2.8 10 5), compared with the rs6457452CC genotype, pies. In the validation set, in which the mean age was 59.65 respectively (Table 2). We did not observe a significant 9.57 years, 242 (73.1%) patients died of lung cancer, 152 (45.9%) interaction of age at diagnosis, sex, and smoking habits underwent surgical operations, 300 (90.6%) received che- with HSPA1B rs6457452CT þ TT genotypes on the risk of motherapies, and 123 (37.2%) received radiotherapies. The lung cancer in the pooled analysis (P ¼ 0.231, 0.982, and 5-year survival rates for the 2 sets were 8% and 10%, respec- 0.291, respectively). tively. The Kaplan–Meier analysis, log-rank test, and univariate Cox analysis revealed that younger patients, patients with Fine-mapping analysis on chromosome 6p21.3 earlier stage, or those who had surgical operations and che- A total of 28 tagSNPs at chr6: 31722081 to 31906010 were motherapy had a significantly longer median survival time successfully genotyped. The rs3115672 was not detected in (MST) and a decreased risk of death (P < 0.05 in Table 3). There any subject. The allele distributions of rs3130617 and were no significant effects of sex, smoking status, and rs805923 deviated from HWE among controls (P < 0.05), radiotherapy. and were excluded from subsequent analysis. Analysis of the In the discovery set, the Kaplan–Meier method and log-rank remaining 25 tagSNPs revealed that only the rs6457452T test showed that patients with the rs1008438AC or allele on HSPA1B 50-UTR was significantly associated with rs1008438CC genotype had a significantly shorter MST than an increased lung cancer risk (P ¼ 0.013; Supplementary patients with the rs1008438AA genotype (log-rank P ¼ 0.047 Table S2). and 0.009, respectively; Table 4). However, this SNP was not

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Table 2. Genotype frequencies of HSPA1L-HSPA1A-HSPA1B polymorphisms among patients and controls and their associations with risk of lung cancer in Chinese populations

Lung cancer Gene SNPs patients, n (%) Controls, n (%) OR (95% CI)a Pa

Discovery set N ¼ 1,152 N ¼ 1,152 HSPA1L (þ2763C>T) rs2075800 CC 464 (40.3) 440 (38.2) 1.00 (Reference) CT 524 (45.5) 550 (47.7) 0.87 (0.72–1.04) 0.131 TT 164 (14.2) 162 (14.1) 0.96 (0.73–1.25) 0.732 HSPA1L (þ2437A>G) rs2227956 AA 674 (58.5) 695 (60.3) 1.00 (Reference) AG 412 (35.8) 411 (35.7) 0.99 (0.83–1.19) 0.919 GG 66 (5.7) 46 (4.0) 1.49 (0.99–2.25) 0.058 AG þ GG 478 (41.5) 457 (39.7) 1.04 (0.87–1.24) 0.665 HSPA1A (110A>C) rs1008438 AA 420 (36.5) 403 (35.0) 1.00 (Reference) AC 530 (46.0) 570 (49.5) 0.92 (0.76–1.12) 0.413 CC 202 (17.5) 179 (15.5) 1.09 (0.84–1.40) 0.518 HSPA1A (þ190G>C) rs1043618 GG 589 (51.1) 564 (49.0) 1.00 (Reference) GC 457 (39.7) 486 (42.2) 0.96 (0.80–1.15) 0.669 CC 106 (9.2) 102 (8.9) 1.02 (0.75–1.40) 0.880 HSPA1B (912C>T) rs2763979 CC 601 (52.2) 611 (53.0) 1.00 (Reference) CT 457 (39.7) 457 (39.7) 1.00 (0.83–1.20) 0.976 TT 94 (8.2) 84 (7.3) 1.18 (0.85–1.64) 0.327 HSPA1B (þ39C>T) r6457452 CC 971 (84.3) 1,007 (87.4) 1.00 (Reference) CT 171 (14.8) 139 (12.1) 1.39 (1.07–1.79) 0.013 TT 10 (0.9) 6 (0.5) 1.46 (0.51–4.18) 0.480 CT þ TT 181 (15.7) 145 (12.6) 1.39 (1.08–1.78) 0.010 a Ptrend 0.026 Validation set N ¼ 1,781 N ¼ 1,038 HSPA1B (þ39C>T) rs6457452 CC 1,435 (80.6) 879 (84.7) 1.00 (Reference) CT 330 (18.5) 151 (14.5) 1.34 (1.08–1.66) 0.009 TT 16 (0.9) 8 (0.8) 1.01 (0.42–2.44) 0.977 CT þ TT 346 (19.4) 159 (15.3) 1.32 (1.06–1.63) 0.012 a Ptrend 0.018 Pooled analysis N ¼ 2,933 N ¼ 2,190 HSPA1B (þ39C>T) rs6457452 CC 2,406 (82.0) 1,886 (86.1) 1.00 (Reference) CT 501 (17.1) 290 (13.2) 1.41 (1.20–1.66) 3.0 105 TT 26 (0.9) 14 (0.6) 1.25 (0.64–2.44) 0.518 CT þ TT 527 (18.0) 304 (13.9) 1.41 (1.20–1.65) 2.8 105 a 5 Ptrend 6.0 10 aData were calculated by logistic regression, adjusted for age, sex, and pack-years smoked.

associated with death risk for patients with advanced NSCLC respectively) and a shorter MST for patients with advanced after adjustment for the confounders. Multivariate proportion- NSCLC (log-rank P ¼ 0.011 and 0.031, respectively); patients al hazards regression models and log-rank test revealed that, with the rs6457452CT þ TT genotypes had a 51% higher death when compared with the HSPA1B rs2763979CC genotype, the risk than those with the rs6457452CC genotype (P ¼ 0.032). rs2763979CT and rs2763979TT genotypes were associated with The HSPA1B rs2763979C>T and rs6457452C>Tpoly- poor survival (adjusted HR ¼ 1.43 and 1.80; P ¼ 0.008 and 0.013, morphisms were further tested in the validation set. In this

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Table 3. Demographic and clinical characteristics of patients with lung cancer in the survival discovery and validation sets

Discovery set (N ¼ 330) Validation set (N ¼ 331)

Lung cancer Deaths Lung cancer Deaths Variables patients, n (%) (n ¼ 257) patients, n (%) (n ¼ 242) MST,a mo Log-ranka P HR (95% CI)a,b

Age, y 65 172 (52.1) 127 228 (68.9) 163 17.9 1.00 (Reference) <0.001 >65 158 (47.9) 130 103 (31.1) 79 13.4 1.39 (1.16–1.66) Sex Male 274 (83.0) 213 247 (74.6) 179 15.3 1.00 (Reference) 0.222 Female 56 (17.0) 44 84 (25.4) 63 19.9 0.88 (0.71–1.08) Smoking Never 66 (20.0) 51 122 (36.9) 93 19.5 1.00 (Reference) Former smokers 160 (48.5) 125 32 (9.7) 24 14.2 0.073 1.29 (1.03–1.63) Current smokers 104 (31.5) 81 177 (53.5) 125 16.5 1.07 (0.86–1.32) Histology Adenocarcinoma 140 (42.4) 106 212 (64.0) 154 18.4 1.00 (Reference) SCC 93 (28.2) 70 98 (29.6) 72 16.5 <0.001 0.94 (0.76–1.15) Othersc 97 (29.4) 81 21 (6.3) 16 11.2 1.55 (1.23–1.96) Stage IIIA 76 (23.0) 49 140 (42.3) 101 19.6 1.00 (Reference) IIIB 85 (25.8) 64 72 (21.8) 46 18.0 <0.001 1.14 (0.89–1.46) IV 169 (51.2) 144 119 (36.0) 95 12.7 1.83 (1.49–2.25) Surgery No 241 (73.0) 188 179 (54.1) 143 13.4 1.00 (Reference) <0.001 Yes 89 (27.0) 69 152 (45.9) 99 21.0 0.58 (0.48–0.70) Chemotherapy No 75 (22.7) 58 31 (9.4) 26 10.0 1.00 (Reference) <0.001 Yes 255 (77.3) 199 300 (90.6) 216 17.8 0.63 (0.50–0.80) Radiotherapy No 178 (53.9) 135 208 (62.8) 153 14.2 1.00 (Reference) 0.053 Yes 152 (46.1) 122 123 (37.2) 89 18.6 0.84 (0.70–1.00)

Abbreviations: HR, hazard ratio; SCC, squamous cell carcinoma. aData are for the combined discovery and validation sets. bData were calculated by univariate Cox regression analysis. cOthers include large cell, bronchioalveolar, mixed cell, undifferentiated and pathologic not otherwise speciﬁed carcinomas.

data set, when compared with the rs2763979CC genotype, Luciferase reporter activity for HSPA1B rs2763979C>T patients with advanced NSCLC with the rs2763979TT geno- and rs6457452C>T type had a 1.61-fold higher death risk (P ¼ 0.036). No Among 4 constructed reporter plasmids with distinct hap- significant association was found on the rs6457452C>T lotypes, the RLAs driven by promoter containing rs2763979C/ polymorphism. Pooled analysis of the 2 cohorts showed rs6457452T, rs2763979T/rs6457452C, and rs2763979T/ that, when compared with the rs2763979CC genotype, the rs6457452T haplotype were significantly lower than that driven rs2763979CT or rs2763979TT genotype had a 1.21- or 1.66- by rs2763979C/rs6457452C haplotype (all P < 0.05); the RLA fold higher death risk (P ¼ 0.045 and 0.002, respectively), driven by promoter containing rs2763979T/rs6457452T hap- whereas the rs2763979TT genotype was associated with a lotype was significantly lower than that driven by rs2763979C/ significantly shorter MST (Table 4; Supplementary Fig. S3). rs6457452T or rs2763979T/rs6457452C haplotype (all P < We did not observe a significant interaction of age at 0.05; Fig. 2B); however, the RLA driven by promoter containing diagnosis, stage, surgical operations, and chemotherapy with the rs2763979C/rs6457452T or rs2763979T/rs6457452C haplo- HSPA1B 2763979CT þ TT genotypes on the death risk among type showed no significant difference (P > 0.05). In 2 reporter patients with advanced NSCLC in the pooled analysis plasmids containing the rs6457452C or rs6457452T allele of (P ¼ 0.302, 0.229, 0.971, and 0.574, respectively). HSPA1B rs6457452C>T polymorphism, the rs6457452C allele

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Table 4. Associations between HSPA1L–HSPA1A–HSPA1B genotypes and survival of patients with advanced NSCLC

Hazard ratio Lung cancer MST, Log-rank Gene SNPs (95% CI)a Pa patients Death mo P

Discovery set N ¼ 330 n ¼ 257 HSPA1L (þ2763C>T) rs2075800 CC 1.00 (Reference) 140 (42.4) 111 11.7 CT 0.79 (0.61–1.04) 0.093 149 (45.2) 113 15.0 0.124 TT 0.87 (0.57–1.31) 0.495 41 (12.4) 33 20.2 0.153 HSPA1L (þ2437A>G) rs2227956 AA 1.00 (Reference) 185 (56.1) 144 14.2 AG 1.08 (0.83–1.40) 0.586 117 (35.5) 94 12.9 0.676 GG 0.75 (0.46–1.23) 0.257 28 (8.5) 19 22.8 0.130 HSPA1A (110A>C) rs1008438 AA 1.00 (Reference) 118 (35.8) 84 18.0 AC 1.15 (0.86–1.54) 0.345 155 (47.0) 126 13.4 0.047 CC 1.37 (0.95–1.98) 0.092 57 (17.3) 47 11.2 0.009 HSPA1A (þ190G>C) rs1043618 GG 1.00 (Reference) 167 (50.6) 123 17.0 GC 1.13 (0.87–1.48) 0.363 139 (42.1) 115 13.3 0.013 CC 1.11 (0.67–1.84) 0.685 24 (7.3) 19 11.7 0.237 HSPA1B (912C>T) rs2763979 CC 1.00 (Reference) 183 (55.5) 132 16.5 CT 1.43 (1.10–1.87) 0.008 120 (36.4) 103 12.2 0.011 TT 1.80 (1.13–2.85) 0.013 27 (8.2) 22 12.7 0.031 HSPA1B (þ39C>T) rs6457452 CC 1.00 (Reference) 291 (88.2) 226 14.5 CT þ TT 1.51 (1.04–2.21) 0.032 39 (11.8) 31 10.7 0.025 Validation set HSPA1B (912C>T) rs2763979 N ¼ 325 n ¼ 239 CC 1.00 (Reference) 142 (43.7) 107 18.6 CT 1.00 (0.75–1.32) 0.982 150 (46.2) 106 16.8 0.948 TT 1.61 (1.03–2.52) 0.036 33 (10.2) 26 15.0 0.270 HSPA1B (þ39C>T) rs6457452 N ¼ 323 n ¼ 236 CC 1.00 (Reference) 256 (79.3) 192 17.3 CT þ TT 0.79 (0.56–1.10) 0.161 67 (20.7) 44 21.4 0.133 Pooled analysis HSPA1B (912C>T) rs2763979 N ¼ 655 n ¼ 496 CC 1.00 (Reference) 325 (49.6) 239 17.8 CT 1.21 (1.00–1.46) 0.045 270 (41.2) 209 15.0 0.128 TT 1.66 (1.21–2.27) 0.002 60 (9.2) 48 14.2 0.026

NOTE: The frequency of rs6457452TT genotype was 0.6% (2 of 322) and 2.2% (7 of 313) in the discovery and validation sets respectively; therefore, we combined rs6457452CT with rs6457452TT for analyses. aThe Cox regression analysis was adjusted for age, sex, pack-years smoked, histology, TNM stage, surgical operation, chemotherapy, and radiotherapy status.

was associated with significantly higher RLA than rs6457452T populations. In this study, we found that the HSPA1B allele in 3 cell lines (P < 0.05). rs6457452T allele was associated with an increased lung cancer risk in 2 independent lung cancer case–control studies com- Discussion prising 5,123 Chinese individuals. Fine-mapping analysis confirmed that only this SNP within the surrounding 185-kb at the To our knowledge, this study is the first to show that genetic previously identified chromosome 6p21.3 locus (chr6: variants of HSPA1B are associated with lung cancer suscepti- 31722081 to 31906010) was associated with lung cancer risk. bility and survival of patients with advanced NSCLC in Chinese In addition, we observed that the HSPA1B rs2763979TT

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genotype was associated with poor survival for patients with at different molecular and cellular levels is the underlying advanced NSCLC in 2 independent cohorts. Further analysis of mechanism for lung carcinogenesis (32), the increased DNA the biologic functions for rs2763979C>T and rs6457452C>Tby damage levels caused by reduced Hsp70 levels may enhance luciferase reporter assay indicated that the rs2763979T and the process for genetic instability and promote cancer rs6457452T alleles led to a significantly lower expression of development. Hsp70 in both normal bronchial epithelial and in malignant Cellular studies and animal models have extensively evalu- cancer cells. ated the functions of Hsp70 in the carcinogenesis process and Hung and colleagues reported an association between in cell proliferation and survival. For example, high Hsp70 rs4324798 at 6p21.3 and lung cancer risk (P ¼ 5.6 10 6; expression was correlated with poor prognosis of breast cancer ref. 5), but this association was not confirmed in a subsequent and endometrial cancer, but with better prognosis of esoph- meta-analysis (21). Wang and colleagues reported that 2 other ageal, pancreatic, and renal cancers (12). The contradictory SNPs at 6p21.3 were significantly associated with lung cancer prognostic implications of Hsp70 might be attributable to its risk (rs3117582 and rs3131379; ref. 3). However, these 3 SNPs do versatile properties. For example, elevated Hsp70 may confer not exist in Asian populations. In contrast, the published resistance to chemotherapy and apoptotic removal of tumor Japanese and Korean GWAS of lung adenocarcinoma did not cells; in addition, Hsp70 can participate in tumor immunoge- find 6p21.3 to be a susceptible locus, possibly because the nicity, because Hsp70 expressed on the surface of tumor cells sample size at the discovery stage was relatively small or can elicit tumor cell–specific immunologic responses through because this locus does not contribute to carcinogenesis for chaperoning and presentation of cancer-specific antigens (33, this specific histology (22, 23). Our fine-mapping analysis of 25 34). Tamura and colleagues found that immunization of mice tagSNPs from the block containing rs3117582 to HSPA1L- with lung carcinoma with cancer-derived Hsp70-peptide com- HSPA1A-HSPA1B cluster found that only the HSPA1B plex led to retarded progression of primary cancer, a reduced rs6457452C>T variant was associated with lung cancer risk. metastasis, and a prolongation of life span (35). In the present However, the associations with other SNPs in the region study, we hypothesize that the low Hsp70 expression levels investigated were not significant, which may be attributable driven by the rs2763979T alleles in lung cancer cells may to the limited sample size. Because the LD region of 6p21.3 contribute to a reduced the immunogenic response, thus is extensive and contains numerous transcripts, dissection conferring unfavorable survival rates for patients with NSCLC. and elucidation of the causal variant require additional Patients with NSCLC are often diagnosed at an advanced investigations with larger sample sizes and different ethnic stage and have a poor prognosis. Individual heterogeneity is a groups. major reason for distinct responses of treatments and out- The HSPA1B þ1267A/G (Gln351Gln, rs1061581) polymor- comes (36). Recently, using high-throughput expression and phism was reported to be associated with susceptibility to genetic microarray platforms, molecular epidemiologic and nasopharyngeal carcinoma (24), non-Hodgkin lymphoma (25), pharmacogenetic studies have been designed that aim to and breast carcinoma (25, 26). Data from the Environmental identify molecular and clinical predictors for outcomes of Genome Project showed that the HSPA1B rs1061581 was in LD patients with lung cancer with specific histology, TNM stage, with rs6457452 (D' ¼ 1.0). Because the rs1061581 is a silent or therapeutic strategy (37–39). Hsp70 has become one of the polymorphism, it is likely to be a surrogate marker for the most exciting anticancer molecular targets, and the Hsp70– rs6457452 in HSPA1B 50-UTR. In our study, the HSPA1B antigen complex has proven effective in the treatment of rs6457452T allele reduced the Hsp70 synthesis levels of nor- rodent lung cancer in preclinical studies (35) and is now mal-status cultured cells. Wang and colleagues previously undergoing clinical trials for treatment of human cancers found that the HSPA1A rs1043618C allele was associated with (40). Therefore, newly established molecular biomarkers as increased lung cancer risk in 159 patients with lung cancer and well as identifiable clinical and pathologic features in patients 202 controls in a Chinese population, but no significant will help pave the way for individualized prevention and association was found for HSPA1B rs1061581 and HSPA1L therapy for lung cancer. rs2227956 polymorphisms (27). Rusin and colleagues reported The present study has several strengths and limitations. that the 1541–1542delGT heterozygous genotype of the con- First, a total of 5,123 Chinese subjects, included in 2 indepen- stitutive heat shock cognate protein 70 (HSC70) gene was dent panels, and 661 patients with advanced NSCLC, included associated with a significantly decreased risk for lung cancer in 2 case-only survival cohorts, consistently confirmed the in 96 paired European patients with lung cancer and controls associations of HSPA1B rs6457452C>T and rs2763979C>T (28). However, the sample sizes for the above 2 studies were polymorphisms with lung cancer risk and survival respectively, small and the results require further validation. which provided an improved statistical power and decreased Polycyclic aryl hydrocarbons (PAH) are the main potent the probability of false positives. Second, the fine-mapping procarcinogens in tobacco smoke that can induce transfor- analysis provided additional confirmation for the potential mation of normal cells, causing malignancies in experimental causal variant of rs6457452T allele. Finally, our functional animals (29). Our previous studies showed that Hsp70 could studies offered a biologically plausible explanation for the protect human bronchial epithelial cells against DNA damage epidemiologic findings. However, the fine-mapping analysis caused by benzo[a]pyrene (30), and could also protect DNA carried out in our study was not suf ficiently extensive. A future, from genotoxic damage introduced by PAHs contained in more well-designed, and comprehensive fine-mapping analysis coke-oven emissions (31). Because accumulation of damage on a longer region surrounding the real hit of 6p21.3, which

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contains a large number of SNPs, is needed to investigate the Acknowledgments real causal variant. Moreover, because all subjects enrolled in this study were ethnic Han Chinese, the associations of these The authors thank all volunteers and medical assistants from cooperating hospitals. They also appreciate the scientific editing of Prof. H. Neumann, SNPs with lung cancer risk and survival in other ethnic groups University of Connecticut, and the helpful comments by Prof. F. Hu, Harvard also needs further validation by larger prospective studies. School of Public Health, and Prof. Q. Wei, University of Texas M.D. Anderson In conclusion, this study provided evidence, for the first Cancer Center. time, that the HSPA1B 50-UTR rs6457452T allele may contrib- Grant Support ute to an increased lung cancer risk, whereas the HSPA1B promoter rs2763979TT genotype may confer poor survival This study was financially supported by the National Basic Research Program outcomes for patients with advanced NSCLC in Chinese popu- grant 2011CB503800, the National High Technology Project grant 2009AA022705, fi lations. Extensive functional evaluations and additional pop- and the National Natural Scienti c Foundation of China grants 30872092 and 30525031 to T. Wu. ulation-based prospective studies with different ethnic groups The costs of publication of this article were defrayed in part by the and well-designed clinical investigations are warranted to payment of page charges. This article must therefore be hereby marked fi fi advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this con rm and extend our ndings. fact. Disclosure of Potential Conflicts of Interest Received April 28, 2011; revised September 26, 2011; accepted October 22, 2011; No potential conflicts of interest were disclosed. published OnlineFirst October 28, 2011.

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Variations in HSPA1B at 6p21.3 Are Associated with Lung Cancer Risk and Prognosis in Chinese Populations

Huan Guo, Qifei Deng, Chen Wu, et al.

Cancer Res Published OnlineFirst October 28, 2011.

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