H.-H. Xu

The 14 bp deletion polymorphisms in HLA-G gene play an important role in the expression of soluble HLA-G in plasma

Soluble human leukocyte antigen-G (sHLA-G) functions as a multiple immunoregulator. A 14 bp insertion (+14 bp)/deletion (-14 bp) polymorphism in exon 8 of the HLA-G gene has been proposed to be associated with HLA-G mRNA stability and the expression of HLA-G. In the current study, a total of 150 normal Chinese Han population had been genotyped for the +14 bp/-14 bp polymorphism, and the expression of plasma sHLA-G was determined with enzyme-linked immunosorbent assay in these case-matched plasma. Data showed that genotype of 14 bp polymorphism was significantly associated with sHLA-G expression. Plasma sHLA-G level with the +14 bp/+14 bp genotype was dramatically lower than that with +14 bp/-14 bp (P = 0.004) and -14 bp/-14 bp genotypes (P = 0.003), while no dramatic difference was observed between the +14 bp/-14 bp and -14 bp/-14 bp genotypes (P > 0.05). In both males and females, plasma sHLA-G with the +14 bp/+14 bp genotype was also significantly lower when compared with other two respective 14 bp genotypes. Data also showed that sHLA-G expression was unrelated to gender. This study suggests that the 14 bp deletion polymorphism in the HLA-G gene plays an important role in sHLA-G expression and that interpretation of the potential biological functions of sHLA-G should be made with caution, taking the polymorphism into consideration.

Aberrant human leucocyte antigen‐G expression and its clinical relevance in hepatocellular carcinoma

The clinical relevance of human leucocyte antigen‐G (HLA‐G) has been postulated in malignancies. Hepatocellular carcinoma (HCC) is a major contributor to cancer incidence and mortality worldwide; however, potential roles of HLA‐G in HCC remain unknown. In the current study, HLA‐G expression in 219 primary HCC lesions and their adjacent non‐tumourous samples was analysed with immunohistochemistry. Correlations among HLA‐G expression and various clinical parameters were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression on NK cell cytolysis was performed in vitro. HLA‐G expression was observed in 50.2% (110/219) of primary HCC lesions, and undetectable in corresponding adjacent normal liver tissues. HLA‐G expression was found in 37.8%, 41.9% and 71.4% of stage I, II and III HCC lesions, respectively. Data revealed that HLA‐G expression in HCC was strongly correlated to advanced disease stage (I versus II, P= 0.882; I versus III, P= 0.020; II versus III, P= 0.037). HLA‐G expression was also more frequently observed in elder patients (≥median 52 years, 57.5%versus 43.4%, P= 0.004). Meanwhile, plasma soluble HLA‐G in HCC patients was significantly higher than that in normal controls (median, 92.49U/ml versus 9.29U/ml, P= 0.000). Functional assay showed that HLA‐G expression in transfected cells could dramatically decrease the NK cell cytolysis (P= 0.036), which could be markedly restored by the blockade of HLA‐G (P= 0.004) and its receptor ILT2 (P= 0.019). Our finding indicated that HLA‐G expression was strongly correlated to advanced disease stage, and more frequently observed in elder patients. Its relevance to HCC progression might be result from the inhibition of NK cell cytolysis.

Clinical relevance and functional implications for human leucocyte antigen-g expression in non-small-cell lung cancer.

HLA‐G has been documented both in establishment of anti‐tumour immune responses and in tumour evasion. To investigate the clinical relevance of HLA‐G in non‐small‐cell lung cancer (NSCLC), expression status and potential significance of HLA‐G in NSCLC were analysed. In this study, HLA‐G expression in 101 NSCLC primary lesions and plasma soluble HLA‐G (sHLA‐G) from 91 patients were analysed with immunohistochemistry and ELISA, respectively. Correlations between HLA‐G status and various clinical parameters including survival time were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression and plasma sHLA‐G form NSCLC patients on natural killer (NK) cell cytolysis were performed. Data revealed that HLA‐G was expressed in 41.6% (42/101) NSCLC primary lesions, while undetectable in adjacent normal lung tissues. HLA‐G expression in NSCLC lesions was strongly correlated to disease stages (P= 0.002). Plasma sHLA‐G from NSCLC patients was markedly higher than that in normal controls (P= 0.004), which was significantly associated with the disease stages (I versus IV, P= 0.025; II versus IV, P= 0.029). Patient plasma sHLA‐G level (≥median, 32.0 U/ml) had a significantly shorter survival time (P= 0.044); however, no similar significance was observed for the lesion HLA‐G expression. In vitro data showed that both cell surface HLA‐G and patient plasma sHLA‐G could dramatically decrease the NK cell cytolysis. Our findings indicated that both lesion HLA‐G expression and plasma sHLA‐G in NSCLC is related to the disease stage and can exert immunosuppression to the NK cell cytolysis, indicating that HLA‐G could be a potential therapeutic target. Moreover, plasma sHLA‐G in NSCLC patients could be used as a prognosis factor for NSCLC.

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia.

Human leukocyte antigen‐G (HLA‐G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA‐G expression in malignant haematopoietic diseases are controversial, and the functions of HLA‐G on this context are limited. In the current study, HLA‐G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B‐ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA‐G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B‐ALL patients. In AML, HLA‐G‐positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA‐G‐negative patients (P < 0.01). Total T‐cell percentage was dramatically decreased in HLA‐G‐positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA‐G‐positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA‐G‐negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA‐G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA‐G expression in AML is of unfavourable clinical implications, and that HLA‐G could be a potential target for therapy.

HLA-G expression is associated with metastasis and poor survival in the Balb/c nu/nu murine tumor model with ovarian cancer

Aberrant HLA‐G expression is associated with tumor invasiveness and poor clinical prognosis; however, there is a lack of preclinical animal model to address whether HLA‐G plays a causal role in the unfavorable prognosis of malignancies. In the current study, ovarian carcinoma cell lines (HO‐8910 and Ovcar‐3) were transfected with HLA‐G gene. HLA‐G expression was analyzed with western blot and flow cytometry. Transwell experiment was performed to analyze the cell migration and invasion capability and/or multicellular spheroid formation was investigated with the 3D culture assay in vitro. The effects of HLA‐G expression for tumor cell organ metastasis and for mouse survival was analyzed with the Balb/c nu/nu mouse model. Our data showed that HO‐8910‐G and Ovcar‐3‐G cells are of higher invasion potential compared with the parental HO‐8910 and Ovcar‐3 cells. Multicellular spheroid formation exists only in HO‐8910‐G cells in a 3D culture assay. In Balb/c nu/nu mouse model, widespread metastasis was observed in mice xenografted with HO‐8910‐G cells, but not in the group with parental cells. Mouse survival was dramatically decreased in HO‐8910‐G and Ovcar‐3‐G xenografted mice than that with HO‐8910 and Ovcar‐3 cells, respectively. In summary, our study provided the first evidence that HLA‐G expression is associated with tumor metastasis and with poor survival in an animal model with ovarian cancer.

Multiple steps of HLA-G in ovarian carcinoma metastasis: Alter NK cytotoxicity and induce matrix metalloproteinase-15 (MMP-15) expression

Previous study showed that aberrant HLA-G expression in cancer cells plays important roles in disease progression and it was associated with tumor metastasis and with poor survival in an animal model with ovarian cancer; however, the mechanisms remain to be explored. In this study, we found that HLA-G expression could dramatically decreased the NK cytotoxicity against the ovarian carcinoma cell line (HO-8910) engineered to express HLA-G (HO-8910-G), and matrix metalloproteinase-15 (MMP-15) expression was up-regulated in HO-8910-G cells. Consistent with this, a strong correlation between HLA-G and MMP-15 expression were observed in a cohort of ovarian cancer samples. Knockdown the HLA-G induced MMP-15 expression by small interfere RNA (siRNA) significantly decreased the HO-8910-G cell migration potential and tumor metastasis. Collectively, our study indicated that HLA-G involved in tumor invasiveness or metastasis may rely on the NK cytotoxicity inhibition and induction of MMP-15 expression in ovarian cancer.

Immunological aspects of human amniotic fluid cells: Implication for normal pregnancy

Amniotic fluid cells (AFCs) are routinely obtained and expanded in vitro for prenatal diagnosis; nevertheless current knowledge about their properties is limited. The detailed mechanisms underlying normal pregnancies are yet to be discovered. The goal of this study was to identify the immunological aspects of AFCs including cytokine production and human leukocyte antigen (HLA) expression, and to discuss its implication for pregnancy. Eighty‐six samples of AFCs were determined for HLA expression before and after culture. Cytokine production was measured with flow cytometry in AFC culture supernatants. Treatment of interferon (IFN)‐γ on induction of HLA‐DR expression in cultured AFCs was also investigated. Data indicated that both fresh and cultured AFCs express HLA‐I, HLA‐G, but not HLA‐DR, and the cultured AFCs predominately produce the cytokine interleukin (IL)‐6. Importantly, we observed that IFN‐γ could induce HLA‐DR expression on cultured AFCs in a dose‐dependent manner. Taken together, our results indicated that AFCs are functionally active cells and are significant in pregnancy.

Analysis of the 14 bp insertion and deletion polymorphism in human leukocyte antigen-G gene in two Chinese ethnic populations.

The biological significance of human leukocyte antigen-G (HLA-G) is now beyond its initial concepts on the fetal-maternal immune regulation. HLA-G in various pathophysical conditions has been investigated, such as autoimmunity, tumor, inflammation and transplantation. HLA-G has also been postulated as a chemotherapy response marker both in protein and in genetic contexts. In the current study, a total of 640 Chinese Han and 258 Chinese She ethnic minority populations had been genotyped for the 14 bp insertion (+14 bp) and deletion (-14 bp) polymorphism in the HLA-G gene. Significant difference was observed for both the +14 bp/+14 bp (15.2% in the Han and 6.6% in the She; P = 0.00048, P(c) = 0.00097) and -14 bp/-14 bp (34.5% in the Han and 50.4% in the She; P = 1.05 x 10(-5), P(c) = 2.1 x 10(-5)) genotypes between the two populations, and similar significance was found for both +14 bp (40.3% in the Han and 28.1% in the She) and -14 bp allele distributions (P = 1.2 x 10(-6), P(c) = 2.4 x 10(-6)). Furthermore, frequencies of the 14 bp genotype and alleles both in the Chinese Han and in the Chinese She populations were compared with other ethnic populations. Data showed that dramatic variations between different ethnic populations were also observed for this polymorphism. In summary, our results indicate that heterogeneity of the 14 bp polymorphism in the HLA-G gene among different ethnic populations is possibly the result of evolution.

Diagnostic accuracy of high-risk HPV genotyping in women with high-grade cervical lesions: evidence for improving the cervical cancer screening strategy in China

Currently, clinical data for primary HPV screening alone are lacking in China. Here, we evaluate cervical cancer screening with primary HPV genotyping, as well as possible future screening strategy. Overall, high-risk HPV (hrHPV) prevalence was 18.2% among hospital-based population in Taizhou area. For cervical intraepithelial neoplasia 2 or worse (CIN2+), the sensitivity of primary hrHPV genotyping strategy and current cervical cancer screening strategy were 93.5%, and 71.1%, respectively; whereas the specificity was 17.5%, and 62.4%, respectively. Current cervical screening strategy had slightly higher positive predictive values (28.4%) for CIN2+ than hrHPV genotyping strategy (21.9%), whereas primary hrHPV genotyping strategy demonstrated higher negative predictive values (94.7%) than current cervical screening strategy (91.1%). Compared to HPV35/39/45/51/56/59/66/68 genotypes, the odds ratios (OR) for CIN2+ in HPV16/18/31/33/52/58 infection women were 3.2 (95% confidence interval [CI] 2.3-4.1). Primary hrHPV genotyping strategy provides a better predictive value than HPV16/18 genotyping alone in guiding the clinical management of the current cervical cancer screening. HPV testing without adjunctive cytology may be sufficiently sensitive for primary cervical cancer screening.

Prevalence of human papillomavirus genotypes and relative risk of cervical cancer in China: a systematic review and meta-analysis

High-risk HPV (hrHPV) is related to cervical carcinogenesis, although clinical data comparing the natural history and carcinogenic potential of type-specific HPV remain limited. Furthermore, the nationwide prevalence rates of overall and type-specific HPV among women with cervical precancerous lesions and cancer have not been reported. Here, a meta-analysis was performed for type-specific HPV distribution among 30,165 HPV-positive women, including 12,094 invasive cervical cancers (ICCs), 10,026 cervical intraepithelial neoplasia grade 2/3 (CIN2/3), 3246 CIN1, and 4799 normal cervices from 45 PCR-based studies. We found that HPV16 was the most common hrHPV type involved in cervical disease. The HPV16 positivity rate varied little across normal (22.7%) and CIN1 individuals (23.6%) but increased through the CIN2 (37.6%) and CIN3 patients (51.9%) to 65.6% in ICC cases. HPV16, 18, 35, 39, 45, and 59 were more frequent in ICC than CIN3, with ICC:CIN3 ratios ranging from 2.3 for HPV18 to 1.1 for HPV35/45. HPV31, 33, 52, and 58 were more frequent in CIN3 compared with normal cervices but less common in ICC compared with CIN3 (ICC:CIN3 ratios ranging from 0.6 for HPV58 and 0.4 for HPV52). The ICC:normal ratios were particularly high for HPV18, 52 and 58 in West China (4.1, 3.9 and 2.9, respectively) and for HPV45 and 59 in North China (1.6 and 1.1, respectively). In summary, this study is the most comprehensive analysis of type-specific HPV distribution in cervical carcinogenesis and could be valuable for HPV-based cervical cancer screening strategies and vaccination policies in China.