Genome-Wide Profiling of Human Papillomavirus DNA Integration into Human Genome and Its Influence on PD-L1 Expression in Chinese Uygur Cervical Cancer Women.

BACKGROUND: The Uygur is the fifth most populous ethnic group in China. Compared to other Chinese population, cervical cancer in them had high incidence, and HPV infection also was particular. Their HPV integration situation has never been reported. We aimed to investigate the integration situation of 20 subtypes of HPV gene into host cell genome in Chinese Uygur cervical cancer patients; meanwhile, we explored the influence of gene integration on PD-L1 expression. METHODS: 40 frozen Chinese Uygur cervical cancer specimens with positive HPV infection were obtained from the cancer prevention and treatment institute of Tumor Hospital Affiliated to Xinjiang Medical University. The integration situation of HPV gene into host cell genome was detected by Agilent SureSelect™ Target Enrichment Chip and Next-Generation Sequencing. The related genes were analyzed by GO functional annotation and KEGG pathway enrichment. The expression levels of PD-L1 in cancer cells were tested by immunohistochemical assay (IHC). Meanwhile, the relationship between PD-L1 levels in cancer cells and gene integration were analyzed. RESULTS: The HPV multiple infection rate by HIVID was as high as 92.5%, much higher than 35.0% by the commercial kit (P < 0.05). There were 13423 integration events in 40 specimens, involving 6867 human genes. These integration events were distributed on all human chromosomes, and chromosome 19 had the excessive concentration phenomenon of integration events. There were some integration hotspots in human genome such as PPP1R37, HECW2, EMBP1, ANKRD50, SPTBN4, LINC00895, LYRM4-AS1, LINC00374, RBFOX1, CSMD1, CDH13, and KLHL4. Insertion breakpoints can be found in all gene regions of the HPV genome. The actual observation of the integration times of E1 and E6 was much higher than the expected value, while the actual observation times of E5 were much lower than the expected value. The result of GO functional analysis showed that binding molecular function and cellular process biological process were the main ways to influence the cell biological behavior of HPV gene integration. The enrichment pathway analysis of KEGG showed that pathways in cancer were the most important enrichment pathways involved in the genomic integration of HPV. The positive PD-L1 rate was 62.5%. Logistic regression analysis showed that 9p24.1 existing integration sites and the number of all gene integration were risk factors for PD-L1 expression (odds ratio 17.313 and 1.012; 95% confidence interval 1.691-177.213 and 1.001-1.023). Conclusions and Relevance. Most high-frequency sites of HPV integration in Chinese Uygur cervical cancer are related to cancer progression, and the gene integration hotspots may be potential HPV carcinogenic targets. The problem of multiple HPV infection in Chinese Uygur cervical cancer patients should be paid attention. L1 and E6 genes are inapposite as the target gene of commercial HPV type detection kit, because of high-frequency breakpoints in these genes. The gene integration especially the integration existing on 9p24.1 could affect the expression level of PD-L1.

Association between PD-L1 and HPV status and the prognostic value for HPV treatment in premalignant cervical lesion patients.

The difference of PD-L1 expression between only HPV-positive patients and premalignant cervical lesion patients did not be reported in present studies. And to test the PD-L1 expression in some cervical cell lesion studies using cervical exfoliated cells sample also was ignored. Meanwhile, the PD-L1 expression as a predictive biomarker still existed controversy. So in the study, first to compare the expression of PD-L1 between only HPV-positive patients and premalignant cervical lesion patients, then to research the association between PD-L1 and HPV status, lastly to explore the possible prognostic value for HPV treatment in premalignant cervical lesion patients.Cervical exfoliated cells samples of 54 premalignant cervical lesion patients with HPV16 infection were collected; meanwhile the cervical exfoliated cells samples from 20 healthy women without HPV infection and 20 patients with only HPV16 infection but cervical cytology normal were collected as 2 control groups. Flow-through hybridization and gene chip (FHGC) was used to detect the HPV type, the PD-L1 expression was tested by Flow cytometry analysis, the methylation-sensitive high-resolution melting (MS-HRM) was used to test the HPV16 L1 gene methylation. The 54 premalignant cervical lesion patients were followed up in 18 months to assess the prognostic value of PD-L1 for HPV treatment.The PD-L1 positive cell rate and mean fluorescence intensity of PD-L1 positive cell in premalignant cervical lesion patients with HPV16 infection were higher than 2 control groups. Mean fluorescence intensity of PD-L1 positive cell were increased in 54 cases when existing multiple HPV status and high HPV16-L1 gene methylation (L1 gene methylation more than 50%). High PD-L1 expression (PD-L1 positive cell rate more than 10%), high HPV16-L1 gene methylation, and multiple HPV infection status could prolong the time to clean HPV infection by Kaplan-Meier analysis. Multivariate Cox proportional hazards analysis also showed that all of high PD-L1 expression, high HPV-L1 methylation, and multiple HPV infection status should increase the risk of HPV unclearance in premalignant cervical lesion patients; the hazard ratio (HR) was 2.043 (CI: 1.050-3.973), 2.797 (CI: 1.277-6.122), and 3.050 (CI: 1.406-6.615).PD-L1 expression only was correction with HPV infection when the infection induced the cervical cells to create the lesion. PD-L1 was the risk factor of HPV unclearance in premalignant cervical lesion patients, so anti-PD-L1 therapy could be a potential effectiveness way of HPV infection in premalignant cervical lesion patients.

Increased HPV L1 gene methylation and multiple infection status lead to the difference of cervical epithelial cell lesion in different ethnic women of Xinjiang, China.

Human papillomavirus (HPV) L1 gene methylation deeply involved in the progression and heterogeneity of cervical cell epithelial lesions. The DNA ploidy also represented the early lesions of cervical cell, and it was associated with different HPV infection status in different ethnic women. So, the research was to explore whether it was possible that HPV L1 gene methylation and HPV infection status as the risk factors to lead to the differences of cervical epithelial cells' lesions in different ethnics women.The flow-through hybridization and gene chip for HPV genotypes test, general characteristics, and cervical exfoliated cell samples were collected from 94 Uygur and 79 Han women with HPV-16 infection. The cases were divided into the single HPV-16 (sHPV-16) infection group and multiple HPV-16 (mHPV-16) infection group in each ethnic women. The DNA ploidy was analyzed by flow cytometry, and the methylation-sensitive high resolution melting (MS-HRM) was used to test the HPV-16 L1 gene methylation, the results of methylation was segmented into mild methylation, moderate methylation, and severe methylation groups. Multifactor logistic analysis explored the relation between DNA heteroploid and HPV-16 infection status, HPV-16 L1 gene methylation in different ethnic women.The higher proportion of mHPV-16 infection in Uygur than Han women (61.7% vs 38.0%). L1 gene methylation had statistic difference between single and mHPV-16 infection under the same ethnic women. The proportion of DNA heteroploid had statistic difference between different HPV-16 infection status or different L1 gene methylation grades in Han or Uygur women. Both L1 gene methylation and HPV infection status were the risk factors of DNA heteroploid. Compared to the sHPV-16 infection, the odds ratio (OR) of mHPV-16 infection were 4.409 (CI: 1.398-13.910) and 3.279 (CI: 1.069-10.060) in Han and Uygur women. Compared the mild L1 gene methylation, the OR of moderate L1 gene methylation were 3.313 (CI: 1.002-10.952) and 5.075 (CI: 1.385-18.603) in Han and Uygur women, the OR of severe L1 gene methylation were 20.592 (CI: 3.691-114.880) and 63.634 (CI: 10.400-389.368) in Han and Uygur women.The study first reported that HPV L1 gene methylation and HPV infection status were the risk factors to the DNA heteroploid of cervical cell in different ethnics women, HPV L1 gene methylation and infection status should be recommended to the existing system of cervical lesion screening in order to provide better serves for the HPV infected women, especially for the ethnic women with high proportion of severe L1 gene methylation and multiple infection status.

The application of CYFRA21-1 in cervical lesions screening in high-risk human papillomavirus infected women.

OBJECTIVES: To investigate the role of cytokeratin-19 fragment (CYFRA21-1) in cervical lesions screening in high-risk hu-man papillomavirus (HR-HPV) infected women. MATERIAL AND METHODS: The study was a retrospective study. First, the results of CYFRA21-1, cytology (TCT), and HR-HPV examinations of 1039 outpatients from gynecology department in Tumor Hospital Affiliated to Xinjiang Medical University were collected. Then, the data was analyzed using a series of statistical methods. RESULTS: There was a correlation between CYFRA21-1 levels and HPV-DNA load in HR-HPV infected women (rs = 0.711, p = 0.015). CYFRA21-1 levels and positive rate increased along with deepening of cervical cell lesions. In HR-HPV infected women, there was a statistically significant difference (t = 6.022, p < 0.001) in CYFRA21-1 levels between the group with cytological lesions (4.87 ± 1.58 ng/mL) and the group with normal cytology (2.52 ± 0.96 ng/mL). Positive rates of CYFRA21-1 in the two groups were 62.06% and 7.83%, respectively, and also exhibited statistically significant differences (χ2 = 74.624, p < 0.001). When diagnosing cytological lesions via CYFRA21-1 in HR-HPV infected women, sensitivity was 62.07%, specificity was 92.17%, positive predictive value was 88.89%，negative predictive value was 70.67%. Compared to CYFRA21-1 negative women, cytological lesions were detected 19.273 times more often in CYFRA21-1 positive women. CONCLUSION: CYFRA21-1 could provide a reference idea for further diagnosis of women who are infected with HR-HPV but whose cytology is normal.

Retrospective Study to Determine Diagnostic Utility of 6 Commonly Used Lung Cancer Biomarkers Among Han and Uygur Population in Xinjiang Uygur Autonomous Region of People's Republic of China.

Early diagnosis was the main way to improve the survival rate of lung cancer patients. At present, the methods to diagnose lung cancer were varied, but early diagnosis of lung cancer was still difficult. In experimental and clinical studies, lung cancer related tumor markers were helpful to the early diagnosis of lung cancer. So far, there were many studies about lung cancer related tumor markers in China, but the subjects in these studies were almost the Han population. There were few studies about the Uygur population. Xinjiang was a multi-ethnic region in China, the ratios of Han and Uygur population were 40% and 45%, respectively. Xinjiang also was a high incidence area of lung cancer in China. The purpose of this study was to research the application of 6 tumor markers in Uygur and Han lung cancer patients in XinJiang, China.The study collected 342 cases who were diagnosed as lung cancer in Tumor Hospital Affiliated to XinJiang Medical University from May 2012 to December 2012. Serum concentrations of squamous cell carcinoma (SCC), cytokeratin fragment 19 (CYFRA21-1), carcino-embryonic antigen (CEA), carbohydrate antigen 125 (CA125), precursor of gastrin-releasing peptide (Pro-GRP), and neuron-specific enolase (NSE) were tested for every patient before radiation, chemotherapy, or surgery. The serum concentrations of SCC, CYFRA21-1, CEA, CA125, and Pro-GRP were assayed using the micro-particle luminescence analysis testing by the Abbott ARHCITECT i2000SR immunoanalyzer. NSE was assayed by the electrochemical luminescence analysis testing using Roche Cobas E601 electrochemical luminescence analyzer.Serum levels of SCC were different between 2 ethnic populations, smoking should be the influence factor to create the difference. Cluster analysis showed that the NSE and Pro-GRP were helpful to identify small cell lung cancer (SCLC), and CEA, CA125, SCC, CYFRA21-1 were beneficial to diagnose non-small cell lung cancer (NSCLC). The compare of diagnosis value about serum tumor markers also proved the result of cluster analysis. No matter SCLC or NSCLC, the positives rate of all tumor markers were increasing as clinical stage advancing. Pro-GRP had higher positive rate than NSE in limited stage of SCLC. CA125 had the highest positive rate in I and II stage of NSCLC, and CYFRA21-1 had the highest positive rate in III and IV stage of NSCLC. CEA and CA125 were beneficial to diagnose adenocarcinoma, CYFRA21-1, and SCC identified squamous cell cancer better.Only SCC level was higher in Han population than Uygur population because of the differences of smoking constituent ratio between 2 populations. So, it could be unified to research the application value of the 6 indicators for the Han and Uygur population. Then, we suggested a primary diagnostic utility of 6 commonly by lung cancer biomarkers in both the Han and Uygur populations in Xinjiang Uygur Autonomous Region of People's Republic of China.