Research Of High-risk Human Papillomavirus Infection On The Poor Prognosis Of Esophageal Squamous Cell Carcinoma

Background:Esophageal squamous cell carcinoma (ESCC) is a common malignancy with high mortality and ranks as the fourth most frequent cause of cancer death in China as well as in worldwide. The Shantou of eastern Guangdong province, in southern China, is only one of coastal area with the high incidence of esophageal carcinoma (EC). The presence of (Human papillomavirus, HPV), mainly for high risk of HPV (HR-HPV), in cases of ESCC has been reported repeatedly about50-80%from Shantou, China, and other regions with a high incidence of esophageal carcinoma (EC). Thus, the role of high-risk HPV types in the carcinogenesis and prognosis of ESCC remains uncertain.Telomeres and Telomerase, as the special structure at the ends of chromosomes, could maintain the stability, and prevent the degradation, abnormal terminal fusion or recombinant of chromosomal DNA. Telomere shortening and activation, which related with DNA methylation of telomere and subtelomere, is crucial in carcinogenesis and tumor progression. It’s helpful for etiology study of EC to research the relationship between HPV infection and epigenetic mechanism.Aim and Hypothesis:Here, we fully explored the association among HR-HPV infection load and integration, telomere length (TL) and DNA methylation of telomerase related gene and submelomere, as well as their significance in the prognosis of ESCC, so as to provide some supplements to the etiology study of EC.Our hypothesis as following:HR-HPV sustain infection in ESCC�'HPV DNA breakage and its E6/7oncogene integration at the "fragile site" of the host chromosomes�'The sustain and continuous expression of HPVE6/7oncoprotein�'The changes of telomerase related gene (hTERT and TRF2) and subtelomere DNA methylation level�'Effect of telomere length�'Promotion of the malignant transformation and immortalization�'Affect the prognosis of ESCC patients. Method:We established a stable and sensitive methodology of real time PCR to assess the prevalence, viral load and physical status of HR-HPV-16,-18and-58, infection the3most common HR-HPVs, as well as telomere length (LT) in patients with70cases of ESCC tumor (T) tissues and paired of paired nontumor (NT) tissues and50cases of normal esophagus (NE). DNA methylation and HPV-16protein expression were carried out by methylation-specific PCR (MSP) and using immunohistochemistry (IHC) assay, respectively. The prognostic value of TL and DNA methylation in ESCC was analyzed using SPSS16.0soft ware (SPSS Inc., Chicago, IL).Results:(1) All typical amplification plots involved HPV-16,-18and-58types for E2and E6/7plasmid DNA (change in the fluorescent signal versus cycle number). Six of standard curve showed a dynamic linear range for quantification across copy number over the entire range of dilution concentrations on bivariate correlation analysis (P<0.05).(2) Widespread, but no difference of HR-HPV-16,-18and-58vial loads were found. The combined infection rates (50.00%vs.33.33%, P=0.045) and viral load (2.55±3.19vs.0.55±0.57copies/cell, P=0.029) for all HR-HPVs in ESCC patients was significantly higher than that of their corresponding controls. HPV DNA integration and mixed forms were more frequently observed in ESCC patients and in controls (91.43%vs.85.00%). The frequency of HR-HPV integration was higher than that for ESCC controls (F=17.832, P=0.001). The integration level was associated with pathological grade of ESCC (r=3.753, P=0.033). However, no significant associations were found between HPV physical status and patient age or lymph-node metastasis. HPV viral loads were not associated with ESCC patient age, lymph-node metastasis or cancer grade.(3) IHC assay indicated that HPV-16protein expression, located mainly in the cytoplasm of cancer cells, was detected in59.26%of ESCC tissues and was positively correlated with pathological grade of cancer (r=0.301, P=0.027) in ESCC patients.(4) TL decreased from normal esophagus mucosa, adjacent NT tissue to T tissue [4.77(4.28-5.33),4.37(3.89-5.09) and4.27(3.79-4.57) kb].(5) T-TL was longer in ESCC samples positive for HPV (represented as low-risk HPV and HR-HPV) or HR-HPV. T-TL was positively correlated with viral load (r=0.410, P=0.037) and integration (r=0.492, P=0.01).(6) Relatively long T-TL and high T/NT-TL ratio were frequently found with high viral load and integrated status. T/NT-TL was higher in ESCC patients with smoking, HPV infection or tumor grade III. T/NT-TL ratio was higher for surviving than non-surviving patients.(7) Using MSP method, we found a hyper-methylation ratio (≥60%) for hTERT,7q,9q and XqYq but a hypo-methylation ratio (<40%) for TRF2,17q, and21q. we also found the concordant methylation level of7q and9q (r=0.304, P=0.011) and18q (r=0.311, P=0.009). The methylation level of hTERT and21q was negatively correlated with that of TRF2(r=-0.281, P=0.025, and r=-0.251, P=0.036, respectively).(8) HR-HPV positivity increased the levels of hTERT,18q and21q methylation and decreased that of TFR2methylation. Furthermore, HR-HPV integration was positively correlated with hTERT methylation (r=0.413, P=0.036) and positively with TRF2methylation (r=0.453, P=0.02). However, HR-HPV viral load did not affect the ratio of hTERT, TRF2and subtelomere DNA methylation.(9) High hTERT and21q methylation was more prevalent with long than short T-TL and was positively correlated with T-TL.(10) Kaplan-Meier analysis and Univariate Cox regression analysis revealed significantly worse outcomes for patients with cancer grade Ⅲ (PHR=0.013), HR-HPV infection (PHR=0.037) and high T/NT-TL ratio (PHR=0.014). Multivariate survival analysis with a Cox proportional-hazards model revealed HR-HPV infection and high T/NT-TL ratio as the poor prognostic factors of survival, independent of age, gender, smoking, alcohol drinking, lymph node metastasis and cancer grade. Summary:(1) We established a stable and sensitive methodology of real time PCR to assess the viral load and physical status of HR-HPV-16,-18and-58DNA, as well as the relative telomere length.(2) A relatively low HR-HPV copy number infection in ESCC is unlikely to play an essential a role in the carcinogenesis of ESCC. However, the mainly presentation of the physical status could sustain the high expression of the HR-HPV types in ESCC, which strongly suggests a possible role of oncogenic HPV infection in carcinogenesis of ESCC.(3) TL gradually decreased in the process of esophageal carcinogenesis. HR-HPV infection could reverse the shortening of LT and maintain the TL in a critical length.(4) DNA methylation levels of hTERT, TRF2and21q were participated in the maintenance of LT by HR-HPV infection.(5) HR-HPV infection and high T/NT-TL ratio was the dependent of prognostic factors for the poor survival of ESCC.Conclusion:HR-HPV integration and expression related to telomere elongation and DNA methylation of hTERT and TRF2, may be a potential biomarker of prognosis in ESCC.