| Background:Esophageal carcinoma(EC)ranks the eighth common cancer and the sixth most lethal cancer worldwide.EC includes two primary histologic subtypes,squamous cell carcinoma(SCC)and adenocarcinoma(AC).Most of the cases are esophageal squamous cell carcinomas(ESCC)in Asia.Causes of ESCC include various genetic and environmental factors,including dietary habits,alcohol tobacco consumption,smoking and probably infection etc.Infection is responsible for tumorigenesis,causing about 16.1%of human cancer in 2008 worldwide.HBV,HCV,HPV,and Hpylori were together responsible for 1.9 million cases worldwide.Human papillomavirus(HPV)are the most prominent oncogenic virus,whose infection accounted for 4.8%of new cancer cases in 2008.HPV infection is associated with more than 90%of cervical cancer(CC)worldwide,which is squamous cell carcinoma.HPV infection is also responsible for tumorigenesis of nongenital cancers such as oropharyngeal squamous cell carcinoma(OSCC),which arises in squamous epithelium as well.HPV is the oncogenic driver in a subset of OSCC patients,who are not cigarette or alcohol consumer.Recently it is found HPV may be associated with esophageal adenocarcinoma(EAC):HPV positivity reaches up to 66.7%in EAC and exhibits a strong association with disease severity.The risk of HPV infection for individuals reaches up to 80%through life.However,only high-risk HPV(hr-HPV)accounts for carcinogenesis and progression of tumors.Hr-HPV includes HPV type 16,18,33,58,etc.,and HPV16 and HPV18 are primary causes of CC and OSCC,which are responsible more than 70%CC and OSCC worldwide.The oncogenic ability of hr-HPV depend on their oncoproteins E6 and E7,which are involved in reactivating DNA synthesis in non-cycling cells,inhibiting cell death and delaying differentiation program.Since ESCC belongs to squamous cell carcinoma,sharing histopathological similarities with CC and OSCC,and esophagus is adjacent to oropharynx,the prevalence of hr-HPV in ESCC and the association between hr-HPV and the tumorigenesis/progression of ESCC is one of the hot topics in ESCC research.However,because of variances in detection methods,detection sensitivity,tested cohorts and quality control of samples,the prevalence of HPV in ESCC remains in controversy.One opinion is that there is no association between HPV and ESCC because of extremely low HPV positivity in ESCC and handling introduced contamination leads to false-positive results.Some other researchers hold the opposite view based on animal model that bovine papillomavirus induces the carcinogenesis of esophageal in bovine and there are strong evidence of an association between HPV and ESCC.In addition,it is reported HPV prevalence is high in ESCC high-incidence area,although the viral load of HPV in ESCC is significantly lower than that in CC.Above all,the prevalence of hr-HPV in ESCC and the association between hr-HPV and ESCC carcinogenesis/progression remains unclear.Objective:In the study we simultaneously investigated hr-HPV(18 types)E6 mRNA,HPV16/18 E6 oncoprotein expression and surrogate marker of hr-HPV transforming infection,p16INK4a expression in a same tested cohort.In addition,we investigated antibodies against HPV16 E6/E7 oncoprotein in the sera from ESCC patients and identified B-cell linear epitopes against HPV16 E6/E7.Our result indicated that there may be a special hr-HPV positive subtype in ESCC with better prognosis,providing preclinical information for precision medicine in ESCC treatment.Materials and Methods:A total of 503 individuals diagnosed as ESCC were provided tumor and adjacent epithelium specimens for in situ detection and HPV E6 oncoprotein andp16INK4a immunohistochemistry.220 ESCC patients and 101 healthy controls provided serum samples for detection of HPV E6/E7 antibodies.An entire tissue sample was divided into different views at 400×magnification and views without epithelial tissues were excluded for further scoring.Then average viral copy number was calculated through dividing the total number of viral signals in all valid views from one sample by the number of valid views.Hr-HPV E6 mRNA level estimation was graded by average HPV viral copy number:<1 signal/view(-,negative),2-20 signal/view(-/+,equivocal),21～40 signal/view(+,positive),and>40 signal/view(++,strong positive).The χ2 test was used for comparison of clinical characteristics between HPV-positive and-negative groups.Rates of overall survival were estimated by means of the Kaplan-Meier method and Cox proportional-hazards models were used to estimate hazard ratios.SPSS v19.0 was used for calculation.Results:In our study,we found that transcriptionally active hr-HPV E6 mRNA exists in ESCC.Positive signals of RNAISH exhibited as dark brown,and most of positive signals in ESCC exhibited "dot" appearance with diameters of most signals smaller than 1.5 μm.A minority of signals were larger,whose diameters were larger than 1.5μm,exhibiting "clot" appearance.In ESCC cells,the hr-HPV E6 mRNA sporadically distributed in both cytoplasm and nuclear.In adjacent epithelium,hr-HPV E6 mRNA was rich in basal cell layer,which are highly proliferating.Classification of hr-HPV E6 mRNA levels in ESCC by defining a cut-off line at 20 signals/view,cases with hr-HPV E6 mRNA not weaker than positive level(+～++)composed 26.52%(96/362)of tumor samples,and 17.22%(52/302)of adjacent epithelium specimens.Notably,hr-HPV E6 mRNA level was higher in tumor tissue than adjacent epithelium.HPV16/18 E6 expressed in ESCC tumors tissues as well as adjacent epithelia,which mainly located in cytoplasm and were higher in tumor tissues than that in adjacent epithelium.The positive rate of HPV16/18 E6 was 44.9%(142/316)in ESCC tumors,whereas it was 32.9%(77/234)in adjacent epithelium.p16INK4a positive rate is 13.6%(46/338)in ESCC tumors,whereas it was 3.7%(12/321)in tumor adjacent epithelium.hr-HPV surrogate marker p16INK4a expression was found correlated with hr-HPV E6 mRNA transcription.The overall survival of patients with positive hr-HPV E6 mRNA(+～++,76/239)was significantly higher than that with hr-HPV E6 mRNA negative(-,75/239,P=0.002.).The middle survival time of patients with positive hr-HPV E6 mRNA was 37.86 months,in contrast to 21.78 months in negative hr-HPV E6 mRNA group.Accordingly,HPV16/18 E6 status,patients expressing HPV16/18 E6 oncoprotein showed a higher overall survival(P=0.045).The middle survival time of patients was 38.23 months in HPV16/18 E6 positive group(87/216)and 21.32 months in HPV16/18 E6 negative group(129/216).ESCC patients both positive in hr-HPV E6 mRNA and p16INK4a(17/223)exhibited a higher overall survival than both negative patients(61/223)(P=0.041.),with middle survival time of 53.97 months versus 22.38 months,respectively.High possibility of hr-HPV infection(RNA(+)PLUS,37/208).Comparing the group with whose hr-HPV E6 mRNA,HPV16/18 E6 and p16INK4a were all negative(Triple Negative,34/208),the overall survival of the Group RNA(+)PLUS was higher(P=0.005.).The middle survival time of Group RNA(+)PLUS was 52.12months,while it was 18.89 months in Group Triple Negative.Compared with hr-HPV E6 mRNA negative(-)group,tumors from ESCC patients with high level of hr-HPV E6 mRNA(++,strong positive)were prone to be poorer histologically differentiated(P<0.001).The group with positiveHPV16/18 E6 expression exhibited a higher proportion of poorer differentiate stage(P<0.001),and a higher proportion of smaller tumor size(P<0.001).Compared with sera from healthy control where barely signal was detected,sera from ESCC patients exhibited signals appeared on the membranes.A total 5 antigen determinants were identified.The seropositivity of HPV16 E6/E7 in ESCC cohort was 29%(29/100),whereas it was 13.58%in Healthy Control cohort(11/81).Conclusion:In conclusion,in this study we discovered a special hr-HPV positive subtype in ESCC via comprehensive information of hr-HPV E6 mRNA level,HPV16/18 E6 expression and surrogate marker of transforming hr-HPV infection,p16INK4a.Notably,the ESCC subgroup showed a better prognosis compared with negative hr-HPV infection patients.We identified 9 B cell linear epitopes of HPV16 E6/E7 oncoproteins in ESCC,providing experimental foundation for the development of therapeutic vaccine targeting the subtype.Our data implies a distinct role of hr-HPV in ESCC progression and highlights the clinical outcome of hr-HPV infection.The main innovation:□ In situ observation of hr-HPV E6 mRNA in ESCC revealed a subtype ESCC cases with positive hr-HPV infection.The higher survival of transcriptionally active ESCC patients suggests hr-HPV contribute to ESCC progression.Epitopes against HPV16 E6/E7 oncoproteins in ESCC were identified. |
PDF Full Text Request