Human leukocyte antigen-G (HLA-G) expression in cervical cancer lesions is associated with disease progression

Human Immunology 73 (2012) 946–949

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Human leukocyte antigen-G (HLA-G) expression in cervical cancer lesions is associated with disease progression Xiang-Juan Li a,b, Xia Zhang c, Aifen Lin c, Yan-Yun Ruan c, Wei-Hua Yan d,⇑ a

Department of Obstetrics and Gynecology, Huangyan Hospital, Wenzhou Medical College, Huangyan, Zhejiang, China Department of Obstetrics and Gynecology, Taizhou First People’s Hospital, Huangyan, Zhejiang, China c Human Tissue Bank, Taizhou Hospital of Zhejiang Province, Wenzhou Medical College, Linhai, Zhejiang, China d Medical Research Center, Taizhou Hospital of Zhejiang Province, Wenzhou Medical College, Linhai, Zhejiang, China b

a r t i c l e

i n f o

Article history: Received 5 March 2012 Accepted 11 July 2012 Available online 20 July 2012

a b s t r a c t The immunotolerant human leukocyte antigen (HLA)-G has direct inhibitory effects on natural killer cells, dendritic cells, T cells and can indirectly induce tolerant regulatory cells. The significance of the aberrant HLA-G expression in malignant contexts has been intensively investigated. In the current study, HLA-G expression in 22 normal cervical tissues, 14 cervical intraepithelial neoplasia (CIN) patients and 129 patients with squamous cell cervical cancer were examined using immunohistochemistry. The association of HLA-G expression with disease progression was calculated with the Pearson Chi-square test. It was found that HLA-G expression was absent in normal cervical tissues, and that HLA-G expression was increased from patients with CIN III (35.7%, 4/14) to patients with cervical cancer (62.8%, 81/129). Among the cervical cancer patients, HLA-G expression in FIGO stage I, II, and stage III+IV was 53.6% (45/84), 76.3% (29/38), and 100.0% (7/7), respectively. Taken together, our findings indicated that HLAG expression was associated with the disease progression in patients with cervical cancer. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Cervical cancer accounts for almost 12% of all cancer cases in women and represent the second most frequent gynecological malignancy in the world [1]. Alteration of human leukocyte antigen (HLA) class I antigen expression is one of the mechanisms most frequently used by tumor cells to escape host anti-tumor immune response [2]. HLA-G, a non-classical HLA class I antigen, its expression in malignancies has been intensively investigated, and the expression and clinical relevance of HLA-G in various malignancies have been addressed [3]. HLA-G has inhibitory effect on immune competent cell responses and induces the generation of suppressor or regulatory cells, providing a potent arm for the malignant cell escaping from host immunosurveillance and attack [4]. In malignancies, aberrant HLA-G expression is preferentially detected in tumor lesions but rare in adjacent non-tumorous tissues, and its expression was associated with disease poor prognosis, raising the hypothesis that HLA-G could contribute to malignant cell immune evasion and tumor progression [5]. Indeed, HLA-G expression could arm the cancer cells with an additional mechanism to overcome the vigilant immune surveil⇑ Corresponding author. E-mail address: [email protected] (W.-H. Yan).

lance and attack by binding to the receptors expression on immune competent cells such as NK cells, both CD4+ and CD8+ T lymphocytes and macrophages [6]. Three HLA-G binding inhibitory receptors have been identified including ILT2/CD85j/LILRB1 (ILT2), ILT4/ CD85d/LILRB2 (ILT4), and KIR2DL4/CD158d (KIR2DL4) [7]. With these receptors, HLA-G could exert immunosuppressive properties including the (a) inhibition of cytotoxic activity of CTL and NK cells, (b) inhibition of allogeneic CD4+ T cell proliferation and naive CD4+ T cell priming, (c) induction of activated CD8+ T cell and CD8+ NK cell apoptosis, (d) induction of tolerogenic dendritic cells associated with inhibition of their differentiation, (e) with the mechanism of trogocytosis, a cell-to-cell contact dependent acquisition of membranes and associated molecules by a cell from another, by acquiring HLA-G from their targets, the functions of activated NK or T cells could switched from cytotoxic effector to regulatory effector [8]. Recently, the significance of HLA-G expression in tumor immunology was strengthened with animal models. A recent study provided the first in vivo data that interaction between HLA-G and the murine receptor PIR-B could expand blood myeloid-derived CD11b(+) Gr1(+) PIR-B(+) suppressor cells, while reduce peripheral T cells and alter cytokine balance toward Th2 [9]. In line with this observation, our study provided the evidence that HLA-G expression is associated with tumor metastasis and with poor survival in an animal model with ovarian cancer [10].

0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.07.041

X.-J. Li et al. / Human Immunology 73 (2012) 946–949

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In this study, HLA-G expression in adjacent tumor-negative cervical tissue, cervical intraepithelial neoplasia (CIN) patients and in patients with squamous cell cervical cancer was analyzed by immunohistochemistry, and its correlation to clinical parameters was evaluated.

T2b, and the remaining 5.4% (7 of 129) were in T3 and T4. All tissue specimens underwent a microscopic confirmation for pathological features prior to their inclusion in this study. The study was approved and monitored by the ethics committee of the Huangyan hospital.

2. Materials and methods

2.2. Immunohistochemistry

2.1. Tissue samples

Four-micrometer thick sections of the paraffin-embedded tissue blocks were cut and mounted on polylysine coated slides. They were dewaxed in xylene and rehydrated through a graded series of ethanol. After de-paraffinization, antigen retrieval treatment was performed at 120 °C for 5 min in a 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by using a 3% hydrogen peroxide solution at room temperature for 15 min. Then, anti-HLA-G mAb 4H84 (1:500, Exbio, Prague, Czech Republic) was applied and incubated overnight at 4 °C. After that, a thorough washing in a 0.01 M phosphate-buffered saline (PBS) solution was performed. Subsequently, binding sites of the primary antibody were visualized using a Dako EnVison kit (Dako, Glostrup, Denmark) according to the manufacturer’s instructions. Finally, sections were counterstained with hematoxylin and mounted with glycerol gelatin. HLA-G staining in samples was determined by three pathologists. The pathologists were blinded to any clinical details related to the patients. Membrane or combined membrane and cytoplasmic expression of HLA-G were interpreted as positive.

The study participants were recruited between June 2005 and December 2010 from the Department of Pathology at Huangyan Hospital affiliated to Wenzhou Medical College, Taizhou, Zhejiang, China. The study participants included 14 patients with CIN III (median age: 50 years; range: 29–74 years) and 129 patients with squamous cell cervical cancer (median age: 48 years; range: 22– 86 years). Furthermore, 32 case-matched adjacent tumor-negative cervical tissue samples were obtained from these cervical cancer patients (median age: 45 years; range: 22–82 years). Samples were formalin fixed and embedded in paraffin. None of the patients had received therapy before tissue collection. The clinicopathological findings for cervical cancer were determined according to the FIGO (Federation International of Gynecology and Obstetrics) classification system [11]. Of the 129 patients, 17.1% (22 of 129) were classified as T1a, 40.3% (52 of 129) were shown to be in T1b, 7.0% (9 of 129) were classified as T2a, 22.5% (29 of 129) were shown to be in

Fig. 1. Immunohistochemistry analysis of HLA-G expression in normal cervical tissues and cervical cancer lesions. (A) negative HLA-G staining in normal cervical tissues; (B) negative HLA-G staining in cervical cancer lesions; (C, D, E, F) positive HLA-G staining in cervical cancer lesions (1+, 2+, 3+ and 4+), respectively. HLA-G mAb 4H84 (1:500) was used to detect the HLA-G expression. Original magnification: 100.

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Cytotrophoblast from first trimester human placenta served as a HLA-G positive control and negative controls were achieved by including isotype matched IgG1 (1:500, Exbio, Prague, Czech Republic) in each of the immunostaining. 2.3. Staining evaluation Membrane or/and cytoplasmic expression of HLA-G were interpreted as positive. Percentage of HLA-G positive tumor cells was determined by each observer, and the average of three scores was calculated. At least 500 tumor cells were scored. Percentage of HLA-G expression was graded as negative for the less than 5% of HLA-G staining, and the positive HLA-G staining was grouped as 1+ (6–25%), 2+ (26–50%), 3+ (51–75%), and 4+ (>75%), respectively. The percentage of positive cells was assigned a value based on the presence or absence of HLA-G staining, irrespective of staining intensity. 2.4. Statistical analysis Statistical analysis was performed with SPSS 13.0 software (SPSS, Inc., Chicago, IL). Correlations between the degree of staining and clinical parameters were calculated with Pearson chi-square test. A p < 0.05 was considered to be significant. 3. Results 3.1. Immunostaining of HLA-G protein in cervical lesion tissues Heterogeneous staining was noted in CIN and cervical cancer samples. In the CIN and cervical cancer lesions, the intensity of HLA-G staining varied from tumor to tumor and from one area to another within the same tumor. Some tumors showed focal patchy positive staining, and others displayed uniform staining pattern in tumor nests. Positive staining was observed in both the cell membrane and the sub-membranous cytoplasm region (Fig. 1). The cytotrophoblasts were used as internal positive control for HLA-G expression (data not shown). Among CIN patients, 45.7% (5/14) of the lesions were determined as HLA-G positive. In cervical cancer samples, 62.8% (81/ 129) of the lesions were HLA-G positive. No staining was detected in corresponding adjacent non-tumorous cervival tissues. When compared, the expression of HLA-G in cervical lesions is significantly higher than that in adjacent normal tissues (p < 0.01). (Table 1).

Table 1 Association of lesion HLA-G expression with clinicopathological parameters. Variables

No. of cases

p*

HLA-G expression Neg. (<5%)

1+

2+

3+

4+

32 14 129

32 (100%) 9 (64.3%) 48 (37.2%)

0 0 6

0 1 14

0 2 9

0 2 52

0.000

CC patient age 6Median (48 years) >Median (48 years)

65 64

22 (33.8%) 26 (40.6%)

3 3

8 6

4 5

28 24

0.905

Figo grade CIN III Ia+Ib IIa+IIb III+IV

14 84 38 7

9 (64.3%) 39 (46.4%) 9 (23.7%) 0 (0.0%)

0 3 3 0

1 9 4 1

2 6 3 0

2 27 19 6

0.020

Tissue type Normal tissue CIN CC

CIN: cervical intraepithelial neoplasia; CC: cervical cancer. Comparison of HLA-G expression status between or among each variable using the Pearson chi-square test.

*

3.2. Association of HLA-G expression with disease progression As shown in Table 1, HLA-G was negative in normal cervical tissues, and was increased from patients with CIN III (35.7%, 4/14) to patients with cervical cancer (62.8%, 81/129). Among the cervical cancer patients, HLA-G expression in FIGO stage I, II, and stage III+IV was 53.6% (45/84), 76.3% (29/38) and 100.0% (7/7), respectively (p = 0.02, Table 1).

4. Discussion Since Paul et al. [12] reported for the first time that HLA-G was expressed in solid tumor lesions, HLA-G expression and its relevance in more than thirty types of malignancies have been investigated. Given the fact that HLA-G is a potent immunotolerant which could inhibit immune component cell functions and induce the generation of various regulatory cells, HLA-G expression with poor prognosis was observed in patients with esophagus cancer, lung cancer, ovarian cancer, etc [13]. Previous studies revealed that HLA-G is selectively expressed in advanced-stage ovarian cancer with high-grade histology, and that HLA-G expression in ovarian cancer was associated with poor survival [14,15]. Clinical relevance was also observed in breast cancer, where lesion HLA-G expression was more frequently observed in advanced disease stage and tumor grade [16]. In hepatocellular carcinoma, HLA-G expression was found to be strongly correlated to advanced disease stage, and more frequently observed in elder patients [17]. Similar correlation was also observed in non-small cell lung cancer [18,19], gastric carcinomas [20], colorectal cancer [21] and esophageal squamous cell carcinoma patients [22,23] that lesion HLA-G expression was strongly correlated to disease stage and poor prognosis and that HLA-G was an independent prognostic factor. In this study, we demonstrated HLA-G expression increased progressively from pre-malignant to malignant cervical lesions. A study by Dong et al. [24] reported that HLA-G expression (mAb HGY) was observed in all tissue sections, and HLA-G expression increasingly progressed from patients with CIN I to CIN II/III and was found highest in patients with cervical cancer. Furthermore, HLA-G expression was significantly higher in CIN and cancer patients with HPV 16/18 than in CIN patients without HPV infection. These results correlate with the reports that both HLA-G mRNA and protein expression in cervical cancer tissues was significantly greater than normal controls and an inverse relationship was observed between FIGO stage and HLA-G mRNA expression, and that HLA-G polymorphism such as homozygous genotypes HLAG⁄01:01:02, –G⁄01:06 and the 14 bp insertion polymorphism in the 30 untranslated region of the HLA-G gene is an independent risk factor for the development of invasive cervical cancer [25,26], supporting a probable role for HLA-G in cervical carcinogenesis. A study by Zheng et al. [27] reported that positive HLA-G expression (mAb 4H84) was observed in 52.8% (19/36) CIN III and 57.5% (23/40) cervical cancer patients, respectively. The percentage of HLA-G positive cervical cancer samples is similar to that of our study, though the staining evaluation method used was different. In that study, they considered the negative as (0–25%), while in our study the negative staining was considered as less than 5%. If we used the staining evaluation method in that study (0–25% as negative), HLA-G expression in cervical cancer samples was 58.1% (75/129). Using the specific mAb 5A6G7 for the HLAG5 isoform, Guimarães et al. [28] found that HLA-G5 molecules were detected in 31.6% (25/79) patients and no HLA-G5 staining was observed in normal cervical specimens. These studies showed that HLA-G expression is negative in normal cervical tissues. Though no normal cervical tissues were included, Rodríguez et al.

X.-J. Li et al. / Human Immunology 73 (2012) 946–949

[29] found that HLA-G protein expression (mAb 4H84) was observed in 27.6% (16/58) of cases. To the contrary, a study by Zhou et al. [30] addressed that a strong and uniform HLA-G expression (mAb MEM-G/1) in normal epithelium, while only a small proportion of CINs and cervical squamous cell carcinoma samples showed reduced expression of HLA-G. The controversial result of the HLA-G expression in normal cervical tissues needs further investigated. In summary, we demonstrated HLA-G expression increased progressively from pre-malignant to malignant cervical lesions. Given its immunotolerant properties, our finding suggested that HLA-G expression induced in cervical cancer cells might be an additional mechanism for tumor cells evading from host immunosurveillance. However, its clinical relevance in cervical cancer needs to be further explored. Acknowledgments This work was supported by grants from National Natural Science Foundation of China (31170879, 81102218), Zhejiang Provincial Natural Science Foundation of China (Y2101323) and by Zhejiang Provincial program for the cultivation of high-level innovative health talents.

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