Persistent HPV infection plays a major role in cervical cancer. This study was undertaken to identify HPV types in a cohort of Indian women with locally advanced cervical cancer as well as to determine the physical state and/or site of viral integration in the host genome. Pretreatment biopsies (n = 270) from patients were screened for HPV infection by a high throughput HPV genotyping assay based on luminex xMAP technology as well as MY09/11 PCR and SPF1/2 PCR. Overall HPV positivity was observed to be 95%, with HPV16 being most common (63%) followed by infection with HPV18. Integration status of the virus was identified using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay in a subset of samples positive for HPV16 and/or HPV18 (n = 86) and with an adequate follow-up. The data was correlated with clinical outcome of the patients. Integration of the viral genome was observed in 79% of the cases and a preference for integration into the chromosomal loci 1p, 3q, 6q, 11q, 13q and 20q was seen. Clinical data revealed that the physical state of the virus  could be an important prognostic marker for cervical cancer. Cervical cancer is the third most common cancer among women worldwide and the most common cancer found in Indian women. HPV infection has been shown to play a critical, though not sufficient, role in the etiology of cervical cancers. Till date more than 200 HPV types have been reported, of which HPV16 is most common, followed generally by HPV18, HPV45, HPV31 and HPV33 Studies report that the integration event is random involving almost all the chromosomes, and accordingly several virus-host integration sites have been mapped till date The aim of this study was HPV genotyping and identification of site of integration of two HR-HPV types (16 and 18), along with evaluation of the prognostic value of integration site, in locally advanced cervical cancers in Indian women.Pretreatment cervical tumor biopsies, predominantly from FIGO stage IIIB, were obtained from patients  undergoing radiotherapy alone or concomitant chemo-radiation at the Radiation Oncology Department, Tata Memorial Hospital, Mumbai, after obtaining IRB approval. A generic consent for basic research was obtained prior to obtaining the biopsies. However, for the current study a consent waiver was obtained from the Hospital Ethics Committee. The biopsies were obtained from histologically proven, primary cervical tumor, before the start of radical radiation therapy and were coded for de-identification by the physician prior to testing. The samples were collected in liquid nitrogen and stored at −80°C until further use. All the samples were assigned a laboratory code to maintain confidentiality.Frozen tissues were cryocut for extraction of DNA and RNA. For DNA extraction, five 30 µm sections were collected in STE buffer  containing 10 mg/ml Proteinase K . DNA was isolated by standard phenol-chloroform method. For isolation of total RNA, RNeasy Mini Kit  was used. Ten 30 µm sections were collected in RLT buffer containing guanidine thiocyanate provided in the kit and processed following manufacturer's instructions. DNAse treatment of the RNA samples was carried out using DNA free kit .2, 200 µM dNTPs, 0.75 unit of Taq DNA polymerase and 1µl primer mix. The amplification steps included an initial DNA denaturation at 94°C for 5 min, followed by 40 cycles of denaturation for 20 s at 94°C, annealing for 30 s at 38°C, and extension for 1 min 20 s at 71°C, before a final extension for 4 min at 71°C. PCR positive samples were then subjected to the luminex run. Ten microlitres of the PCR product was mixed with the luminex bead mix containing distinct bead populations coupled to 24 HPV types. After thermal denaturation, the target sequences were hybridized to bead-bound probes. The hybridized PCR products were labeled by binding to R-phycoerythrin conjugated streptavidin. The read-out was obtained in the luminex bioanalyzer . HPV types were discerned according to the unique bead signature, whereas the presence of PCR products was determined by phycoerythrin fluorescence. An analytical sensitivity cut-off was calculated based on the negative control which was deducted from each of the read-out.Genotyping of 24 HPV types which included 15 high-risk types , 3 putative high-risk types , and 6 low-risk types  +/6+ primers (n = 92). These were further screened for HPV by PCR using MY09/11 L1 primers  was done to determine association between infection with these HR-HPV types and recurrence of disease. Disease free survival was considered from start of radiation therapy to the time when recurrence occurred or till last follow-up. Statistical significance was evaluated using the log-rank test (SPSS 15.0).et al2, 200 μM dNTPs, 25 µM of each primer and 1 unit of Taq DNA polymerase. The reaction comprised of an initial denaturation step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 4 min. This was followed by a final extension at 72°C for 20 min. Next, 7 µl of the PCR product was used as template for nested PCR using forward primers p2–16 specific for HPV16 or p2–18 specific for HPV18 and (dT)17–p3 as reverse primer (Samples (n = 86) positive for HPV16, HPV18 or both and with an adequate clinical follow-up  were taken to study viral integration using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay, as described by Klaes e primer . The PCRAmplicons other than the major episomal transcripts (∼1050 bp for HPV16 and ∼1000 bp for HPV18) were suspected to be derived from the integrated HPV genomes. These were excised from the gel and DNA isolated using GFX PCR DNA and Gel Band Purification Kit . The isolated DNA was either sequenced directly or following cloning into pTZ57R/T vector using the InsTA PCR Cloning Kit , on DNA sequencer . The chromosomal integration loci were determined using National Centre for Biotechnology Information (BLAST) and the University of California, Santa Cruz (UCSC) hg19 (Feb. 2009) (BLAT) human genome assemblies. Further, the integration sites were checked for the presence of fragile sites and any genes of known identity by using NCBI fragile site map viewer and the UCSC Blat tool respectively.The data obtained was compared with the clinical outcome of the patients. Kaplan-Meier analysis (SPSS 15.0) was done to determine the association of the viral state  with recurrence of the disease. Disease free survival was considered from start of radiation therapy to the time when recurrence occurred or till last follow-up (median follow-up for 86 cases was 44 months). Statistical significance was evaluated using the log-rank test (SPSS 15.0).+/GP6+ primers. Using this primer set for PCR, we obtained 178 out of 270 samples positive for HPV . Further genotyping of the 79 samples, using HPV 16/18 specific primers, showed 49 samples to be positive for HPV16. None of these 79 samples were positive for HPV18. Therefore, the prevalence of HPV16 and/or HPV18 in this cohort was 69% (185/270) .Kaplan-Meier survival analysis data for 125 patients with HPV type16, 18 and dual infection and with adequate clinical follow-up (median follow-up for 125 cases was 54 months), revealed that there was no significant difference between infection with these two HR-HPV types in terms of disease outcome .Out of 125 HPV16, HPV18 or dual HPV positive samples, a sub-set of 86 samples with good quality RNA, were taken to study the physical state and/or site of viral integration by APOT assay. In most of the cases, the viral genome was found to be integrated (n = 68), whereas in 21% (n = 18) only episomal transcripts could be identified. In 12 cases with integrated viral genome, episomal form of HPV was also detected . The phyIn order to understand whether the integration event is random or there is some preference for certain sites within the chromosomes, the sequencing data for 68 cases derived from APOT assay were investigated by Blast and/or Blat. The site of integration could be predicted with a high score in 48 cases , for theUsing NCBI Fragile site Map Viewer it was observed that 60% of integrations (29/48) were located in or close to a common or rare fragile site. The rest of the integration sites were not associated with any fragile sites . Using tIt is proven beyond doubt that infection by HPV plays a major role in the etiology of cervical cancer. Reports from different parts of the sub-continent indicate a prevalence of HPV ranging from 73–99% Integration of the virus is common in late stage cervical cancers and is considered an important event in the progression of the disease. Integration generally occurs downstream of the early genes E6 and E7, often in the E1 or E2 region. The E2 gene is transcriptionally inactivated once the virus gets integrated due to disruption of its open reading frame. Viral E2 gene has been extensively studied and is known to play a role in viral replication as well as negative regulation of E6 and E7 genes + tumors + tumors + samples where the virus was integrated and one HPV18+ sample where the virus was episomal. The incidence of integration in HPV16+ samples was higher. The mechanism of HR-HPV integration is not fully understood. It is speculated that integration might represent a chance occurrence, the probability of which increases with the frequency of double-strand breaks (DSBs) in host and viral DNA. Chromosomal fragile sites could represent the hotspots for HR-HPV integration.Various techniques have been used to study integration of the virus, such as Ligation-mediated PCR The physical state and/or site of integration was studied in 86 cervical tumour samples infected with either or both of two HR-HPVs, HPV16 and HPV18, and with an adequate clinical follow-up. Episomal form of HPV was observed in 18 cases. Presence of only episomal form in these patients with advanced disease stage (predominantly FIGO stage IIIB), could indicate that either HPV integration is not solely responsible for the progression of the disease; or it could be a limitation of the technique resulting in failure of amplification of the integrant derived transcript. Recent studies have confirmed presence of only episomal form of the virus in advanced cervical squamous cell carcinomas trans to modulate the expression levels of oncogenic E6 and E7 et al., loss of episomes is as much important as integration of the virus into the host genome for progression of lesions to cervical neoplasia In the present study, we observed 12 samples where both integrated as well as episomal forms of the virus were present. In such cases, E2 may be available in There are reports that HR-HPV proteins other than E6/E7 induce chromosomal instability and transformation Although integration sites were distributed throughout the genome in different samples, there was a preference for certain chromosomal loci such as 1p, 3q, 6q, 11q, 13q, 6q and 20q. Certain specific regions in some of these loci such as 1p36.23, 1p36.33, 3q26, 3q28 and 20q11.21 showed repeated integrations, indicating that integration might not be a random event. Reports from studies involving western populations indicate that integration of the virus occurs most commonly at 8q chromosomal locus The 3q, 13q and 20q loci besides being preferential target for HPV integration have been reported to be sites for genomic instability associated with cervical cancer. Gain of 3q and 20q, while loss of 13q has been reported in various stages of the disease et al wherein they have shown involvement of tumour related genes (myc and TP63) in HPV integration process Most of the integrations (28/48) were found to be located within or near certain genes. This could indicate that the virus prefers transcriptionally active regions for the integration event. Such genes included oncogenes such as myc, transcription factors like TP63, MECOM, etc. This observation is supported by previous report by Wentzensen Integration of the virus near or within fragile sites has frequently been reported Figure S1Kaplan-Meier analysis for two HR-HPV types – HPV16 and/or HPV18 and disease outcome. Kaplan-Meier survival analysis for HPV16 and/or HPV18 in 125 patients who had a good clinical follow up was carried out. Patients with HPV16 infection alone showed a trend towards better disease free survival as compared to HPV18 infection alone and dual infection with HPV16/18.(TIF)Click here for additional data file.Figure S2Genomic DNA PCR of the recurrent integration sites. Representative gel images  showing HPV integration at the genomic level. Recurrent integrations at chromosomal loci 1p36.23, 3q28, 6q23.3, 8p11.21 and 11q13.1 is depicted.(TIF)Click here for additional data file.Table S1Primer sequences.(DOCX)Click here for additional data file.Table S2Clinicopathological data for all cases where the viral integration was studied.(DOCX)Click here for additional data file.Table S3Sequence of HPV integration sites in the genome in 68 cases. a) Integration sites for 48 cases with a high prediction score. b) Integration sites for 20 cases with a low prediction score.(DOCX)Click here for additional data file.


As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk , heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV-negative and HPV-positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV-positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high-risk (hr)HPV versus low-risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV.The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens ( Although cervical cancer is a model for early cancer detection, it remains one of the most important causes of mortality affecting women worldwide. Molecular and epidemiological data proved that human papillomavirus (HPV) is the aetiological agent of cervical cancer In this context, some investigations have been focused on the role of folate in carcinogenesis, including cervical one C677T  and A1298C . A1298C polymorphism has a lower effect in reducing enzyme activity, compared with the C677T mutation, which determined different enzyme thermolability degree . The enzyme residual activity is almost 18–22% in 677TT individuals compared with heterozygous 677CT (56%) and normal 677CC (66–67%) Although multiple polymorphisms in MTHFR gene were described, epidemiologic studies have shown that only two of them affect the enzymatic activity and are related to different disorders including malignancies 677CT heterozygote patients were reported to have mild elevation levels of plasma homocysteine and non-methylated folate in red blood cells (both conditions involving impairment in 5-methylTHF synthesis) 677TT genotype was frequently associated with different types of cancer  and cardiovascular diseases A1298C polymorphism correlates with ovarian cancer and lymphoblastic acute leukaemia Whereas et al. performed an integrated analysis of gene expression related to CpG islands methylation. They concluded that differentially expressed genes which exhibited differential methylation pattern of one or more CpG islands within the region promoter of the gene, presented an inversely correlation between mRNA abundance and CpG methylation status It is already known that MTHFR polymorphisms can modulate DNA methylation status and the alteration of DNA methylation pattern is associated with carcinogenesis This study aimed to investigate MTHFR polymorphisms and the methylation pattern of MTHFR promoter in relationship with susceptibility to cervical lesions and cervix papillomaviruses infection in Romania, which has one of the highest rates of cervical cancer in Europe .n = 77) from women with dysplastic cervical lesions were collected by a gynaecologist, and conventional cytological screening or histological exams of the samples were performed by a trained pathologist. According to cytological investigation, the cervical samples were divided as: ASCUS  , LGSIL   and HGSIL  . Tissue specimens (n = 23) were obtained from patients with squamous cervical carcinoma (SCC) who underwent surgery for cervix tumour mass removal . Tumours were immediately stored at −80°C.Investigated patients were selected based on suggestive cervical pathology for human papillomavirus infection. Cervical cytology specimens (n = 50) with negative cytological smears attending gynaecological clinic for regular investigations . For nucleic acids preservation all samples were collected in sterile tubes with ESwab Collection and Transport System medium . Blood samples used for the detection of MTHFR polymorphisms were collected on EDTA from all the women enrolled in this study. No age or ethnicity exclusion criteria were used.Control group consisted of women  was performed with High Pure PCR Template Preparation Kit , according to manufacturer's recommendations. Isolated DNAs were subsequently stored at −20°. The concentration and purity of each DNA sample was evaluated with NanoDrop spectrophotometer .Human papillomavirus genotyping was performed with Linear Array HPV Genotyping Test , according to the manufacturer's instructions. A pool of biotinylated primers amplify near 450 base pairs from L1 gene of 37 HPV genotypes including 13 high-risk types . An additional primer pair targets the human β-globin gene to provide a control for cell adequacy, extraction and amplification.Bisulphite treatment for unmethylated C residues conversion was realized with EpiTect Bisulfite kit . Seven-hundred nanogram of each isolated DNA  in a maximum volume of 20 μl was bisulphite treated along with positive  and negative  controls .Primers to distinguish methylated and unmethylated status of MTHFR following bisulphite treatments were designed using Methprimer  and were synthesized by Invitrogen Corporation :MTHFR methylated FW 5′-TAGATTTAGGTACGTGAAGTAGGGTAGAC-3′.MTHFR methylated R 5′-GAAAAACTAATAAAAAACCGACGAA-3′.MTHFR unmethylated FW 5′-TTTAGGTATGTGAAGTAGGGTAGATGT-3′.MTHFR unmethylated R 5′-CAAAAAACTAA TAAAAAACCAACAAA-3′.Taq DNA Polymerase (1 U), 1× enzyme buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 0.3 μM of each specific primers, 5 μl target DNA. The PCR conditions were as follows: 95°C – 5 min., 35 cycles of 95°C – 30 sec., 56°C – 30 sec., 72°C -30 sec. and a final elongation of 72°C – 7 min. The methylated/unmethylated status of the target gene was estimated in 2% agarose gel electrophoresis.Methylation-specific PCR was performed in a final volume of 25 μl using Platinum Direct Q-MSP of genomic DNA was used for evaluating the degree of sample methylation. Standard curves were designed using serial dilution  of positive (DNA fully methylated) and negative controls (DNA fully unmethylated), according to Applied Biosystem Guide . The quantity of unmethylated and methylated DNA for each patient sample was extrapolated using the standard curves.Direct Q-MSP was performed in 50 μl final volume comprising 50 ng/μl DNA, 25 μl FastStart Universal SYBR Green Master  and 0.3 μM primers. PCR parameters we used were as follows: 95°C/10 min. (1 cycle), 40 cycles at 95°C/15 sec. and 60 sec./specific annealing temperature.qPCR experiments were performed in duplicate and mean values were used for calculations. To control the quality of DNA samples we used ACTB gene as reference et al. (% M = 100× [ng methylated gene A/(ng methylated gene A+ unmethylated gene A)]) Methylation percentage was calculated according to method described by Fackler C677T and A1298C) were performed in PCR – RFLP technique, according to Frosst et al. C677T mutation were as follows: FW 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and R 5′-AGGACGGT GCGGTGAGAGTG-3′ whereas for A1298C polymorphism we used: FW 5′-GGGA-GGAGCTGACCAGTGCAG-3′ and R 5′-GGGGTCAGGCCA GGGGCAG -3′. The PCR conditions were as follows: for C677T polymorphism, 30 cycles of 94°C/45 sec., 65°C/45 sec., 72°C/45 sec.; for A1298C polymorphism, 35 cycles of 94°C/45 sec., 65°C/45 sec., 72°C/45 sec. 198 bp amplicons for C677T polymorphism and 138 bp amplicons for A1298C polymorphism were subjected to enzymatic restriction with Hinf I and Fnu4HI, respectively, according to manufacturer's instructions (New England Biolabs). Following digestion, the samples were evaluated in 3% agarose gel electrophoresis. Heterozygote (CT) presented three lanes of 198 pb, 175 bp and 23 bp whereas heterozygote (AC) displayed 138, 119 and 19 bp lanes.Methylenetetrahydrofolate reductase polymorphisms  was used to compare the variance between the investigated groups. The biological samples were provided with patient's agreement and the study protocol was approved by the Ethics Committee.Statistical analysis was performed with GraphPadInstat3. The association between MTHFR polymorphisms and Pap test was analysed using Fisher's exact test, wherever appropriate. The confidence interval was established at 95%. Viral test results for each category of investigated patients are presented in C677T polymorphism in all patients included into the study revealed that 75 cases were homozygote for normal allele (CC), 65 were heterozygote (CT) and 10 were mutant homozygote (TT). The calculated frequency of C allele in population was 0.72, whereas the frequency of T allele was 0.28. The χ2 test was performed with 1 degree of freedom and 5% level of significance (3.84). The value of χ2 test was 0.674 (below 3.8), the population being in Hardy–Weinberg equilibrium. The calculated frequency of A allele for 1298 polymorphism in our patients was 0.75, whereas the frequency of C allele was 0.25. Eighty-one of the investigated patients were normal homozygous (AA), 63 were heterozygous (AC) and 6 were mutant homozygous (CC). The population was in Hardy–Weinberg equilibrium, the value of χ2 test being 2.16.The analysis of MTHFR T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk , heterozygote 14/51(0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). By contrast, the MTHFR polymorphism A1298C was not associated with cervical disease development , heterozygote 19/44 (0.38/0.44), mutant homozygote 1/5 (0.02/0.05); OR = 1.441, P = 0.2278).When comparing patients with cervical lesions  with the normal group, a statistically significant association was found between T allele of C677T polymorphism (P = 0.0287). The results of MTHFR polymorphisms in the investigated groups are presented in C677T: wild homozygote 21/5, heterozygote 36/5, mutant homozygote 3/3, P = 0.07; A1298C: wild homozygote 34/6, heterozygote 22/3, mutant homozygote 3/0, P = 0.44).Furthermore, we evaluated a possible link between MTHFR polymorphisms and HPV infection. Using chi-square test for control group, HPV-negative and HPV-positive patients with cervix lesions, we found a significant correlation between viral infection and R2) around 0.999 providing evidence of linearity over the entire range of template concentration.The methylation percentage (% M) value for negative standard was approximately 0 and no amplification of methylated standard with unmethylated primers was obtained. We validated our data regarding quantitative methylation analysis based on the similar differences between ΔCt values (ΔCt = CtM − CtU) for all dilutions (ΔCt ≍ 0.726). On the other hand the slope value for all the standard curves were around the −3.16 value that means a twofold increase in PCR product per cycle during the linear phase of real-time PCR. The average of calculated efficiencies was around 107.3. The standard curves showed a correlation coefficient . Moreover, statistic differences (P = 0.0026) between samples from patients with hrHPV infection versus lrHPV were obtained  in our samples of around 6.67%, which is in agreement with data published by Osian et al. C677T polymorphism in correlation with the severity of cervix lesion associate the presence of T allele with cervical cancer susceptibility (P = 0.0035). Our results are similar with the study of Goodman et al. et al.et al. reported that women with CT genotype had twice the risk of cervical LGSILs, whereas TT genotype women had almost three times the risk of LGSILs compared with women with the homozygous MTHFR CC genotype The impairment of MTHFR enzymatic activity through  P = 0.00. Our res P = 0.00. Our reset al. 677T allele of MTHFR is a protective factor against cervical carcinogenesis. Moreover, the results from our investigation support the hypothesis that individuals carrying the T allele are susceptible for acquiring HPV cervix infection (P = 0.0287). The susceptibility of C667T mutation carriers individuals to HPV infection is due to genomic instability We investigated patients with different cytology, including cancer. Overall, for both CT and TT genotypes we found a threefold increased risk for cervical lesion development. These observations are in contrast with Agodi T allele (C677T polymorphism) had a 14-fold increased risk  for cervical dysplasia compared with HPV-negative women with the CC genotype. On the other hand, the T allele is almost sixfold more frequent in patients with tumours than in control group . No association between HPV types (high or low risk) and MTHFR polymorphisms was found. Mei et al. highlighted that the association between C677T MTHFR polymorphism and cervical cancer could be affected by HPV status. C677T polymorphism might lead to cervical cancer by impairing DNA methylation in both host and viral genomes In our study, HPV-positive women who had at least one variant of et al. A1298C polymorphism and cervical cancer. Gathering these findings, we support the idea that MTHFR polymorphism presence could represent a risk factor for cervical HPV acquisition and persistence. Furthermore, it is assumed that MTHFR plays an important role in DNA methylation, an important epigenetic mechanism which modulates gene expression. Differences in genome-wide methylation are linked with pathological conditions, including cervical neoplasia On the other hand, our findings concerning the Romanian women assert the results of Kang P < 0.0001). These findings support the involvement of papillomaviruses in oncogenesis, a process associated with aberrant methylation. No association between MTHFR genotype and MTHFR methylation was found. Therefore, we concluded that MTHFR polymorphism is a factor for promoting cervical dysplasia, but promoter methylation might result from cervical lesion severity linked to viral infection. This scenario is supported by the fact that the methylation level of MTHFR seems to vary between high-risk and low-risk HPV types. Moreover, the most powerful methylator HPV type is HPV16, in single- and co-infections. To our knowledge, these data has not been reported before.We found that MTHFR hypermethylation is rare in LGSIL lesion, but more frequent in HGSIL and cancer lesions. Significant differences were observed between HPV-positive samples, control group and HPV-negative dysplastic lesions (C677T and not the A1298C MTHFR polymorphism represents a risk factor of cervical dysplasia. MTHFR promoter methylation varies between high-risk and low-risk HPV types, with HPV 16 being linked to most methylation changes in the promoter. No link between MTHFR polymorphism at C677 and MTHFR promoter methylation was observed. Given that in Romania a high prevalence of HPV infection is found, our patient cohort might prove valuable for studies of the dynamics of MTHFR promoter methylation.Our study shows that the


While immunosuppressive regimens improve the overall survival of renal transplant recipients, they also contribute to the long-term complications of post-transplant malignancies. Chronic immune suppression in renal transplant recipients (RTR) increases the risk of viral-associated cancers. In male RTR, human papillomavirus (HPV) is implicated in the development of penile, anal, oropharyngeal, and non-melanoma skin carcinomas. Despite the significance of this virus in RTR, there is an overall deficiency in the understanding of the natural history of HPV infection in male RTR. In the next 20 years, it is believed that cancers will be the leading cause of death in kidney transplant recipients. HPV-associated carcinomas are of particular interest since they are sexually transmitted and in theory may be preventable diseases. This commentary highlights some of the progress made in understanding how HPV is transmitted amongst couples in the general population. It also summarizes the current knowledge of HPV infection in male RTR and describes the deficiencies in published medical literature. Renal transplant recipients (RTR) have an increased incidence of human papillomavirus (HPV)-related malignancies, however, studies on the natural history of HPV infection are lacking especially in male RTR. While immunosuppressive regimens improve the overall survival of renal transplant recipients, they also contribute to the long-term complications of post-transplant malignancies -6. In main-situ hybridization, PCR, or histopathology. We excluded case reports and studies that evaluated men that had sex with men or anal HPV infection because while it is important to look at all groups of men in the RTR population, heterosexual men have been extremely under-represented in studies, yet they make up the majority of male RTR. Through our search, only 19 citations were identified examining anogenital infection in male RTR; however, they were excluded based on our criteria. To our surprise, there are no published studies that have been done reporting the prevalence of genital HPV in heterosexual male RTR, despite the fact that thousands of men have undergone kidney transplantation.Our group attempted to establish the prevalence of genital HPV infection in males post kidney transplantation by conducting a systematic review of the published medical literature in the Medline database. Our inclusion criteria consisted of studies that examined HPV by et al.[et al.[In addition to urogenital malignancies in RTR, HPV infection has been implicated by some investigators to be associated with having an increased risk of squamous cell carcinoma (SCC) of the skin -13. Unliet al., investil.[et al., investil.[et al., studiesl.[et al.,16.When looking at screening tests for HPV in men, the options are limited. Unlike women where established commercial tests are available, HPV testing in men in the clinical setting is not done and published reports in the general population use only the research methods. This is another factor which has limited research for studying HPV infection in men. In women, a localized sample of cervical mucosa is easily collected to test for HPV DNA, while in men the cornified epithelium of the penis and scrotum does not always provide a reliable collection of relevant cells . TherefoIn the next 20 years, it is believed that cancer will be the leading cause of death post transplantation . HPV-assThe authors declare that they have no competing interests.All authors participated in writing, reading, editing, and approving the final manuscript.


Human Papillomavirus (HPV) in cervical epithelium has been identified as the main etiological factor in the developing of Cervical Cancer (CC), which has recently become a public health problem in Mexico. This finding has allowed for the development of vaccines that help prevent this infection. In the present study, we aimed to determine the prevalence and HPV type-distribution in Mexican women with CC, high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL), and Normal cytology (N) to estimate the impact of the HPV vaccines.The PubMed database was used to identify and review all articles that reported data on HPV prevalence in CC, precursor lesions, and normal cytology of Mexican women.A total of 8,706 samples of the tissues of Mexican women were stratified according to diagnosis as follows: 499 for CC; 364 for HSIL; 1,425 for LSIL, and 6,418 for N. According to the results, the most prevalent genotypes are the following: HPV16 (63.1%), -18 (8.6%), -58, and −31 (5%) for CC; HPV-16 (28.3%), 58 (12.6%), 18 (7.4%), and 33 (6.5%) for HSIL; HPV-16 (13.1%), 33 (7.4%), 18 (4.2%), and 58 (2.6%) for LSIL, and HPV-16 (3.4%), 33 (2.1%), 18, and 58 (1.2%) for N.Taken together, genotypes 58 and 31 (10%) are more common than type 18 (8.6%) in CC. Therefore, the inclusion of these two genotypes in a second-generation vaccine would provide optimal prevention of CC in Mexico. Cervical cancer (CC) is one of the main causes of death in women in developing countries . In 2008There are previous meta-analysis papers, which report information on the HPV-type prevalence distribution in precursor lesions and CC worldwide -8. HowevIn order to have a possible HPV status in Mexico, the most complete of the already published data on cervical lesions and normal cytology were reviewed.In the present report, we analyzed the HPV prevalence from 12 already published reports that met the aforementioned criteria. There were a total of 8,706 cases, of which 499 were identified as CC, 364 as HSIL, 1,425 as LSIL, and 6,418 as N. Overall, HPV prevalence was 95, 75.5, 42, and 15%, respectively  (Table p = 0.000), while in HPV18 and 31 it was not (p >0.05) (Table p <0.05) (Table p <0.05), but in HPV33, the difference was not statistically significant (p >0.05)  were assessed by contingency tables and the 5) Table . The difThe relative risk  for HPV-16 infection between CC and HSIL was 4.3, and increased between CC and LSIL (11.9) and between CC and normal (49.1)  and late activity genes . The molecular evidence indicates that the oncogenic HPV proteins E6 and E7 are responsible for the inactivation of certain cell cycle regulatory proteins, such as p53 and pRb, respectively. The uncontrolled proliferative cellular activity that characterizes cells transformed by oncogenic HPV infection in CC and other cancers associated with this infection is due to the loss-of-function of proteins that arrest the cell cycle. Thus, this infection with oncogenic HPV results must play a central role in the developmental model of the CC +direct sequencing, or 2) PCR+restriction fragment length polymorphisms, or 3) PCR+reverse line blot.A search of the literature identified 51 studies, of which only 12 could be included in the final meta-analysis. Exclusion criteria were the following: a) only HPV16 and −18 genotypes were studied; b) HPV type was not specified (hybrid capture); c) number of cases was carried out for <10; d) a method other than PCR was used to detect HPV types, e) the histological category was not reported as normal, LSIL, HSIL, or CC.Overall, HPV prevalence was estimated by dividing the total number of subjects for each histological group by the number of HPV-positive cases. Type-specific HPV prevalence is presented for each histological classification for all HPV types identified. Multiple infections were not considered in this analysis.x2 test was conducted and considered of statistical significance with a level of p <0.05. Type-specific HPV prevalence was compared between HSIL/LSIL/normal and CC by Prevalence ratios (PR) and Relative risk (RR) with 95% Confidence intervals (95% CI). The analyses were performed by SPSS v15 software.The differences in HPV prevalence for each group  were assessed by contingency tables. The The authors’ declare that they have no competing interests.RP, BG and MS conceived and designed the study, analyzed data and drafted the manuscript, PR, VV, MM, KT and DM helped with statistical analysis. All authors read and approved the final manuscript.


Human papillomavirus (HPV) vaccine prevents cervical pre-cancers and cancers caused by HPV types 16 and 18. This study provides information on the HPV types detected in cervical cancers of Alaska Native (AN) women.Cases of invasive cervical cancer diagnosed in AN women aged 18 and above between 1980 and 2007 were identified from the Alaska Native Tumor Registry. A representative formalin-fixed, paraffin-embedded archived pathology block was retrieved and serially sectioned to allow histologic confirmation of lesion (first and last sections) and PCR testing of intervening sections. Extracted DNA was tested for HPV using Linear Array HPV Genotyping Test (Roche Diagnostics) with additional INNO-LiPA HPV Genotyping Assay (Innogenetics) testing on negative or inadequate specimens. All specimens were tested for a minimum 37 HPV types.Of 62 cervical cancer specimens evaluated, 57 (91.9%) contained one or more HPV types. Thirty-eight (61.2%) cancers contained HPV types 16 or 18, and 18 (29%) contained an oncogenic type other than type 16 or 18.Overall, almost two-thirds (61.2%) of the archived cervical cancers had detectible HPV types 16 or 18, a finding similar to studies of US women. As expected, a proportion of cancers would not be prevented by the current vaccines. HPV vaccination and cervical cancer screening are important prevention strategies for AN women. Cervical cancer rates were  average .Infection with HPV causes virtually all cervical squamous cell carcinomas and cervical adenocarcinomas –5. StudiOur study assessed HPV genotypes found in invasive cervical cancer specimens from AN women diagnosed from 1980 to 2007. The purpose was to characterize the preventable burden of HPV-associated cervical cancers in AN women to assess the potential impact of HPV vaccination on cervical cancer in this population.Invasive cervical cancer diagnosed from 1980 to 2007 in AN women living in Alaska aged 18 years and older were identified through the Alaska Native Tumor Registry . One repFrom 1980 to 2007, a total of 139 AN women were diagnosed with invasive cervical cancer while a resident of Alaska. Among these, a total of 136 were ≥18 years of age and 90 were diagnosed at the ANMC in Anchorage, Alaska. Archived tissues were located for 71 (79%) of ANMC cases >18 years and 62 (69%) contained representative cervical tumor.The median age of the 62 women with an archived specimen available was 41 years, the same as the median age for all AN women diagnosed with invasive cervical cancer during the period 1980–2007 . Among wAll 62 tissue specimens available for HPV typing yielded adequate results: 57 (91.9%) had HPV detected; 56 (90.3%) specimens were positive for one or more HPV types considered oncogenic . HPV 16 The histologic types identified from 62 specimens were: squamous cell carcinoma (84%), adenocarcinoma (11%), and carcinoma, not otherwise specified (5%). Among the squamous cell carcinomas, HPV 16 was detected in 48% and HPV 18 in 12%. Among the adenocarcinomas, HPV 16 was detected in 29% and HPV 18 in 29%.This study of cervical cancers in AN women found that 61% of tumors had evidence of HPV 16 or HPV 18. This is similar to findings from other studies on US women, in which approximately 70% of tumors were attributed to HPV 16 or 18 , 7. An HOur study found some differences in HPV types in cervical cancer compared to the only prior study in AN women. In the prior study of cervical cancer specimens from AN women diagnosed from 1980 to 1989, 53 formalin-fixed and paraffin-embedded cervical cancer biopsies were assayed by PCR for 6 HPV genotypes  .Studies in which HPV genotypes are detected in invasive cervical cancer provide important information to understand the impact HPV vaccines might have on reducing cervical cancer in defined populations. To date, most studies of different regions/ethnicities find similar proportions of cancers caused by HPV 16 or 18. Our study on AN women found a lower prevalence due to HPV 16 or 18, but given smaller numbers this is consistent with other studies. Evaluations of cancers due to HPV 16 and 18 in specific populations as well as vaccine impact monitoring on cancer reduction is warranted. One-third of all cervical cancers are caused by HPV types not prevented by vaccine, and provide support for continued Pap screening.Overall, almost two-thirds (61.2%) of the archived cervical cancers had detectible HPV types 16 or 18, a finding similar to studies of US women. As expected, a proportion of cancers would not be prevented by the current vaccines. HPV vaccination and cervical cancer screening are important prevention strategies for AN women.


Human papillomavirus (HPV) vaccine recommendation by a health care provider (HCP) is an important predictor of vaccine receipt. We examined whether being of a minority race/ethnicity, having lower income and education, and the lack of health insurance and a regular HCP are each associated with a lower likelihood of a discussion on HPV vaccine occurring between a woman and her HCP.A sample of 1,631 women aged 18 years and older was drawn from the 2007 Health Information National Trends Survey. Given that only a subgroup of women who were aware of the HPV vaccine were asked if they had a discussion with their HCPs, we estimated a probit model correcting for sample selection.Among those aware of the HPV vaccine, 17.3% of respondents reported having discussions on the vaccine with their HCPs. Compared with Whites, African Americans were less likely to be aware of the HPV vaccine but more likely to have discussions with their HCPs concerning the vaccine. A statistically significant association between lower income and education levels and a lower likelihood of HPV vaccine awareness was observed, but low levels of income and education did not appear to affect the probability of having HPV vaccine discussions with HCPs.Socioeconomically disadvantaged women did not show a lower propensity to have vaccine discussions with their HCPs, suggesting that HCPs can be a major catalyst in increasing vaccine receipt among the higher risk group. The results of the study suggest a two-pronged approach that seeks to raise vaccine awareness among socioeconomically disadvantaged women at the population level and encourages HCPs to intensify discussions about the HPV vaccine with patients. Women at high risk of cervical cancer in the United States tend to have less income, less education, and are likely to belong to racial/ethnic minorities, namely African Americans, Hispanics, and Asians-4. This The unequal adoption of HPV vaccination can exacerbate the socioeconomic disparities in cervical cancer rates. Many studies have identified predictors of HPV vaccine uptake to address this disparity; most have found HPV vaccine recommendation from a health care provider (HCP) to be an important predictor of vaccine receipt-21, bothDespite the potentially important implications for both clinical and public health practice, research regarding HPV vaccine discussions between patients and their HCPs is currently lacking. This study aimed to identify socioeconomic factors associated with having a patient- or HCP-initiated discussion about the HPV vaccine among a nationally representative sample of American women. As women of lower socioeconomic status are less likely to have adequate access to health care, we postulated that interaction with HCPs follows the same disparate trajectory. Specifically, we hypothesized that being in a minority race/ethnicity, having lower income and education, and the lack of health insurance and a regular HCP are each associated with a lower likelihood of a discussion on HPV vaccine occurring between a woman and her HCP.The data for this study were drawn from the National Cancer Institute’s 2007 Health Information National Trends Survey (HINTS). The HINTS routinely collects nationally representative data on the US public’s use of and access to cancer-related information, health communication trends and practices, and the public’s perceptions of health risks. HINTS respondents were recruited via two modes: (1) random digit dialing of telephone numbers for recruiting respondents in computer-assisted telephone interviews (CATI) and (2) using mail surveys from a national listing of addresses available from the United States Postal Service. Response rates for the CATI and mail surveys were 24.2% and 31.0%, respectively. Detailed information on the HINTS survey methods can be found elsewhere. BecauseThe outcome of interest was a binary indicator variable derived from the question, “Has a health care provider such as a doctor or nurse ever talked to you about a cervical cancer vaccine or HPV shot?” This question did not differentiate between a respondent-initiated and a HCP-initiated discussion. Only the respondents who reported having heard of the cervical cancer vaccine or HPV shot before taking part in the survey were asked this question.household income , education , race/ethnicity , and age group , as well as any health care coverage, and any regular HCP. Health care coverage in the HINTS 2007 referred to health insurance, prepaid plans such as health maintenance organizations (HMOs), and government plans such as Medicare. Likewise, HINTS 2007 defined a HCP as a doctor, nurse or other health professional but not a psychiatrist or other mental health professional. For two of these variables – any health care coverage and any regular HCP – responses of ‘yes’ were coded ‘1’ while responses of ‘no’ were coded ‘0’.This study used socioeconomic and demographic characteristics as explanatory variables. These variables included In this study, only women who reported being aware of the HPV vaccine were asked to answer the question on having any discussions with their HCPs about the vaccine. However, HPV vaccine awareness may favor women with more advantageous socioeconomic characteristics. Thus, in terms of having discussions on HPV vaccine with a HCP, a statistical model that only includes respondents who were aware of the vaccine may result in sample selection bias. This issue compromises the study’s internal validity resulting in an underestimation or overestimation of the effect of socioeconomic factors on the likelihood of patients having a discussion on HPV vaccine with their HCPs. External validity is likewise compromised because estimates from the sample selected are not generalizable to the population.To address sample selection bias, we used the heckprob command in Stata , which simultaneously estimates the main probit model of HPV vaccine discussion and the selection model of being aware of the HPV vaccine, while correcting for the potential sample selection bias,24.Discuss* is a latent variable measuring the propensity of a vaccine discussion occurring between a respondent and her HCP and Discuss is a dichotomous variable indicating whether or not an HPV vaccine discussion has occurred between a respondent and her HCP. X and β1 refer to the matrix of explanatory variables and corresponding coefficients, respectively; ε1 is the error term. A positive coefficient in β1 indicates that the propensity of a woman to have a discussion on the HPV vaccine with her HCP increases with a higher value in the corresponding variable. Discuss is only observed when respondents were aware of the HPV vaccine (Aware = 1).where Aware* is a latent variable measuring the propensity of being aware of the HPV vaccine and Aware is a dichotomous variable indicating whether or not a respondent is aware of the HPV vaccine. X and β2 respectively denote the same matrix of explanatory variables and corresponding coefficients, and ε2 is the error term. A negative coefficient in β2 indicates that the propensity of a woman to be aware of the HPV vaccine decreases with a higher value in the corresponding variable.where ρ) between ε1 and ε2 to test for sample selection bias. A p<0.05 cutoff was used to determine statistical significance for all analyses. Data were weighted to adjust for survey design.Our model used the correlation coefficient  – women aged 45 years and above, belonging to an ethnic minority group, with household income below $50,000 or with education levels lower than or equivalent to high school were less likely to be aware of the HPV vaccine.ɛ2) in the model of awareness of HPV vaccine were correlated with unexplained factors (ɛ1) that predict discussion with HCPs. The negative sign indicates that ɛ1 and ɛ2 are negatively correlated.The statistically significant correlation coefficient of the probit model , who did not find evident socioeconomic disparities in the odds of reporting HCPs as previous sources of HPV vaccine information[The primary goal of this study was to examine socioeconomic differences in the likelihood of a discussion on HPV vaccine occurring between a woman and her HCP using a model that accounts for potential sample selection bias. Our analysis suggested that African American women were ormation.These results are encouraging although not intuitively predictable. Many studies demonstrate that the socioeconomically disadvantaged tend to have less access to health care-28, whicSecondly, both the psychosocial factors that affect a woman’s behavior on seeking health information and the predictors of a HCP recommending the HPV vaccine influence whether a woman discusses the HPV vaccine with her HCP. Individual psychosocial factors such as having a higher internal locus of control,33 and aHowever, similar to other studies,26,41,42The results of this study have a number of important implications for research, practice and policy. In terms of research, our statistical approach provides a fuller picture of socioeconomic disparities in HPV vaccine by simultaneously examining two related indicators. However, the eventual uptake of the vaccine depends on a complex interplay of factors in a woman’s psychosocial milieu as well as other demographic and socioeconomic factors. Thus, future research that assesses the impact of socioeconomic status on the integrated continuum of HPV vaccine awareness, acquisition of information, and vaccine uptake will greatly inform policy makers and health care providers in guiding education and practice.Regarding the implications for clinical practice, HCPs may be the preferred source of health care information for the socioeconomically disadvantaged because of the lack of other reliable options; this preference results in a higher likelihood of having discussions on HPV vaccine. Thus, HCPs can play a pivotal role in dispensing accurate, objective information about the vaccine, and help dispel any myths or negative attitudes about HPV vaccination, thereby allowing socioeconomically disadvantaged women to make informed decisions for themselves and for their daughters.Lastly, our findings that lower socioeconomic status is associated with lower awareness but not with lower opportunities for discussions on HPV vaccine with HCPs has important public health and policy implications. Primarily, social disparities in HPV vaccine uptake will be greatly reduced if discussions between patients and their HCPs become more prevalent. Targeted outreach programs should also be promoted in order to raise awareness about the vaccine among socioeconomically disadvantaged women and should be tailored to address attitudes and perceptions that are specific to each ethnic group. In addition, given the potential for HCPs to ameliorate social disparities in HPV vaccine uptake and its importance, public health efforts can be directed toward giving full support to HCPs for HPV vaccination promotion. This support can come in the form of informative pamphlets in various vernaculars to enhance communication and provide easily accessible, timely, and up-to-date information on HPV vaccine research.Notwithstanding these implications, the findings presented herein should be considered in the context of a few limitations. A key limitation of this study was the short interval between the FDA approval of the quadrivalent HPV vaccine in 2006 and the launch of the HINTS in early 2008. Thus, the small number of respondents who reported having HPV vaccine discussions with their HCPs may not be reflective of the corresponding number today. Future research involving HCPs who are more familiar with the vaccines may offer a more accurate picture of the prevalence of vaccine discussions between women and their HCPs. A second limitation, which is related to the first, is that four years have elapsed since the launch of HINTS in early 2008; thus, the results may not reflect the current situation today. However, while average awareness of the vaccine may have increased over the years, the increment of increase is unknown and its rate differs among populations. Hence, this study is still pertinent in underserved populations whose awareness of the HPV vaccine and accessibility to HCPs remains low, and in which identifying ways to best target patient education remains crucial. Additionally, the self-reported status of having discussions on HPV vaccine with a HCP is subject to recall bias. Moreover, the content and extent of the discussion was unknown, and a standard definition for what qualifies as a “discussion on HPV vaccine” was not described in the survey. This could have resulted in an underestimation or overestimation of the number of respondents who reported having discussions on HPV vaccine with their providers. Lastly, the HINTS was not developed to include constructs that aim to capture HCP factors and individual psychosocial factors that could have influenced the likelihood of women having discussions on HPV vaccine with their HCPs. Future research surveys designed to capture all of these factors could give a more comprehensive analysis of socioeconomic disparities in HPV vaccine discussions.Numerous studies have demonstrated that socioeconomically disadvantaged women are at higher risk of HPV-related mortality and morbidity. However, the comparable tendencies to have discussions on HPV vaccine with HCPs amongst women across different socioeconomic groups differed from the wide disparities in awareness found in our study, suggesting that HCP involvement in broader public health efforts is of paramount importance in reducing socioeconomic disparities in HPV infection and cervical cancer.The authors declare that they have no competing interests.KYW conceived of the study, analyzed the data, drafted and completed the manuscript. YKD supervised data analysis and helped with revisions. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6874/12/33/prepub


Human papillomavirus (HPV) is associated with different malignancies but its role in the pathogenesis of ovarian cancer is controversial. This study investigated the prevalence, genotyping and physical state of HPV in ovarian cancer Saudi patients.Hundred formalin fixed paraffin embedded (FFPE) ovarian carcinoma tissues and their normal adjacent tissues (NAT) were included in the study. HPV was detected by nested polymerase chain reaction (PCR) using degenerated HPVL1 consensus primer pairs MY09/MY11 and GP5+/GP6 + to amplify a broad spectrum of HPV genotypes in a single reaction. The HPV positive samples were further genotyped using DNA sequencing. The physical state of the virus was identified using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay in the samples positive for HPV16 and/or HPV18.High percentage of HPV (42%) was observed in ovarian carcinoma compared to 8% in the NAT. The high-risk HPV types 16, 18 and 45 were highly associated with the advanced stages of tumor, while low-risk types 6 and 11 were present in NAT. In malignant tissues, HPV-16 was the most predominant genotype followed by HPV-18 and -45. The percentage of viral integration into the host genome was significantly high (61.1%) compared to 38.9% episomal in HPV positive tumors tissues. In HPV18 genotype the percentage of viral integration was 54.5% compared to 45.5% episomal.The high risk HPV genotypes in ovarian cancer may indicate its role in ovarian carcinogenesis. The HPV vaccination is highly recommended to reduce this type of cancer. Human papillomavirus (HPV) belongs to Papillomaviridae family that consists of small double stranded DNA viruses associated with cutaneous and mucosal squamous epithelial lesions. HPV infOvarian carcinoma is the most lethal gynecological malignancy because it is detected in advanced stages with 5-year survival rate is <40%,13. The The genetic alterations associated with ovarian carcinomas are well known,18. The The integration site of HPV is extensively investigated in cell lines and clinical samples of HPV related cancers at various sites of the body. The integration site for HPV is random throughout the genome but the Integration mechanisms are not fully understood. IntegraGenomic DNA was isolated from 100 FFPE ovarian carcinoma and their normal adjacent tissues. All samples were positive for β-globin gene amplification. The amplified HPV DNA with MY09/MY11 followed by GP5+/GP6+ were considered HPV positive and were subjected to DNA sequencing. The negative samples by nested PCR were subjected to HPV-type specific PCR to confirm that the samples were negative. Forty two out of 100 tumor samples and 8 out of 100 NAT were positive for HPV by nested PCR as shown in Figure The patients’ mean age was 50 ± 11 years . The patients’ age distribution was analyzed and the prevalence of HPV genotypes was detected among them. There was no significant difference (P > 0.5) observed in HPV infection among patients with age <45 compared to >45 years old, in which 15/35 (42.8%) of the patients with age <45 years old and 27/65 (41.5%) were positive for HPV. The HPV infection in relation to histological grade, HPV was detected in 25% (7/28) of cases with grade I, 50% (21/42) of cases with grade II and 46.7% (14/30) of cases with grade III.By using sequencing technique, the most common detected HPV genotype was HPV-16 in 18/42 (42.9%), followed by HPV-18 in 11/42 (26.2%), finally HPV-45 in 7.1% as in Figures As the sequencing technique failed to identify the specific mixed HPV genotypes, therefore type specific PCR assay was used. Mixed infection with HPV-16/18, HPV-16/45, HPV-18/45 or HPV = 16/18/45 were observed in 7/42 (16.6%), 2.4%, 2.4%, 2.4% respectively as showed in Table The clinical stages were determined according to the International Federation of Gynecology and Obstetrics system (FIGO). The ovarian cancer samples were classified as followed: stage 1-23%, stage II-37%, stage III-29% and stage IV-11%. HPV was detected in 21.7% (5/23) stage 1, 40.5% (15/37) stage II, 41% (16/39) stage III and 75% (9/11) stage IV as showed in Figure The HPV genotypes were distributed among different FIGO stages as followed: HPV-16 were (7/37) 18.9%, (9/39) 23% and (2/11) 18.2% in stage II, III and IV respectively as showed in Figure E1 or E2 open reading frames and deletion of viral early-region from the viral oncogene-encoding sequences. Thirty six HPV-16, HPV-18 or HPV-16/-18 positive samples with good quality RNA, were subjected to study the physical states by APOT assay. The viral genome was found to be integrated in 22/36 (61.1%), whereas the episomal transcripts were found in 38.9%. Out of the 22 cases with integrated viral genome the episomal form of HPV was also detected in 5/22 (22.7%). In HPV-18 genotype positive samples the percentage of viral integration was 54.5% compared to 45.5% episomal. On the other hand in HPV-16 genotype positive samples the percentage of viral integration was 65% compared to 35% episomal. The integrated state of the virus was significantly found in 90% in advanced stages III and IV compared to 10% in localized stages I and II (p < 0.05) showing that the integrated form was associated with the advanced stages of cancer.The APOT assay is based on the structural differences among the 3′-ends of viral oncogene transcripts. The integration of HR-HPV genomes into the host genome results in both disruption of the The role of HPV infection in cervical cancer and otheHPVs are classified as high and low risk, according to their relationship with benign or malignant proliferative lesions. The oncIn many studies, molecular assays were used to identify different types of HPV in cells and tissues-47. The In the current study, the high-risk HPV types 16, 18 and 45 were associated with advanced stages of the disease, while low-risk types 6 and 11 were associated with normal tissues adjacent to the tumor. This finding was in agreement with other study found that the presence of high-risk types of HPV was found in the serous histological subtype and advanced stages of the disease (FIGO stages III and IV). Other sThere is no statistical significant difference in HPV infection in relation to age group more/or less than 45 years. The current study are consistent with those reports based the detection of HPV in relation to age, they found that the median age of diagnosis of ovarian cancer patients infected with HPV was 57 years for patients with HPV and 59 years for patients without HPV infection for HPV16 and [P1-18] for HPV18 as forward primers and linker p3 as the reverse primer. The PCR was performed in 50 μL reaction volume containing 5 μL of the RT reaction mixture (cDNA), 2.5 units Taq polymerase , 1× PCR buffer , 200 μM each of the deoxyribonucleotide triphosphate and 0.25 μM of each primer. The reaction mixture was subjected to initial denaturation for 2 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 58°C for 45 s, elongation at 72°C for 2 min, and a final elongation step at 72°C for 7 min. Five μl of the amplified products were used as template for nested PCR under the same conditions at annealing temperature 65°C using forward HPVE7-specific primer [p2-16] specific for HPV16 and [P2-18] specific for HPV18 as forward primers and (dT)17-p3 as reverse primer.To identify the HPV genotypes and the integration, all positive PCR products were analyzed by direct DNA sequencing. PCR products were purified using QIAquick Purification Kit according to manufacturer’s instructions . Purified PCR products were labeled with fluorescent dyes using BigDye Terminator v3.1 Cycle Sequencing Kit Applied Biosystem. Labeled oligonucleotides were purified using BigDye X Terminator Purification Kit .The samples were sequenced by automatic ABI 3500 genetic analyzer . Chromatograms with sharp peaks and quality values ≥20 with little or no background noise consider as single HPV infection. When the samples contain more than one HPV genotype, direct sequencing gave mixed chromatograms, with overlapping peaks or two or more fluorescent signals. Samples with mixed chromatogram were subsequently subjected to type-specific PCR. The nucleotide sequences were subsequently subjected to Basic Local Alignment Search (BLAST) provided by the National Cancer Institute, USA.Chi-square test used to test prevalence differences in HPV genotypes and integration frequencies between different HPV types in cancerous tissues and NAT in relation to age, tumor stage and tumor grade. A p value of < 0.05 was considered statistically significant. SPSS, version 17.0 was used for these analyses.The authors declare that they have no competing interests.MMH and ZK shared in molecular studies and their statistical analysis and draft the manuscript. MMH, OA, MS, WN, SM and AA participated in study design, sample and data collection and shared in revising the manuscript. SS shared in sample and data collections. All authors read and approved the final manuscript.


Only a portion of the US population is willing to consider HPV vaccination to date. The primary aim of this study is to determine the decisional satisfaction associated with HPV vaccination.This is a prospective survey conducted at an urban college where women 18–26 years old completed a decisional satisfaction survey about their HPV vaccine experience.Regardless of the decision to accept or reject HPV vaccination, the decisional satisfaction was very high (mean 5-item score = 21.2 (SD 3.8)). Women without HPV vaccination were decisionally neutral significantly more often than those already vaccinated; 22% were decisionally neutral for the option to accept HPV vaccination at that visit. Cervical cancer prevention was preferred significantly more often than genital wart prevention in all analyses.Targeting those who are decisionally neutral about HPV vaccination may result in a higher uptake of HPV vaccination. Many women’s health screenings including cancer prevention involve patient education, patient participation and shared decision making. In order to reach a high-quality decision, the benefits and harms of the alternative options must be clearly discussed. More importantly, the choice depends on how patients value benefits versus harms. The eventual goal is a strong match between the chosen option and the features that matter most to the informed patient Much of human papillomavirus (HPV) vaccination in the US has been presented through the eyes of public health with an overriding sense of community obligation to be vaccinated for whatever herd immunity may occur The primary aim of this paper is to quantify the satisfaction of the decision to receive at least one dose of HPV vaccine among young adult women. The secondary aim was to determine which demographic and medical history items influenced the value of cervical cancer prevention vs. genital wart prevention followed by choice of HPV vaccine.This prospective study was approved by the University of Missouri Kansas City (UMKC) Social Sciences Adult Institutional Review Board (SSIRB) (#SS10-56X) and is part of a larger study on women’s preferences for HPV vaccination.All women, 18–26 years old, being seen at UMKC Student Health and Wellness, an urban college health service, were offered the opportunity to participate in the survey between January 2011 and August 2012. All members of the health care staff were educated on the purpose of the study; and provided scripts to introduce it at check-in. Women were only allowed to complete the survey once, and they self-reported demographic and medical history. For those who had received an HPV vaccine, we did not ask number or timing of doses. The study was open to both those with prior HPV vaccination and those who had not yet started the series. Women completed the survey prior to being seen by the health care provider, and could ask questions about HPV vaccination during the visit.The cervical cancer prevention education information was constructed in a format similar to a decision aid The decisional satisfaction scale was adapted for cervical cancer prevention from the original Holmes-Rovner model Three items were included in the survey modeled on the O’Connor’s decisional conflict scale Statistics: Descriptive statistics included means testing with Student’s t-test for significance testing. Chi-square comparison and Fisher’s exact testing were used for ratios as appropriate and p-values less than 0.05 were considered significant Over five academic terms, 10,562 patients  were seen at the Student Health and Wellness clinic. Of these, 306 women without prior HPV vaccination and 299 vaccinated women agreed to participate in the survey. The average age did not differ between the two groups, but for the vaccinated women the average age of first intercourse was significantly lower and the average number of lifetime sexual partners was significantly higher . Over 90Among the women 21 years and older, age-appropriate for cervical cancer screening, 24% of those without any doses of HPV vaccine had never had a Pap test, significantly higher than those with at least one dose of HPV vaccine. There were similar proportions of women with abnormal Pap tests and who underwent colposcopy in both the vaccinated and unvaccinated groups. Significantly more women with prior HPV infections had been vaccinated. A past history of genital warts or of other sexually transmitted infections (STIs) was equally distributed between the vaccinated and unvaccinated groups.The decisional satisfaction scores on the five item survey were high regardless of HPV vaccination status  indicatiThe “expectation to be vaccinated at this visit” choice showed low satisfaction scores for those not yet vaccinated (mean 2.1 (SD 1.0)), meaning that there was substantial disagreement with the intent to start HPV vaccination among those not yet vaccinated.A decisional satisfaction score of 3 indicates neutrality and potential decisional conflict where certainty about choice does not yet exist. The highest decisional neutrality occurred among those without HPV vaccination regarding expectations of being vaccinated before leaving the office (22%). The lowest decisional neutrality occurred for the item “I am informed about the cervical cancer prevention options” indicating that only 9% of women regardless of HPV vaccine status were uncertain about their cervical cancer prevention options.Whereas a certain amount of decisional neutrality was evident among all women regardless of HPV vaccine status, women with no prior HPV vaccination had significantly greater decisional neutrality on the remaining four qualities of decisional satisfaction than those with at least one HPV vaccine dose.Women with diverse medical histories ranked the currently perceived value of preventing different HPV associated diseases differently . CervicaWomen who experienced HPV related disease had higher preferences for cervical cancer and genital wart prevention than inexperienced women. For instance, women with at least one dose of HPV vaccine valued cervical cancer and genital wart prevention more than women with no vaccination  vs. mean 4.2 (SD 0.9), p<0.001); genital warts: mean 4.6 (SD 0.8) vs. mean 4.1 (SD 1.1) p<0.001). Similarly, women with a prior STI valued cervical cancer and genital wart prevention more than women without a prior STI. Among women 21 years and older, those with a prior Pap, with a prior abnormal Pap and a prior HPV infection valued cervical cancer and genital wart prevention more than those women without the respective medical histories. A past colposcopic experience only enhanced the women’s value of cervical cancer prevention, not genital wart prevention.Genital wart prevention was never more highly valued than cervical cancer prevention in any sub-population. Significantly more women would trade off genital wart prevention for more cervical cancer prevention (57% (312/547) vs. 43% (235/547), p<0.001); this preference remained when stratifying by prior HPV vaccine receipt (no vaccine: 57% (160/279) vs. 43% (119/279), p<0.001; vaccine: 56% (152/268) vs. 44% (116/268), p = 0.002).When asked to make a choice between vaccines, significantly more women chose HPV2 over HPV4 (61% (325/537) vs. 39% (212/537), p<0.001, Our work is the first to explore decisional satisfaction with HPV vaccination. Some have studied how normative messaging from peers, family and health professionals influence HPV vaccine choice Recently discrete choice experimentation (DCE) has provided a health economics metric to understand how adults trade off risks and benefits among competing options for similar health care outcomes These theoretical models have resulted in using strong health care provider messaging for vaccination, promoting the commonness of HPV infections associated with multi-organ cancers, and emphasizing no out-of–pocket expenses as motivators to increase HPV vaccination rates. However, these strategies have not led to an increase in uptake, but rather a plateauing of those receiving HPV vaccinations Decisional satisfaction offers the perspective of determining which set of young women are still neutral about their HPV vaccine choice; and hence more likely to be open to more discussion about how the benefits and risks of HPV vaccination play into their value for cervical cancer prevention. While Healthy People 2020 anticipates 80% vaccination of the young adult women during their adolescence, the current trends support a large portion that will still have the opportunity to make a HPV vaccination decision as young adult women. Increased vaccination may be more likely if this decisionally neutral set of young women is targeted.Women with a clear sense of what their personal value of HPV vaccination was (accept/reject) were highly satisfied with their decision. The women who have had at least one dose of HPV vaccine who expressed neutral satisfaction with their decision to be vaccinated may be indicating decisional regret about a decision made for them at an earlier time. Future research will use decisional conflict and decisional regret scales to attempt to understand these values Our work shows that young women have greater value for cervical cancer prevention than for genital wart prevention. This was also seen in Oteng’s work among adult women who were willing to trade genital wart protection for cervical cancer protection Limitations: Our work is limited by several factors. In the time course of making a medical decision, the decision aid  is presented followed by a survey of the satisfaction associated with the choice made. In our work half the population had already made the choice to receive at least one dose of HPV vaccine. This limitation offers a potential hypothesis-generating benefit, though, in that the decisional neutrality expressed might provide insight into decisional regret they may be experiencing up to five years after initial vaccination.Within the decisional framework of HPV vaccination, we did not offer the choice of no HPV vaccination; therefore, we do not know how an option of no vaccination would change these results.Separately, while our study population resembled most young adult HPV studies in that the number of lifetime sexual partners and age at first intercourse is historically similar Finally, the developmental capacity to make medical decisions is recognized to be more complicated in adults than in adolescents Targeting young adult females who are decisionally neutral about HPV vaccination may be a more direct method of increasing HPV vaccination rates in a targeted population than spending resources on those already highly satisfied with their decision not to vaccinate.


The aim of the study was to test for human papilloma virus (HPV) infection and human telomerase RNA component (hTERC) gene amplification in tissues derived from esophageal cancer, in esophagus displaying atypical hyperplasia and in normal tissue, and to analyze the relationship between them and discuss whether HPV infection and hTERC gene amplification play a role in the duration of survival of esophageal cancer patients.in situ hybridization technique.To test for HPV infection, surface plasma resonance was used after extracting and subjecting the DNA to PCR amplification. Measurement of hTERC gene amplification was performed by the fluorescence P < 0.01. The hTERC gene amplification rate in normal tissue, grade I atypical hyperplastic tissue, grade II/III atypical hyperplastic tissue and esophageal cancer tissue were 0% (0/89), 15.38% (4/26), 47.06% (8/17) and 89.13% (82/92), respectively, with a statistically significant difference of P < 0.01. On follow-up of 92 patients, survival curves of the HPV-positive and HPV-negative groups were not significantly different (P > 0.05). Survival curves of the hTERC gene amplification-positive and hTERC gene amplification-negative groups were statistically significant (P < 0.05). A matching chi-square test showed that there was no correlation between HPV infection and hTERC gene amplification (P > 0.05).The rates of HPV infection in the normal group, the atypical esophageal hyperplasia group and the cancer group were 0% (0/40), 10.00% (1/10) and 20.65% (19/92), respectively, with a statistically significant difference of HPV infection may be one of many factors contributing to the development of esophageal cancer, but it does not influence prognosis. Amplification of the hTERC gene appears to influence certain features associated with postoperative survival in esophageal carcinoma patients. China has the highest incidence and mortality of squamous cell carcinoma of the esophagus (ESCC) in the world. Patients with ESCC are most commonly diagnosed with locally advanced tumor stages. Early metastatic disease and late diagnosis are common reasons responsible for the poor clinical outcome of this tumor. The prognosis of ESCC is very poor because of the paucity of symptoms seen in these patients at the earliest stages of disease. Progressively increasing epidemiologic and biologic evidence indicates that human papilloma virus (HPV) infection is associated with the occurrence of ESCC -3. In ceNinety-two specimens of freshly frozen ESCC were collected from December 2007 through December 2008 at the First Affiliated Hospital of Henan University of Science and Technology. Esophageal cancer tissue, esophageal cancer side tissue (approximately 2 cm) and normal tissue were selected and made into sectioned wax pieces. Ninety-two cases of esophageal cancer tissue, 26 cases of grade I esophageal atypical hyperplastic tissue, 17 cases of grade II/III esophageal atypical hyperplastic tissue and 89 cases of esophageal normal tissue were selected, and their pathology was confirmed. Ten cases of esophageal atypical hyperplastic tissue and 40 cases of normal tissue were collected by endoscopic examination over the same period. This study was conducted in accordance with local ethics guidelines compliance with the Helsinki Declaration, the patients’ esophagus tissues used in this study were approved by the Medical Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China, reference no. 2010006.g, the filtrate discarded and the spin column placed in the microcentrifuge. Buffer GD (500 μl) was added and again the mixture was centrifuged for 1 minute at 13,400 × g, the filtrate discarded and the spin column placed in the microcentrifuge. Buffer PW (600 μl) was added, and the mixture was centrifuged for 1 minute at 13,400 × g, the filtrate discarded and the spin column placed in the microcentrifuge. TE (50 μl) was then centrifuged for 2 minutes at 13,400 × g, and the filtrate collected. The DNA concentration was measured using a Nanodrop 1000 , an A260:A280 ratio of 1.75 to 2.05 indicating an absence of contaminating proteins. The isolated DNA was amplified by the HPV L1 consensus primers MY11 and MY09 [DNA was isolated from frozen esophageal mucosa using the TIANamp Genomic DNA Kit (Tiangen Biotech (Beijing) Co., Ltd, Beijing, China). Excised tissue (20 μg) in liquid nitrogen was ground until the tissue was powder. Liquid nitrogen was allowed to evaporate and the powdered tissue added to buffer GA (200 μl), and then RNAase was added to a final concentration of 2 μg/μl, mixed and incubated for 30 minutes at 37°C. Proteinase K was added to a final concentration of 100 μg/μl and placed in a water bath for 3 hours at 56°C. Buffer GB (200 μl) was added, mixed and incubated for 10 minutes at 70°C. Ethanol was then added, and the solution mixed and transferred to a spin column that was closed and placed in the microcentrifuge. The mixture was centrifuged for 1 minute at 13,400 × 2+), 2.5 mM MgCl2, 200 μM each dNTP, 0.2 μM my11/my09, 2.5 U Taq and 2 μl template DNA. Amplification without DNA template was used to monitor contamination in both the HPV and β-actin reactions. An HPV-positive sample was considered the positive control . The mixtures were incubated for 3 minutes at 94°C; then for 20 seconds at 94°C, 30 seconds at 55°C, 30 seconds at 72°C for 40 cycles; for 5 minutes at 72°C; and at 4°C on hold. A 10 μl aliquot of the PCR mixture was visualized by ethidium bromide staining after agarose gel electrophoresis. HPV-positive samples reveal a band of approximately 450 base pairs.The PCR processes were monitored through amplification of part of the human β-actin gene in replicate tubes with related primers . Each PCSurface plasma resonance (SPR) technology is a novel method to detect the HPV genotype. SPR exploits surface plasmons  that can be excited at certain metal interfaces, most notably silver and gold. When incident light is coupled with the metal interface at angles greater than the critical angle, the reflected light exhibits a sharp attenuation (SPR minimum) in reflectivity owing to the resonant transfer of energy from the incident light to the surface plasmon. The incident angle (or wavelength) at which the resonance occurs is highly dependent on the refractive index in the immediate vicinity of the metal surface. Binding of molecules at the surface changes the local refractive index, resulting in a shift of the SPR minimum angle. By monitoring changes in the SPR signal, it is possible to measure binding activities at the surface in real time.in situ hybridization kit were obtained from Beijing GP Medical Technology Co., Ltd. Microscopic images were acquired using an Olympus B Microscope equipped with custom optical filters for 4,6-diamidino-2-phenylindole, spectrum green and spectrum red, as well as a Plan Apo objective. The fluorescence in situ hybridization IMSTAR 3.0 image analysis system was used . All procedures were conducted in accordance with the product instruction manual.The SPR instrument and the PCR and SPR kits were obtained from Beijing GP Medical Technology Co., Ltd. The peltier-based thermal cycler was obtained from the Hangzhou LongGene Scientific Instruments Co., Ltd . A probe panel based on the hTERC gene sequence was designed to evaluate the gain of chromosome 3q. The probes and the fluorescence The SPR gold chip was immobilized with 24 types of specific HPV probes targeting the L1 region of HPV genome. In addition, one positive control probe (specific to the human β-actin gene) and one negative control probe (unrelated to both HPV and human genome sequences) were also spotted on the gold chip. For an informative reaction, the positive control probe spot should yield a positive hybridization resonance unit, the negative control probe spot a negative hybridization resonance unit and the rest of the HPV probe spot different hybridization resonance units, depending on the HPV status existing in each specimen. A positive control signal value <40 or a negative control signal value ≥40 were indicative of obtaining an invalid result. A positive control signal value ≥40 and negative control signal value <40 were indicative of obtaining a valid result. For the 24 types of specific HPV probes, a probe signal ≥40 was considered HPV-positive and a signal <40 was considered HPV-negative. The cutoff value was set at 17.3 per 100 random nuclei displaying increased hTERC signals and/or tumor ploidy (for more detail see Results).P < 0.05 was considered statistically significant.For the evaluation of the data, SPSS 17.0 statistical software  was used to perform the chi-square test. The follow-up data obtained from different subgroups of patients with esophageal cancer were assessed by Kaplan–Meier survival analysis and use of log-rank hypothesis testing of the survival curves for the different groups. P < 0.01. There was no significant correlation found between HPV infection and either gender, age, history of alcohol or tobacco use and family history (Table P > 0.05). The survival curves for the HPV-negative group are shown in Figure Of 92 cases of cancer tissue, 10 cases of atypical hyperplasia tissue and 40 cases of normal esophageal mucosa, only 20 cases were found to test positive for HPV. The infection type included the high-risk types HPV18, HPV35, HPV51, HPV56, HPV66, HPV58, HPV68, HPV39, HPV81, HPV59 and HPV16, and the low-risk types HPV42 and HPV54. Mixed infection was found to exist in five specimens, and four specimens contained the HPV18 type. The main infection types were found to be HPV18, HPV35 and HPV51. The HPV infection rates of normal tissue, atypical hyperplasia of the esophagus and cancer tissue were found to be 0% (0/40), 10.00% (1/10) and 20.65% (19/92), respectively, with a statistically significant difference of The signals were visually evaluated by screening the entire slide for the chromosome 3q-specific probe . Cells with normal 3q signal numbers were recorded as diploid. Cells with abnormal 3q signal numbers were recorded in patterns in the relocation charts of the whole probe panel. Abnormal nuclei were defined by the recording of more than two hTERC signals and more than two centromeric region of chromosome 3 (CSP3) signals. This cutoff value was based on 35 normal tissues:n = 35), the numbers of hTERC gene amplification cells  were counted in 100 cells of random view, and the mean value for the 35 normal tissues was 7.1 (standard deviation 3.4). The cutoff value was set at 17.3. A case was considered hTERC gene amplification-positive for the 3q assay when >17.3% of the cells exhibited >2 hTERC signals.For normal tissue slides (P < 0.01). The gene amplification of hTERC in different differentiation grades, infiltration depth, pathology type and lymph node metastasis was found to not be significantly different (P > 0.05) (Table P < 0.05) , 15.38% 4/26), 47.06% (8/17) and 89.13% (82/92), respectively, and were statistically significantly different .In 92 cases of esophageal carcinoma, 19 cases were found to be infected with HPV, and in the remaining 73 patients HPV infection could not be detected. In addition, hTERC gene amplification was detected in 82 cases, and in 10 cases hTERC gene amplification was not detected. There were also 15 cases in which not only the existence of HPV infection was confirmed but also hTERC gene amplification was detected. In 67 cases, the existence of hTERC gene amplification in the absence of HPV infection was found. By contrast, four cases were negative for hTERC gene amplification in the presence of HPV infection, and in six cases neither hTERC gene amplification nor HPV infection was found. The matching chi-square test showed that there was no correlation between HPV infection and amplification of hTERC genes , suggesting that HPV infection may not be of prognostic utility in the evaluation of factors contributing to esophageal cancer.The infection rate of HPV in the esophagus of normal subjects, patients presenting with atypical hyperplasia and patients presenting with esophageal cancer were found to be 0%, 10.00% and 20.65%, respectively. It is possible that HPV plays a role in the occurrence of esophageal cancer. In the normal esophagus group, the infection rate was found to be 0%, which was concordant with prior observations in the literature for which the infection rate ranged from 0 to 59.76%. The survival curves of the positive and negative HPV infection groups were not significantly different . Historically, telomerase activity was thought to be closely associated with human telomerase reverse transcriptase. In recent years, hTERC has been of particular importance in regulating telomerase activity . The hTEerplasia ,17.P > 0.05). The hTERC gene may participate in the malignant transformation of esophageal epithelial cells, but it does not influence tumor invasion and metastasis. This finding might indicate that amplification of the hTERC gene is an early event in the development of esophageal cancer.The level of gene amplification of hTERC under conditions of different grades of differentiation, invasion depth, pathology type and lymph node metastasis was found to be statistically nonsignificant (P < 0.05). This observation suggested that hTERC gene amplification might be an independent risk factor for the prognosis of esophageal cancer. The hTERC gene is anticipated to become a new target to assist in the treatment of esophageal cancer.The survival curves for the hTERC gene amplification-positive and hTERC gene amplification-negative groups were statistically significant (There was no correlation between HPV infection and hTERC gene amplification in our study. Infection with HPV and the hTERC gene may have different pathogenic mechanisms in the development of esophageal and cervical cancer.HPV infection may be one of many factors contributing to the development of esophageal cancer, but it does not influence prognosis. Amplification of the hTERC gene appears to influence certain features associated with postoperative survival in esophageal carcinoma patients.ESCC: Squamous cell carcinoma of the esophagus; HPV: Human papilloma virus; hTERC: Human telomerase RNA component; PCR: Polymerase chain reaction; SPR: Surface plasma resonance.The authors declare that they have no competing interests.X-SF conceived and designed the experiments. Y-FW designed the experiments, prepared reagent and materials, performed the experiments, analyzed the data and wrote the paper. X-SW participated in analyzing the data and drafted the manuscript. S-GG analyzed the data. QC carried out the experiments. Y-TY carried out the experiments. Z-YX prepared reagent and materials. X-QP prepared reagent and materials. X-FH prepared reagent and materials. Q-YW prepared reagent and materials. All authors read and approved the final manuscript.