Yan-Yun Ruan

Human leukocyte antigen‐G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma

Human leukocyte antigen (HLA)‐G inhibits functions of immune component cells and promotes malignant cells evading from antitumor immunity. We investigated the clinical relevance of HLA‐G expression in esophageal squamous cell carcinoma (ESCC). In our study, HLA‐G expression in 79 primary ESCC lesions and corresponding adjacent normal tissues were analyzed with immunohistochemistry. Soluble HLA‐G (sHLA‐G) in plasma was detected with enzyme‐linked immunosorbent assay (ELISA) in 41 ESCC patients (including 19 case‐matched lesions and plasma samples) and in 153 normal healthy controls. HLA‐G expression was observed in 65.8% (52/79) of the ESCC lesions but not in adjacent normal esophageal tissues. HLA‐G expression was more frequently observed in patients with advanced disease stage (III/IV vs. I/II, p = 0.01). Patients with HLA‐G expression had a significantly worse survival, and HLA‐G could be an independent prognostic factor. sHLA‐G levels in plasma were significantly increased in patients compared to normal controls (median: 152.4 U/ml vs. 8.9 U/ml, p < 0.001). The area under receiver‐operating characteristic (ROC) curve for sHLA‐G in plasma was 0.992. However, no significant correlation was found between sHLA‐G in plasma and clinical parameters studied. In conclusion, our findings indicated that HLA‐G expression in ESCC is associated with poor survival and could be a prognostic indicator. Furthermore, increased levels of sHLA‐G in plasma might be a useful preoperative biomarker for diagnosis.

HLA-F expression is a prognostic factor in patients with non-small-cell lung cancer

Human leukocyte antigens (HLA)-E, -F and -G are referred to as non-classical HLA class I antigens. Among them, the clinical relevance of HLA-E and HLA-G has been intensively investigated, but that of HLA-F remains unknown. In this study, HLA-F expression in 83 primary non-small-cell lung cancer (NSCLC) lesions and corresponding adjacent normal tissues were analyzed with immunohistochemistry. Relevance of HLA-F expression with clinical parameters and patient survival was evaluated. Data revealed that HLA-F expression was observed in 24.1% (20/83) of the NSCLC primary lesions but not in adjacent normal lung tissues. HLA-F expression was not significantly relative to clinicoparameters including patient age, gender, tumor histological type, grade of tumor differentiation and TNM stage. Unexpectedly, patients with HLA-F positive expression had a significantly worse prognosis (p=0.017). The median overall survival for the patients with HLA-F positive was 10.0 months (range: 4.4-18.3 months) and with HLA-F negative was 17.0 months (range: 10.4-23.6 months), respectively. Multivariate analysis revealed that HLA-F could be an independent prognostic factor with the hazard ratio of 5.12 [95% confidential Intervals (CI): 1.8-14.3]. Summary, this study was for the first time to provide the evidence that HLA-F expression was of clinical significance in tumor patients and that its expression was associated with a poor survival and could be a prognostic indicator in patients with NSCLC.

Alteration of HLA-F and HLA I antigen expression in the tumor is associated with survival in patients with esophageal squamous cell carcinoma.

Alteration of human leukocyte antigen (HLA) expression, such as decreased HLA I (HLA‐A, ‐B and ‐C) antigens and elevated nonclassical HLA I antigens (HLA‐E, ‐F and ‐G), was reported to have an unfavorable prognosis in various cancers. In our study, HLA‐F expression in 105 primary esophageal squamous cell carcinoma (ESCC) lesions and 62 case‐matched adjacent normal tissues, and HLA I antigens among 68 cases were analyzed by immunohistochemistry. Data revealed that HLA‐F expression was observed in 58.1% (61/105) of the ESCC lesions and in 54.8% (34/62) of the normal esophageal tissues. Among the 62 case‐matched samples, HLA‐F expression (lesion vs. normal tissue) was upregulated, unchanged and downregulated in 13 (21.0%), 6 (9.6%) and 43 (69.4%) cases, respectively. Patients with HLA‐F positive had a worse survival than those with HLA‐F negative (p = 0.040). Patients with upregulated HLA‐F expression (lesion vs. normal tissue) had significantly worse survival than those with HLA‐F unchanged and downregulated (p = 0.010). Furthermore, decreased HLA I expression was observed in 41.2% (28/68) patients and was with worse prognosis in comparison to those with preserved HLA I expression (p = 0.001). Multivariate analysis using Coxs proportional hazards model revealed that upregulated HLA‐F expression (p = 0.026) and downregulated HLA I expression (p = 0.013) could be an independent unfavorable prognostic factor. In conclusion, our study provided the evidence that alteration of HLA I and HLA‐F antigen expression was associated with survival in patients with ESCC.

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.

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

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 HLA-G expression was associated with the disease progression in patients with cervical cancer.

HLA-G1 and HLA-G5 isoforms have an additive effect on NK cytolysis.

The immunotolerant HLA-G could generate seven isoforms including HLA-G1--G7. The suppressive function of either HLA-G1 or HLA-G5 isoform to NK cell cytolysis has been well established. Whether HLA-G1 and HLA-G5 isoform have an additive effect on the cytolysis of NK cells remain to be explored. In this study, effects of expression of HLA-G1 and HLA-G5 isoforms and their combination on NK cytolysis was investigated. NK cell cytolysis was analyzed by detecting the NK cell surface CD107a expression. In this study, data showed that the inhibition capacity is dependent on the level of both HLA-G1 and HLA-G5 expression, but the HLA-G5 isoform has a more potent inhibition effect on the NK cytolysis (p<0.01). Furthermore, HLA-G1 and HLA-G5 have an additive effect on the suppression of NK cell cytolysis. Our study provided further understanding for the roles of HLA-G1 and HLA-G5 isoform expression in target cells immune escaping from NK cells.

HLA-G expression is irrelevant to prognosis in patients with acute myeloid leukemia

Human leukocyte antigen (HLA)-G could contribute to escape of cancer cells from host anti-tumor responses, and its potential clinical relevance in various malignancies was also addressed. However, the prognostic value of HLA-G in acute myeloid leukemia (AML) remains debated. In this study, HLA-G expression in malignant blasts was analyzed from 77 de novo AML patients (AML-M2, n=26; AML-M3, n=24; AML-M4, n=10; AML-M5, n=17) with flow cytometry. The proportion of HLA-G expressing blasts varied from 0% to 93.96% (median: 0.42%; 95% CI: 0-89.0%). Blasts with 0.5% or fewer HLA-G expressing were defined as negative according to its expression in normal CD34(+)CD45(+) cells (n=20, range: 0-0.5%; median: 0.13%; 95% CI: 0-0.42%). HLA-G expression status on leukemic blasts was not associated with the clinical parameters such as patient age at diagnosis, sex, sub-type of AML, percentage of blasts at diagnosis. Survival analysis revealed that HLA-G expression status on leukemic blasts is unrelated to the prognosis (p=0.884). The mean overall survival time for the HLA-G negative and positive patients was 20.7 months (95% CI: 16.1-25.3) and 20.1 months (95% CI: 14.3-25.8), respectively. Taken together, our findings indicated that HLA-G expression is of no significance for the prognosis of patients with AML.

Lesion HLA-G5/-G6 isoforms expression in patients with ovarian cancer

HLA-G is an immune tolerant with seven isoforms. HLA-G expression was observed to be associated with tumor cell immune escaping, invasion and metastasis, and with poor prognosis in cancer patients. Different types of HLA-G isoforms could be expressed in clinical settings when meet different cellular and environmental conditions. Lesion total HLA-G expression detected by the monoclonal antibody (mAb) 4H84 was widely investigated in previous studies, while specific HLA-G isoforms such as HLA-G5/-G6 remains to be clarified. In this study, 118 primary ovarian cancer lesions were probed with mAb 5A6G7 which recognizes HLA-G5/-G6 was performed by immunohistochemistry. Data showed that HLA-G5/-G6 was expressed in 79.7% (94/118) of these ovarian cancer lesions, where HLA-G5/-G6 expression was observed in 75.7% (53/70) serous, 63.6% (7/11) mucinous cystadenocarcinoma and in 100% (11/11) endometrioid adenocarcinoma, in 85.7% (6/7) clear cell carcinoma, 100% (10/10) sex cord-stromal tumor and 77.8% (7/9) germ cell tumors. However, lesion HLA-G5/-G6 expression was unrelated to histological type, patient age, FIGO stage and patient survival. Unlike total HLA-G expression, no clinical significance of HLA-G5/-G6 expression in ovarian cancer lesion was observed in this study. Our findings indicated that different HLA-G isoforms might have different biological functions in malignancies.

VNTR polymorphism of human IL1RN in Chinese Han and She ethnic populations

Interleukin 1 receptor antagonist (IL‐1Ra) is an important anti‐inflammatory molecule encoded by the IL1RN gene. The polymorphism of IL1RN characterized by variable numbers of an 86 bp tandem repeat (VNTR) sequence in intron 2 has been described. Moreover, frequencies of the IL1RN alleles vary among different ethnics. In the present study, we analysed the IL1RN polymorphism in intron 2 in 256 Chinese Han and 252 Chinese She individuals. Four alleles including IL1RN*1, *2, *3 and IL1RN*4 were identified in this study. Data revealed that the distribution of the IL‐1RN genotypes and allele was significantly different between the two Chinese populations (P < 0.001). Among them, 66.8% (171/256) and 86.5% (218/252) were homozygous for the allele IL‐1RN*1 in Chinese Han and She individuals respectively. Homozygosity for allele IL‐1RN*2 was only observed in Chinese Han with the percentage of 0.8% (2/256). Heterozygosity for IL‐1RN*1/2, IL1RN*1/3 and IL1RN*1/4 was 30.9% (79/256), 0.4% (1/256) and 1.2% (3/256) in Chinese Han, whereas only heterozygosity for IL‐1RN*1/2 was found in Chinese She (13.5%, 34/252). Frequencies of the most common allele IL‐1RN*1 and IL‐1RN*2 were 83.0% and 16.2% for Chinese Han and 93.3% and 6.7% for Chinese She respectively. The rare allele IL‐1RN*3 and IL‐1RN*4 was only observed in the Chinese Han population with the frequency of 0.2% and 0.6% respectively. Our findings suggested that the ethnic background plays an important role in IL‐1Ra gene variation in different populations.

Importance of the plasma soluble HLA-G levels for prognostic stratification with traditional prognosticators in colorectal cancer

An increased peripheral soluble HLA-G (sHLA-G) expression has been observed in various malignancies while its prognostic significance was rather limited. In this study, the prognostic value of plasma sHLA-G in 178 colorectal cancer (CRC) patients was investigated. sHLA-G levels were analyzed by specific enzyme-linked immunosorbent assay. Data showed sHLA-G levels were significantly increased in CRC patients compared with normal controls (36.8 U/ml vs 25.4 U/ml, p = 0.009). sHLA-G in the died were obviously higher than that of alive CRC patients (46.8 U/ml vs 27.4 U/ml, p = 0.012). Patients with sHLA-G above median levels (≥ 36.8 U/ml, sHLA-Ghigh) had a significantly shorter survival time than those with sHLA-Glow (< 36.8 U/ml, p < 0.001), and sHLA-G could be an independent prognostic factor for CRC patients. With stratification of clinical parameters in survival by sHLA-Glow and sHLA-Ghigh, sHLA-G exhibited a significant predictive value for CRC patients of the female (p = 0.036), the elder (p = 0.009), advanced tumor burden (T3 + 4, p = 0.038), regional lymph node status (N0, p = 0.041), both metastasis status (M0, p = 0.014) and (M1, p=0.018), and clinical stage (I + II, p = 0.018), respectively. Summary, our data demonstrated for the first time that sHLA-G levels is an independent prognosis factor and improves the prognostic stratification offered by traditional prognosticators in CRC patients.