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ORIGINAL ARTICLE
Year : 2018  |  Volume : 55  |  Issue : 4  |  Page : 340-343
 

Correlation between human leukocyte antigen-G expression and clinical parameters in oral squamous cell carcinoma


Department of Stomatology, Taizhou Hospital, Wenzhou Medical University, Linhai 317000, China

Date of Web Publication28-Feb-2019

Correspondence Address:
Gengsheng Shi
Department of Stomatology, Taizhou Hospital, Wenzhou Medical University, Linhai 317000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijc.IJC_602_17

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 » Abstract 


BACKGROUND: Human leukocyte antigen-G (HLA-G) is a tumor-associated molecule, whose expression may help the cancer cells to escape the immune response. AIMS: The aim of this study was to evaluate the diagnostic value of HLA-G level in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: A total of 52 patients who had definite pathological diagnosis and 20 cases of healthy controls were enrolled in this clinical trial. Immunohistochemisty (IHC) and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis were considered for HLA-G identification and multilevel validations. Statistical analysis was performed using SPSS and statistical significance was determined at P < 0.05. RESULTS: IHC results demonstrated that the expression of HLA-G in OSCC was strongly positive and the rate of positive expression was 55.77% (29/52), but the expression of HLA-G in healthy controls was negative (0/20). Furthermore, RT-PCR results showed that the positive expression rate of HLA-G messenger RNA was weak in healthy controls, but strong in OSCC. Besides, HLA-G expression in the tumors was significantly correlated with histological grade. CONCLUSIONS: Our results suggested that HLA-G is associated with the prognosis of OSCC and may serve as a novel therapeutic target.


Keywords: Clinical parameters, human leukocyte antigen-G, oral squamous cell carcinoma


How to cite this article:
Shen X, Wang P, Dai P, Jin B, Tong Y, Lin H, Shi G. Correlation between human leukocyte antigen-G expression and clinical parameters in oral squamous cell carcinoma. Indian J Cancer 2018;55:340-3

How to cite this URL:
Shen X, Wang P, Dai P, Jin B, Tong Y, Lin H, Shi G. Correlation between human leukocyte antigen-G expression and clinical parameters in oral squamous cell carcinoma. Indian J Cancer [serial online] 2018 [cited 2019 Mar 20];55:340-3. Available from: http://www.indianjcancer.com/text.asp?2018/55/4/340/253299





 » Introduction Top


Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck,[1] and the 5-year overall survival rate still remains 40–50% over a 40-year period.[2] Conventional oral examinations are the routine methods for the screening of oral lesions, but it is difficult to make a distinction among benign, premalignant, and malignant lesions. Although much progress has been made, the lack of high sensitivity biomarkers for OSCC diagnosis is still a serious public health problem.[3] Therefore, there is an urgent need to discover potential specific biomarkers and their utility for oral cancer detection.

Human leukocyte antigen-G (HLA-G), as a nonclassical HLA-I antigen molecule, was originally found in extravillous trophoblast cells of the placenta at the maternal–fetal interface and with immune suppression.[4] Subsequent studies had found that HLA-G acted as an immune tolerance molecule and its upregulation may promote tumor growth as well as impair antitumor immunity.[5],[6] It is widely accepted that expression of HLA-G is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction.[7] In general, tumor cells can secrete a variety of immunosuppressive molecules such as IL-10 and IFN-γ, which increase HLA-G gene expression. The upregulation of HLA-G in tumor cells can suppress alloreactive CD4+ T-cell proliferation, inhibit natural killer- or T-cell-mediated cytolysis, and impede maturation and function of dendritic cells.[8] Furthermore, numerous studies indicate that abnormal expression of HLA-G is found in malignant cancers, including colon, gastrointestinal, breast, and kidney.[7],[9],[10] However, the prognostic value of HLA-G in OSCC has not been defined clearly. Therefore, we investigate the correlation between HLA-G expression and disease progression in OSCC and examine whether the expression of HLA-G can be used as a reliable prognostic immune indicator for patients with OSCC.


 » Materials and Methods Top


Patients and samples

Fifty-two OSCC patients including 28 males and 24 females underwent operative treatment and were enrolled between January 2012 and January 2016 at Taizhou Hospital in Linhai, Zhejiang, Republic of China. At the same time, 20 normal patients without tumors were used as a matched group. None of the patients had received therapy before tissue collection. Patients with OSCC were clinically staged at the time of diagnosis based on the tumor, node, and metastasis (TNM) and histological-grade stages. Medical information of the OSCC patients, including TNM clinical staging, lymph node involvement, and histological grade, was obtained from their medical records. In accordance with the TNM Classification of Malignant Tumors, patients were classified into stages I (n = 13), II (n = 11), III (n = 7), and IV (n = 21). Meanwhile, based on the World Health Organization Classification of Tumors, patients were divided into grade I (well differentiated, n = 24), II (moderately differentiated, n = 25), and III (poorly differentiated, n = 3). All tissue specimens were fixed in 10% formalin and embedded in paraffin. The fresh tissue specimens were stored in liquid nitrogen until use.

Immunohistochemistry

For immunohistochemistry (IHC) analysis, sections of 4-μm thickness were cut. Then, the tissue sections were mounted on polylysine-coated slides, dewaxed in xylene, and rehydrated using a graded ethanol series, before being washed with phosphate-buffered saline (PBS). Deparaffinized tissue sections were subjected to antigen retrieval treatment at 100°C for 10 min in citrate sodium buffer (pH 6.1) and then brought to room temperature for cooling. The sections were then blocked with 10% normal goat serum (Invitrogen, Carlsbad, CA, USA) for 20 min and incubated with primary anti-mouse HLA-G antibody (1:200) (Exbio, Prague, Czech Republic) overnight. Immunostained sites were visualized by using a DAKO EnVision kit (DAKO, Glostrup, Denmark), in accordance with the manufacturer's instructions. Normal tissues were used as negative controls. Trophoblast tissue after abortion at 6-week pregnancy was employed as a HLA-G-positive control, whereas matched sections that were treated with PBS instead of primary antibody were used as blank control.

Analyzing tissue staining

When evaluating the HLA-G-positive staining results, ten random fields were observed. The staining was determined by the occurrence of reddish brown particles in the cell cytoplasm and membrane of the stained OSCC or normal tissues. The staining of tissues was quantified based on staining intensity and percentage of the cells stained. Finally, the staining results were categorized into four grades: 0 if 0–5% of cells stained positive, 1 if 5–25% of cells were positive, 2 if 25–50% were positive, or 3 if >50% of cells were positive. The intensity was scored as 0 for negative staining, 1 for weak intensity (light yellow), 2 for moderate intensity (light brown), or 3 for strong staining (brown). Accordingly, the 0 groups were assigned as HLA-G negative and the 1–3 groups as HLA-G positive.

Real-time reverse transcription-polymerase chain reaction

For messenger RNA (mRNA) expression analysis, tissues were harvested, snap-frozen in liquid nitrogen, and kept at 80°C until use. Total RNA was extracted from normal or OSCC tissues using Tirol Reagent (Invitrogen Life Technologies, Carlsbad, CA) and quantified by measuring the absorbance at 260 and 280 nm. DNA sequences of human HLA-G and β-actin were obtained from GeneBanK as follows: HLA-G sense primer: 5'-CTG ACC CTG ACC GAG ACC T-3', antisense primer: 5'-CTC GCT CTG GTT GTA GTA GCC-3'; β-actin sense primer: 5'-AAC TCC ATC ATG AAG TGT GAC G-3', and antisense primer: 5'-GAT CCA CAT CTG CTG GAA GG-3'. The cDNA-specific SYBR Green Mix was incorporated into the PCR buffer provided in the QuantiTect SYBR PCR kit to allow for quantitative detection of the PCR product in a 25-μl reaction volume. The temperature profile of the reaction was 50°C for 2 min followed by 95°C for 10 min and 40 cycles of denaturation at 94°C for 15 s and annealing and extension at 60°C for 1 min. Each treatment was performed in quadruplicate, and data were normalized to β-actin RNA of the corresponding sample.

Ethics

The present research was screened and approved by the Ethics Committee of Taizhou Hospital.

Statistical analysis

Statistical analysis was performed using SPSS statistical software (SPSS 17.0, SPSS Inc., Chicago, IL, USA). Data shown are mean ± standard deviation for comparisons among groups, the Pearson Chi-square test or Fisher's exact test was used to evaluate the association between categorical variables. The unpaired Student's t-test or the one-way analysis of variance was applied for continuous variables. P < 0.05 was considered statistically significant.


 » Results Top


Human leukocyte antigen-G expression in oral squamous cell carcinoma

Using IHC analysis, we first examined HLA-G expression in OSCC compared with normal tissues. As shown in [Figure 1], HLA-G expression was indicated by brown staining at the cell membrane, the cytoplasm or both, and varied from tumor to tumor, or from one area to another within the same tumor. In addition, HLA-G-immunopositive staining in OSCC was significantly higher than that of control group. Overall, HLA-G expressed in 55.77% (29/52) of the lesions and, among them, 62.07% (18/29) of the lesions were classified as 2 or 3, whereas 37.93% (11/29) were classified as 1. On the contrary, no HLA-G was found in adjacent nontumorous tissues or normal oral mucosa tissues.
Figure 1: Immunohistochemistry analysis of human leukocyte antigen-G expression in normal oral lesions and oral squamous cell carcinoma lesions. (a) Normal oral lesions; (b) Moderate staining in oral squamous cell carcinoma lesions; (c) Strong staining in oral squamous cell carcinoma lesions. Original magnification, ×200

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To provide further evidence, we evaluated the mRNA expression level of HLA-G in each group. The results showed that expression of HLA-G mRNA in OSCC group was much higher than that of control group [Figure 2] (P < 0.05), as 64% (16/25) of OSCC tissue sections were HLA-G-positive and almost no HLA-G were found in normal oral mucosa tissues. On the whole, these data suggested that HLA-G was involved in OSCC.
Figure 2: Expression of human leukocyte antigen-G messenger RNA in the control group and oral squamous cell carcinoma sample measured by real-time reverse transcription-polymerase chain reaction. O: Oral squamous cell carcinoma sample; C: Normal tissue

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Correlation between human leukocyte antigen-G expression and clinical parameters

To evaluate the role of HLA-G in early diagnosis in OSCC, we analyzed the relations between HLA-G expression and clinicopathological parameters, such as gender, histological grade, lymph nodal metastasis, and TNM stage [Table 1]. We found that there was a trend that the association between the tumor clinical TNM stage and the expressions of HLA-G was positive (P < 0.05), which showed that the higher TNM stages' patients had higher level of the protein expression. In addition, consisted with TNM stage, histological grade and lymph nodal metastasis had positive correlation with HLA-G expression (P < 0.05). However, no significant associations were demonstrated between gender and HLA-G expression (P > 0.05). Then, we evaluated the correlation between HLA-G mRNA level and various clinicopathological parameters of patients [Table 2]. Except histological grade, no obvious associations were demonstrated between HLA-G mRNA level and the clinicopathological parameters including gender, TNM stage, and lymph nodal metastasis.
Table 1: Association of HLA-G expression with clinicopathological parameters

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Table 2: Association of HLA-G mRNA expression with clinicopathological parameters

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 » Discussion Top


Previous studies have shown that the development of OSCC is in relation to host immune systems. The tumor cells usually express a variety of immunosuppressive cytokines to protect cells from destruction by the immune system. HLA-G is a tumor-associated molecule, whose expression may help the cancer cells to escape the immune response. In addition, the expression of HLA-G has been found in colorectal cancer, gastric cancer, esophageal squamous cell carcinoma, non-small cell lung cancer, and other common malignant tumors in the world.[11] In this study, we investigated whether HLA-G could act as a reliable biomarker of OSCC growth and evolution. We found that HLA-G was expressed in OSCC and was markedly higher than that in normal control or adjacent nontumorous tissues. Moreover, the intensity of HLA-G expression in OSCC was significantly correlated with histological grade, which was proved by both IHC and PCR.

HLA-G is a nonclassical MHC class I molecule and accumulated evidence has suggested its role of inactivation of immune response. HLA-G gene is located on the short arm of chromosome 6 and encodes seven isoforms, including membrane-bound HLA-G1, HLA-G2, HLA-G3, and HLA-G4 and soluble HLA-G5, HLA-G6, and HLA-G7. The possible mechanisms of HLA-G in immunosuppression may through direct interaction with immunoglobulin (Ig)-like receptors (LIR1 and LIR2) and the killer cell-Ig-like receptor KIR2DL4, which are receptors on the immune cell surface.[12] Upon HLA-G binding, these receptors initiate a cascade of signals that reduce cellular proliferation and compromise the immunological functions on the immune cells.[13] Numerous studies have demonstrated an association between high levels of HLA-G expression in biopsy specimens or high blood sHLA-G levels and cancers. Lin et al. found that 50.2% (110/219) of the primary hepatocellular carcinomas (HCC), as revealed by IHC, was stained heterogeneous.[14] Similarly, Wang et al. detected HLA-G by Western blot analysis and showed that 66.7% (24/36) of the HCC was positive.[15] Both Wang and Lin found that blood sHLA-G concentration was much higher in patients with HCC than in patients with liver cirrhosis and healthy controls, but there was no correlation between sHLA-G concentration and the staining of the tumor for HLA-G. Besides HCC, HLA-G expression was found upregulated in a wide variety of human neoplasms, including trophoblastic tumors, ovarian carcinoma, renal cell carcinoma, glioma tumors, lung carcinoma, breast carcinoma, colorectal carcinoma, cervical carcinoma, and esophageal squamous cell carcinoma.[7],[16],[17],[18],[19],[20],[21]

Recently, several studies started to focus on the role of HLA-G in oral biopsies. It was advocated by Fregonezi et al. for the first time. They found high expression of HLA-G in benign oral lesions, with low expression in premalignant and OSCC lesions.[22] In contrast, Gonçalves et al. found that HLA-G expression was significantly higher in OSCC than in potentially malignant lesions and it was also higher in metastatic OSCC than nonmetastatic. Moreover, they also found that patients with lower HLA-G expression exhibited a tendency toward longer survival compared with those with higher HLA-G expression.[23] Then, they further compared the salivary concentrations of sHLA-G in patients with OSCC to control group, though no correlation was observed.[24] Afterward, Gonçalves et al. also reported similar upregulation of HLA-G expression in lip carcinogenesis,[25] oral precancerous lesions,[26] and intraoral mucoepidermoid carcinoma.[27] Although divergence among these results was still controversial, they have one thing in common: the presence of HLA-G may contribute to the escape of altered cells and correlate with malignant transformation. Consistent with Gonçalves et al.'s reports, we demonstrated that both protein and DNA levels of HLA-G were upregulated in OSCC. Therefore, HLA-G overexpression is very possibly associated with OSCC.

However, Fregonezi et al. did not assess the correlation between HLA-G expression and clinical prognostic factors.[22] Meanwhile, Gonçalves et al. only assessed the correlation between HLA-G expression and benign, premalignant, and malignant oral lesions.[24],[25],[26],[27] A study by Yie et al. observed that HLA-G expression cancer cells were significantly correlated with metastasis, clinical stage, histologic grade, depth of invasion, and host immune response in ESCC.[28] On the contrary, Lin et al. showed that HLA-G expression in primary ESCC was only associated with TNM stage and was not associated with other parameters such as patient gender, age, and grade of tumor differentiation.[21] In this study, we, for the first time, evaluated the relations between HLA-G expression and clinicopathological parameters, such as gender, histological grade, lymph nodal metastasis, and TNM stage in OSCC. Results showed that intensity of HLA-G expression in situ was significantly correlated with TNM stage, histological grade, and lymph nodal metastasis. Meanwhile, HLA-G mRNA level was only related to histological grade. In conclusion, we believed that HLA-G could be associated with histological grade of OSCC. The divergence between our data and these studies might be caused by the staining evaluation method, antibodies used, and different tissue types.

As the molecular mechanism accounting for the role of HLA-G in OSCC remains largely unknown, it still needs further exploration. In summary, our study showed that both protein and DNA levels of HLA-G were dramatically increased in OSCC. Therefore, HLA-G could be used as a clinical marker in the early-stage diagnosis of OSCC.

Financial support and sponsorship

This work was supported by the Health Science and Technology Project of Zhejiang Province (2015C33261).

Conflicts of interest

There are no conflicts of interest.



 
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