Different Impact of IL10 Haplotype on Prognosis in Lung Squamous Cell Carcinoma and Adenocarcinoma

Abstract.

Background: Polymorphisms (1082A>G,

-819C>T, and -592G>A) in the interleukin-10 (IL10)

promoter are associated with its transcriptional activity. IL10

induction by cigarette smoking plays a role in smokingrelated

lung tumor progression. We therefore expected to find

a difference in impact of IL10 haplotypes on overall survival

(OS) and relapse-free survival (RFS) between squamous cell

carcinomas (SCC) and adenocarcinomas (ADC) of lung.

Materials and Methods: Normal lung tissues adjacent to

resected tumors from 439 lung cancer patients were collected

to determine IL10 haplotypes (ATA and non-ATA) by direct

sequencing and polymerase chain reaction-restriction

fragment length polymorphism (PCR-RFLP). Kaplan-Meier

and multivariate Cox regression models were used to assess

the impact of IL10 haplotype on OS and RFS. Resluts: The

non-ATA haplotype was more prevalent in patients with

nodal metastatic tumors (N1 and N2) than in those with nonnodal

metastatic tumors (N0). This observation was only

made for patients with SCC and not ADC. Patients with SCC

with the non-ATA haplotype had poorer OS and RFS when

compared to those with the ATA haplotype, whereas IL10

haplotype was not associated with the clinical outcome of

patients with ADC. Conclusion: The IL10 haplotype may

independently predict survival and relapse in patients with

surgically resected SCC, but not ADC.

interleukin-10 (IL10) promoter are associated with its

transcriptional activity (1, 2). The involvement of IL10

expression in cancer development is explained by its blockage

of tumor immune surveillance via suppressed T-cell immunity

(3-7). Several reports have indicated that IL-10 promoter

polymorphisms are associated with various cancer risks

including lung cancer (8-15). Further studies showed that

IL-10 promoter polymorphisms were correlated with tumor

progression and clinical outcome in a number of cancers, such

as diffuse large B-cell lymphoma, non-Hodgkin’s lymphoma,

advanced melanoma, breast cancer, skin squamous cell

carcinoma after renal transplantation, and NSCLC (16-20).

Non-small cell lung cancer (NSCLC) is categorized into

two major histological types: squamous cell carcinoma

(SCC) and adenocarcinoma (ADC). SCC is predominantly

associated with cigarette smoking, and induction of IL10

production has been reported in response to cigarette smoke

extracts and nicotine-derived nitrosamine ketone (NNK), a

major carcinogenic compound of cigarette smoke extract

(21-25). Recently, we reported that IL10 mRNA levels were

significantly higher in lung tumors with the non-ATA IL10

haplotype than with the ATA IL10 haplotype (26). Therefore,

we further hypothesized that IL10 haplotype might play a

more important role in tumor progression in cases of SCC

than in ADC. To this end, we determined the IL10 promoter

polymorphisms at -1082A>G, -819C>T, and -592G>A in

439 patients with NSCLC. We analyzed normal lung tissues

obtained from areas adjacent to tumors in 245 patients with

SCC and 194 with ADC by direct sequencing and

polymerase chain reaction-restriction fragment length

polymorphism (PCR-RFLP) in order to verify whether IL10

*These Authors contributed equally to this work, as did these Authors≠.

Correspondence to: Professor Huei Lee, Ph.D., Graduate Institute

of Cancer Biology and Drug Discovery, Taipei Medical University, Room 5, 12F, No. 3, Park Street, Nankang District 115, Taipei, Taiwan, R.O.C. Tel: +886 227361661ext.7616, e-mail:

[email protected]

Key Words: IL10, haplotype, prognosis, squamous cell carcinoma,

adenocarcinoma, lung cancer.

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Different Impact of IL10 Haplotype on

Prognosis in Lung Squamous Cell

Carcinoma and Adenocarcinoma

YAW-CHENG WANG

, WEN-WEI SUNG

, LEE WANG

, YA-WEN CHENG

,

CHIH-YI CHEN

, TZU-CHIN WU

, SHWN-HUEY SHIEH

and HUEI LEE

1

Institute of Medicine,

School of Medicine and

Department of Public

Health Chung Shan Medical University, Taichung, Taiwan, R.O.C.;

4

Department of Internal Medicine, Chung Shan Medical University Hospital,

Taichung, Taiwan, R.O.C.;

R.O.C.;

6

Department of Health Services Management, China Medical University and

Hospital, Taichung, Taiwan, R.O.C.;

7

Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical

University, Taipei, Taiwan, R.O.C.

0250-7005/2013 $2.00+.40

promoter polymorphisms could have a different impact on

overall survival (OS) and relapse-free survival (RFS)

between patients with SCC and with ADC.

Materials and Methods

Patients. This study included 439 patients with NSCLC. All patients

were unrelated ethnic Chinese and residents of central Taiwan. Patients had been diagnosed with ADC (194; 44.2%) or SCC (245; 55.8%) and underwent surgical resection at the Department of Thoracic Surgery, Taichung Veterans General Hospital, between 1993 and 2004. Samples were immediately frozen at surgery and kept at −80˚C 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 (86 patients had no relapse data) and the Taiwan Cancer Registry, Department of Health, Executive Yuan, ROC.

Genomic DNA extraction. Genomic DNA was extracted by

conventional methods. Surgically resected normal tissues adjacent to the lung tumor were prepared by proteinase K digestion and

phenol-chloroform extraction, followed by ethanol precipitation, as described previously (27).

PCR-RFLP analysis for IL10 -592C/A genetic polymorphism.

Genotypes of IL10 -592C/A were determined by PCR-RFLP as described by Rad et al. (28). PCR amplification products from 50 samples were randomly selected for direct sequencing to confirm the genotype analysis by PCR-RFLP.

Direct sequencing for IL10 -1082A/G and -819C/T genetic polymorphisms. Polymorphisms of IL10 -1082G/A and -819C/T were determined by direct sequencing of PCR products amplified from the DNA of normal tissues adjacent to the tumors. Primers used for DNA amplification and direct sequencing were: 5’-ATCCAAGACAACACTACTAA-3’ (forward) and 5’-TAAATAT CCTCAAAGTTCC-3’ (reverse). The PCR cycle conditions

consisted of an initial denature step at 94˚C for 10 min followed by 35 cycles of 30 s at 94˚C; 45 s at 56˚C; 45 s at 72˚C; and a final elongation at 72˚C for 10 min. The PCR products were sequenced using an Applied Biosystems 3100 Avant Genetic Analyzer (Applied Biosystems, California, USA).

Statistical analysis. Student’s t-test and Chi-square test were applied

for continuous or discrete data analysis. The associations between

IL10 promoter polymorphisms and patient survival were estimated

using the Kaplan Meier method and assessed using the log-rank test. Potential confounders were adjusted by Cox regression models, with

IL10 promoter polymorphisms fitted as indicator variables. All

software program (version 13.0) (SPSS, Inc., Chicago, IL, USA).

All statistical testing was conducted using two-sided tests and pvalues <0.05 were considered to be statistically significant.

Results

The non-ATA haplotype in NSCLC is more prevalent in nodal

metastatic tumors than in non-nodal metastatic tumors. As

previously described, two IL10 haplotypes (ATA and

non-ATA) were categorized by three IL10 promoter

polymorphisms (-1082A>G, -819C>T, and -592G>A). Our

previous study has indicated that patients with non-ATA

haplotype had higher IL10 mRNA expression levels than

those with ATA haplotype (26). The association of IL10

haplotype with clinical parameters is shown in Table I.

Patients with nodal metastatic tumors more frequently had

the non-ATA haplotype when compared to patients with nonnodal

metastatic tumors (N0) (59.7% vs. 48.4%, p=0.017).

The IL10 haplotypes were not associated with other clinical

parameters including age, gender, smoking status, stage, T

classification, and tumor histology (Table I). When stratified

by tumor histology, having a non-ATA IL10 haplotype was

not only associated with nodal metastasis but also correlated

with tumor stage in SCC (63.0% vs. 40.5%, p<0.001 for

nodal metastasis; 59.2% vs. 46.3%, p=0.047; Table II);

however, no association was observed with ADC. These

results suggest that non-ATA haplotypes might be associated

with the clinical outcome in patients with SCC.

Non-ATA haplotypes may be used as a biomarker for poor

prognosis in surgically resected NSCLC. IL10 expression has

been shown to promote tumor progression via suppressed

tumor immune surveillance (3, 5, 6). We therefore expected

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Table I. Correlations between interleukin-10 (IL10) haplotype and

clinical characteristics in 439 patients with non-small cell lung cancer.

IL-10 haplotype

Parameter No. cases ATA Non ATA p-Valuea Age, year <65 182 83 (45.6) 99 (54.4) 0.949 ≥65 257 118 (45.9) 139 (54.1) Gender Female 117 58 (49.6) 59 (50.4) 0.337 Male 322 143 (44.4) 179 (55.6) Smoking status Non-smoker 200 97 (48.5) 103 (51.5) 0.296 Smoker 239 104 (43.5) 135 (56.5) Stage Ι and Ⅱ 249 119 (47.8) 130 (52.2) 0.334 Ⅲ 190 82 (43.2) 108 (56.8) T Classification 1 and 2 331 149 (45.0) 182 (55.5) 0.570 3 and 4 108 52 (48.1) 56 (51.9) N Classification

0 213 110 (51.6) 103 (48.4) 0.017 1 and 2 226 91 (40.3) 135 (59.7) Tumor type

SCC 245 119 (48.6) 126 (51.4) 0.188 ADC 194 82 (42.3) 112 (57.7)

SCC, Squamous cell carcinoma; ADC, adenocarcinoma. aTwo sided Chi-square test.

that in patients with non-ATA haplotypes tumor, it might

show higher IL10 expression to explain its enhanced tumor

progression and consequent with poor patient outcome (1,

2). Kaplan-Meier and Cox regression analysis showed that

patients with the non-ATA haplotypes had poorer OS and

RFS when compared with patients with the ATA haplotype

in this study population [hazard ratio (HR)=1.522, 95%

confidence interval (CI)=1.191-1.945, p=0.001 for OS;

HR=1.611, 95% CI=1.247-2.082, p<0.001 for RFS; Table

III]. The median survival duration and 5-year survival rate of

patients with non-ATA haplotypes were significantly shorter

when compared to those of patients with the ATA haplotype

(median OS=25.8 vs. 42.9 months; median RFS=16.8 vs.

30.9 months; 5-year OS=28.6% vs. 44.7%; 5-year

RFS=22.2% vs. 36.2%). As expected, tumor stage had a

prognostic value for OS and RFS in this study population.

After adjusting for various parameters, including age, gender,

smoking status, stage, and tumor histology, IL10 haplotype

and tumor stage retained their prognostic value for OS and

RFS (Table III). These results were consistent with a recent

report indicating that IL10 haplotype may be a useful

biomarker for predicting survival and relapse of NSCLC.

Differential impacts of the IL10 haplotype on OS and RFS

between patients with SCC and those with ADC. Differences

in the prognostic value of IL10 haplotype as a predictor of

clinical outcome were analyzed by dividing the study

population into SCC and ADC groups. As shown in Table III,

patients with SCC with the non-ATA haplotype had poorer

OS and RFS than did patients with the ATA haplotype

(HR=1.727, 95% CI=1.227-2.429, p=0.002 for OS;

HR=2.108, 95% CI=1.440-3.087, p<0.001 for RFS).

However, no prognostic value of the IL10 haplotype was

observed for OS and RFS in patients with ADC (Table III).

As expected, the tumor stage was associated with prognosis

in both SCC and ADC. Multivariate Cox regression analysis

further showed that the IL10 haplotype could independently

predict OS and RFS in SCC, although not in ADC. These

results suggest that the IL10 haplotype has different impacts

on OS and RFS in patients with SCC from those with ADC.

Discussion

The IL10 haplotype has been associated with lung cancer

risk (9). Our recent repot showed an association of IL10

haplotype with survival and relapse in lung cancer. In the

current study, prognostic value of non-ATA IL10 haplotypes

were observed for SCC, but not for ADC. In addition,

non-ATA IL10 haplotypes were associated with nodal metastasis

and tumor stage in SCC. This finding seemed to be

consistent with previous reports indicating that serum IL10

levels were increased with advanced T classification, nodal

metastasis, and tumor stage in head and neck SCC

(HNSCC) (29-32).

Wang et al: IL10 Haplotypes Predict Prognosis in Lung Cancer 2731

Table II. Correlations between interleukin-10 (IL10) haplotype and clinical characteristics in non-small

cell lung cancer according to tumor type.

Squamous cell carcinoma Adenocarcinoma IL10 haplotype IL10 haplotype

Parameter No. cases ATA Non-ATA p-Value No. cases ATA Non-ATA p-Value Age, year <65 85 38 (44.7) 47 (55.3) 0.378 97 45 (46.4) 52 (53.6) 0.245 ≥65 160 81 (50.6) 79 (49.4) 97 37 (38.1) 60 (61.9) Gender Female 24 32 (53.3) 28 (46.7) 0.396 93 42 (45.2) 51 (54.8) 0.434 Male 221 87 (47.0) 98 (53.0) 101 40 (39.6) 61 (60.4) Smoking status Non-smoker 60 32 (53.3) 28 (46.7) 0.396 140 65 (46.4) 75 (53.6) 0.059 Smoker 185 87 (47.0) 98 (53.0) 54 17 (31.5) 37 (68.5) Stage I and II 147 79 (53.7) 68 (46.3) 0.047 102 40 (39.2) 62 (60.8) 0.365 III 98 40 (40.8) 58 (59.2) 92 42 (45.7) 50 (54.3) T Classification

1 and 2 170 83 (48.8) 87 (51.2) 0.905 161 66 (41.0) 95 (59.0) 0.427 3 and 4 75 36 (48.0) 39 (52.0) 33 16 (48.5) 17 (51.5)

N Classification

0 126 75 (59.5) 51 (40.5) <0.0001 87 35 (40.2) 52 (59.8) 0.604 1 and 2 119 44 (37.0) 75 (63.0) 107 47 (43.9) 60 (56.1)

Cigarette smoking plays a key role in the development of

both SCC and HNSCC (5, 21, 23, 33-36). The induction of

IL10 expression by cigarette smoking may have a partial

involvement in the progression of SCC and HNSCC,

especially in advanced stages (37, 38). Extensive studies

have shown an association of IL10 with tumor cell growth

and an inhibition of antitumor responses by IL10 through

modulation of the functions of antigen-presenting cells and

induction of regulatory T-cell generation (38-42). These

tumor microenvironmental findings may suggest an

underlying mechanism for the association between IL10

haplotype and NSCLC prognosis.

In Taiwan, the majority of smokers with lung cancer are

men, because only 4% of women are smokers. Among lung

cancer cases, over 70% of male smokers have SCC;

conversely, fewer than 30% of the female non-smokers have

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Table III. Univariate and multivariate analysis of the influence of interleukin-10 (IL10) haplotype on

overall survival (OS) and relapse-free survival

Parameter OS RFS

No. Median 5-Year HR 95% CI p-Value No. Median 5-Year HR 95% CI p-Value cases survival survival cases survival survival

(month) (%) (month) (%) All patients

IL10 haplotype

ATA 201 42.9 44.7 1.000 Referent 154 30.9 36.2 1.000 Referent

Non-ATA 238 25.8 28.6 1.522 1.191-1.945 0.001 199 16.8 22.2 1.611 1.247-2.082 <0.001 Stage

I and II 249 49.8 44.4 1.000 Referent 196 36.5 36.1 1.000 Referent

III 190 20.1 24.8 1.973 1.552-2.509 <0.001 157 12.6 18.8 1.979 1.541-2.541 <0.001 IL10 haplotype*

ATA 201 42.9 44.7 1.000 Referent 154 30.9 36.2 1.000 Referent 0.001

Non-ATA 238 25.8 28.6 1.405 1.097-1.799 0.007 199 16.8 22.2 1.556 1.200-2.018 Stage*

I and II 249 49.8 44.4 1.000 Referent 196 36.5 36.1 1.000 Referent <0.001 III 190 20.1 24.8 2.226 1.736-2.854 <0.001 157 12.6 18.8 2.205 1.704-2.851 SCC

IL10 haplotype

ATA 119 66.5 51.1 1.000 Referent 77 42.8 46.4 1.000 Referent

Non-ATA 126 26.1 30.3 1.727 1.227-2.429 0.002 97 15.1 23.6 2.108 1.440-3.087 <0.001 Stage

I and II 147 53.0 45.2 1.000 Referent 101 34.9 36.8 1.000 Referent

III 98 26.8 32.6 1.656 1.187-2.310 0.003 73 13.8 29.5 1.563 1.086-2.250 0.016 IL10 haplotype*

Non-ATA 126 26.1 30.3 1.665 1.177-2.357 0.004 97 15.1 23.6 2.039 1.390-2.992 <0.001 Stage*

I and II 147 53.0 45.2 1.000 Referent 101 34.9 36.8 1.000 Referent

III 98 26.8 32.6 1.747 1.239-2.462 0.001 73 13.8 29.5 1.636 1.125-2.379 0.010 ADC

IL10 haplotype

ATA 82 29.3 35.8 1.000 Referent 77 20.5 25.6 1.000 Referent

Non-ATA 112 23.7 26.4 1.243 0.873-1.768 0.227 102 17.3 20.7 1.197 0.846-1.695 0.309 Stage

I and II 102 42.2 43.0 1.000 Referent 95 38.4 35.4 1.000 Referent

III 92 17.2 15.6 2.533 1.775-3.615 <0.001 84 12.1 8.2 2.658 1.865-3.788 <0.001 IL10 haplotype*

ATA 82 29.3 35.8 1.000 Referent 77 20.5 25.6 1.000 Referent

Non-ATA 112 23.7 26.4 1.195 0.831-1.717 0.336 102 17.3 20.7 1.160 0.809-1.664 0.420 Stage*

I and II 102 42.2 43.0 1.000 Referent 95 38.4 35.4 1.000 Referent

III 92 17.2 15.6 3.051 2.100-4.432 <0.001 84 12.1 8.2 3.085 2.133-4.462 <0.001

*Adjusted with age, gander, smoking status and stage for multivariate analysis. HR: Hazard ratio; CI: confidence interval.

SCC (43-45). In the present study population, the distribution

of SCC and ADC, when stratified by gender, was similar to

that described in previous reports (46, 47). Gender

stratification revealed that the prognostic value of IL10

haplotype on OS and RFS was observed in male, but not in

female patients (Table IV). This finding further supports the

differential impact of IL10 haplotype on OS and RFS of

patients with SCC compared to those with ADC.

Many immune cell types are found in the tumor

microenvironment, including dendritic cells (DCs) and

macrophages in myeloid cells show constitutive activation of

signal transducer and activator of transcription 3 (STAT3),

which allows efficient cluster of differentiation 8 (CD8

) T

cell infiltration into tumors while inhibiting accumulation of

regulatory T-cells (42, 48, 49). Activated STAT3 suppresses

antitumor immunity by inhibiting the expression of many

cytokines and chemokines important for stimulating

antitumor immunity, and by up-regulating production of

several immunosuppressive factors, including IL10 (49). The

activation of the IL6/STAT3 pathway has been shown to play

a crucial role in the pathogenesis and progression of lung

cancer (48). In addition, IL6 and STAT3 were activated by

mainstream cigarette smoke in the lungs of subchronically

exposed mice (50). Previous reports indicated that IL6

expression levels were significantly higher in SCC cells than

in ADC cells (37). T-cells stimulated by DCs treated with

lung tumor cells showed a greater increase in IL10 secretion

when the DCs were SCC-DCs rather than ADC-DCs (37).

As expected, SCC-DCs were less effective stimulators of Tlymphocyte

proliferation when compared with ADC-DCs

(37). However, this phenomenon was diminished by

neutralization by IL6 (37). Therefore, we suggest that the

switch in monocyte differentiation from DCs to macrophagelike

cells promoted by SCC, but not by ADC, may occur

predominantly through the IL6/STAT3 pathway.

In summary, IL10 may more strongly influence tumor

progression in SCC than in ADC. The differential impacts of

the IL10 haplotype on the clinical outcome between SCC

and ADC observed in the current study seem to support this

possibility. However, the underlying mechanisms of IL10

expression in tumor progression of SCC need further

investigation.

Acknowledgements

This work was jointly supported by grants from the National Science Council (NSC99-2628-B-038-016-MY3 and NSC100-2314-B-038-043-MY3).

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Wang et al: IL10 Haplotypes Predict Prognosis in Lung Cancer 2733

Table IV. Multivariate analysis of the influence of interleukin-10 (IL10) haplotype on overall survival

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Received April 16, 2013

Revised May 2, 2013

Accepted May 9, 2013

Wang et al: IL10 Haplotypes Predict Prognosis in Lung Cancer 2735