HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals.1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results.HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests’ positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used.Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results. Human Papillomavirus (HPV) infection is estimated to be the most common sexually transmitted infection Although the life-long risk for HPV infection is 80%, only a fraction of that percentage of women will eventually develop cervical cancer The expression of the two viral genes E6 and E7 is responsible for the transformation of cells by HPV, resulting in continuous morphological changes and finally cervical neoplasia In the present study the clinical performance of flow cytometric in-situ hybridization for E6 and E7 mRNA transcript detection was evaluated, in women attending two colposcopy clinics of two University Hospitals in Greece.rd Department of Gynecology and Obstetrics in the tertiary care “ATTIKON” University General Hospital and the Department of Gynecology and Obstetrics in the University Hospital of Ioannina were enrolled in the present study. The study population did not represent a normally screened population, since most patients attended the outpatient clinics after a referral abnormal cytology and or colposcopy. All patients signed an informed consent form, while the study was approved by the Bioethics comity of the hospitals. A liquid-based cytology  sample was collected and a monolayer smear was prepared on a TP 2000 Processor and stained according to Papanicolaou technique. A trained cytopathologist diagnosed each case, according to the Bethesda 2001 system A total of 1173 women that attended the colposcopy clinics of the 3HPV DNA detection was performed on the first aliquot using a commercially available kit, CLART® HPV 2 kit  that allowed the detection of 35 different HPV genotypes, 20 HR-HPVs  and 15 LR-HPVs  as categorized by Dunne et al. The other 1 ml aliquot of the LBC specimen was used for whole cell detection of E6/E7 HPV mRNA over-expression by means of a commercially available kit . Briefly, cells were washed in PBS to remove the preservative, fixed for 1 h at room temperature and washed with two pre-hybridization buffers. HPV mRNA was labeled after a 30′ hybridization at 43°C with a FAM labeled probe cocktail, followed by two stringency washes of the cells in order to remove the unbound probe. Cells were resuspended in 1 ml PBS containing 2% fetal calf and analysed on a Partec CyFlow SL with a 488 nm argon laser, with front-scatter (FSS) and side-scatter (SSC) set on logarithmic scale. Cells of interest were gated as previously described, and the cut-off for positive samples was set 1.5% of green fluorescent cells. Two cell lines commercially obtained by IncellDx (HPV Positive control Cells and HPV Negative Control Cells) were used to verify gating of cells and the cut-off threshold .2 for trend for other tables. Comparison of results of the two molecular tests was performed using the McNemar test and their agreement was accessed using the κ value. Comparison of molecular testing with cytology was performed using the McNemar test in CIN2- for specificity and in CIN2+ for sensitivity. Sensitivity, specificity, positive and negative predictive values  and odds ratios were calculated using a 95% confidence interval. Finally correlations between assessed parameters were calculated using the Spearman test.Both morphology and molecular results were independently collected and both pathologists and biologist were blinded from the results of the other part. Statistical analysis of the data was performed using SPSS19 . Analysis of results was performed using fishers exact test for 2 by 2 tables and χStudy participants were between ages of 19–81 years with a mean of 38.2 years of age. Out of 1173 women 538 had a LSIL+ colposcopy result and 6 women with referral cytology of HSIL+ had a non-satisfactory colposcopy result . 53 womeResults of molecular testing according to histology findings are summarized in mRNA and DNA testing showed significantly different results as depicted in Clinical efficiency of morphology and molecular tests was evaluated with classic statistic measures using as golden standard histology results . MorpholCLART displayed higher positivity rates in CIN2+ cases that mRNA , as mirrored by higher sensitivity values . This diCombination of molecular testing and cytology was analyzed using three distinct scenarios . These iThe study population was divided in 9 age groups with the two extreme categories (<20 and >60) having the least cases and all other containing about the same number of samples. Positivity for morphology and molecular tests peaked at different age groups . DNA posComparison of molecular testing efficiency was performed in dichotomized groups with cut-off either 25 or 30 years of age . The mosPositivity was higher in women under 30 years of age for IncellDx, CLART and CLART HPV16/18  while the same result didn’t reach statistical significant in ages under 25. In CIN3+ lesions positivity rate for HPV16/18 in women under 30 was statistically higher than in CIN3+ cases of women over 30 . Positive mRNA samples were statistically more often CIN3+ in women over 25 or over 30 than in younger women . The same was observed for HPV DNA and HR HPV DNA with weaker strength of association . HR DNA or HPV16/18 positivity was more often a CIN1+ in women under 30 .Even though, there was an obvious bias for cytology due to the clinically negative cases, a sub-analysis was performed comparing ASCUS+ cytology diagnosis to molecular testing, triggered by the observation that CIN2+ cases were correlated with higher spearman values to mRNA testing (0.552) than ASCUS + cytology (0.486). This difference was more profound when analysing only histological confirmed cases (0.582 for mRNA vs. 0.232 for ASCUS+). DNA testing had lower correlation co-efficiencies than ASCUS + ranging from 0.33 to 0.40.ASCUS+ displayed higher sensitivity in CIN2+ cases than HPV DNA (McNemar p = 0.064), HR HPV DNA (p = 0.007), HPV16/18 DNA (p<0.001) and mRNA (p<0.001)  and 6. OBy including the age variable in the analysis, significant differences of sensitivity and specificity were identified in the different age groups  and 6. BIn the present study 1173 samples from women visiting colposcopy units of two tertiary hospitals in Greece were used to identify molecular testing efficiency. Most studies designed to evaluate efficacy of molecular testing, are large scale randomized screening trials, while retrospective analysis of histological confirmed cases has greatly aided in connecting HPV with cervical neoplasia The study population included many samples with abnormalities of either low or high grade and an increased percentage of carcinomas. Molecular testing had increased positivity as lesion grade progressed, as expected. However mRNA positivity was significantly lower, especially in clinically negative, histological negative and CIN1 samples, in concordance with previously studies Since both colposcopy clinics have experienced colposcopists and all cytologic diagnoses were set by experienced cytopathologists, morphology yielded excellent results with highest sensitivities and NPVs calculated when using as cut-offs ASCUS for cytology and LSIL for colposcopy and highest specificities and PPVs for ASCH+ cytology and HSIL+ colposcopy. mRNA HPV displayed characteristics between the two cut-offs with higher PPV than ASCUS+ and higher sensitivity than ASCH+. On the other hand DNA testing displayed much worse specificity apart from when considering presence of HPV16 or HPV18. It has been previously shown that mRNA testing has significantly higher specificity and PPV than DNA testing More recently, many have either proposed replacement of cytology with molecular testing for either HPV DNA or HPV RNA, or their combined use is specific categories of lesions. For instance, it has been shown that combination of LSIL+ cytology and HPV DNA cases positive for HPV16/18 in women over 25 displayed significantly higher sensitivity and similar PPV that ASCUS+ cytology alone Since HPV positivity has been shown to peak in ages under 30 years old with a steady decline in older women Even though the population of the study was not a screening population, HPV DNA positivity followed the same profile with its peak in ages 20.1–25 years of age but with a second peak at 30.1–35 years of age. The same profile was followed by mRNA positivity. On the other hand only CIN1+ cases were statistically more often in ages under 30, while CIN2+ cases were similar for both age groups, CIN+3 cases were more often in women over 30 and cancers were only present in the group of women over 30. Surprisingly, mRNA results were better for the under 25 years of age group, an observation that could be result of the limited number of samples (n = 140). As far as HPV DNA is concerned CIN3+ cases of women under 30 years of age had significantly higher positivity for HPV16 or 18, suggesting that these two types are the most important types for lesion progression in younger women and that type specific DNA testing for these women could have potential benefits.In sum, mRNA testing for E6/E7 outperformed DNA testing either when used alone in younger ages or overall in combination with cytology results. Since the study population originated from a colposcopy clinic, a larger scale screening population is needed to verify mRNA testing’s predominance. Furthermore, a longitual study with timed follow-up of patients is necessary in order to track the behavior of cytology negative samples with positive mRNA tests.


TNF-α and TNFRSF1B genes that alter gene expression are likely to be associated with risk and clinical outcomes of cancers. However, few reported studies have investigated the association between potentially functional SNPs in both TNF-α and TNFRSF1B and prognosis of NSCLC patients treated with chemoradiotherapy.The dysregulation of gene expression in the TNF-TNFR superfamily has been involved in various human cancers including non-small cell lung cancer (NSCLC). Furthermore, functional polymorphisms in TNF-α and TNFRSF1B genes  in 225 NSCLC patients treated with chemoradiotherapy or radiotherapy alone. Kaplan-Meier survival analysis, log-rank tests and Cox proportional hazard models were used to evaluate associations between these variants and NSCLC overall survival (OS).We genotyped five potentially functional polymorphisms of TNFRSF1B +676 GG genotype was associated with a significantly better OS of NSCLC . Further stepwise multivariate Cox regression analysis showed that the TNFRSF1B +676 GG was an independent prognosis predictor in this NSCLC cohort , in the presence of node status  and tumor stage .We found that the TNFRSF1B +676 T>G (rs1061622) in the prognosis of NSCLC. Further large and functional studies are needed to confirm our findings.Although the exact biological function for this SNP remains to be explored, our findings suggest a possible role of  Lung cancer is the most common tobacco-induced cancer and the leading cause of cancer-related deaths worldwide, with an estimated 1.61 million new cases and 1.38 million deaths in 2008 ; howeverTNF and TNFRSF1B may play a role in patients' treatment response, toxicity, and survival. Thus, genetic variations in TNF and TNFRSF1B that alter gene expression and/or protein production may be potential candidates for prognosis predictors of NSCLC patients.Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine produced by activated macrophages and exerts its action through binding to its two cognate cell surface receptors, TNFRSF1A/TNFR1 p55/60) and TNFRSF1B/TNFR2 (p75/80). It is well known that TNF and its superfamily members have both beneficial and harmful activities, playing a role as a "double-edged sword" 5/60 and . For exaTNF-α and TNFRSF1B genes are highly polymorphic, and several functional single nucleotide polymorphisms (SNPs) in these two genes have been identified, which may contribute to differences in expression levels of the genes or protein products [TNF-α SNPs (SNP -308 G>A and -1031 T>C in the promoter region) and one TNFRSF1B SNP (+676 T>G in exon 6), which have been widely investigated for their associations with susceptibility to and progression and prognosis of various cancers [TNF-α, and +676 T>G) as well as the other two potentially functional SNPs (-1709A>T and +1663A>G in TNFRSF1B) are associated with overall survival of NSCLC.products -20. Of a cancers -37. HoweEpidemiological and clinical data were available from a larger dataset of 576 NSCLC patients who were treated with definitive radiation at The University of Texas M.D. Anderson Cancer Center  between 1998 and 2006. The detailed information about these subjects has been described elsewhere . After thttp://www.ncbi.nlm.nih.gov/, build 131) and Hapmap database  for common, potentially functional SNPs with a minor allele frequency (MAF) ≥ 0.05 in CEU populations, i.e., those SNPs located in the 5' near gene, 5'- and 3'-untranslated regions and coding region of TNF-α and TNFRSF1B. Meanwhile, linkage disequilibrium (LD) analysis and bioinformatics prediction by SNPinfo (http://snpinfo.niehs.nih.gov/) [TNF-α -308 G>A, TNF-α -1031 T>C, and TNFRSF1B +676 T>G, TNFRSF1B -1709A>T and TNFRSF1B+1663A>G) were selected for genotyping. Among these, three SNPs are located in the promoter regions of the genes , which are predicted to affect the binding of some transcription factors. One SNP (TNFRSF1B +676 T>G) causes a missense change and the other (TNFRSF1B +1663A>G) is located in the 3'-untranslated region, which may affect miRNA binding.We screened the NCBI dbSNP database (ih.gov/)  were alsTNF-α, and -1709A>T in TNFRSF1B ) were determined by the primer-introduced restriction analysis (PIRA)-PCR assay. The primers used for TNF-α -308 G >A, TNF-α -1031 T>C and TNFRSF1B -1709A>T were 5'-GCAATAGGTTTTGAGGGCCATG-3' (sense) and 5'-TTTGGAAAGTTGGGGACACACA-3'(anti-sense); 5'-GGGAAGCAAAGGAGAAGCTGAGAACA-3' (sense) and 5'-GGGGGGTCCCCATACTCGACTT-3' (anti-sense); and 5'-CCACACACCTCAAGACCAATGAG-3' (sense) and 5'-CTTGAATTCGTTCCCAGGATGG-3' (anti-sense), respectively. The mismatched G, C and A were respectively introduced into the sense primers at 6 bp, 2 bp and 2 bp from these three polymorphic sites to create NcoI, NlaIII and MlyI restriction sites. Additionally, TNFRSF1B +676T>G and +1663A>G were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with the primers 5'-CTCCTCCTCCAGCTGTAACG-3' (sense) and 5'-CCAACTGGAAGAGCGAAGTC-3' (anti-sense), and 5'-AGGCCCCCACCACTAGGACTCT-3' (sense) and 5'-GTTGTGGAAAGCCTCTGCTGC-3' (anti-sense), respectively. The following PCR conditions were performed: 5 min of initial denaturation at 95°C followed by 35 cycles of 30 s at 95°C; 45 s at 64°C (TNF-α -308 G >A) or 59°C (TNF-α -1031 C>T) or 64°C (TNFRSF1B +676 T>G) or 59°C (TNFRSF1B -1709A>T) or 62°C (TNFRSF1B +1663A>G), respectively; 45 s at 72°C; and a final 10-min step at 72°C for final extension.Genomic DNA was isolated from the buffy coat fraction of each blood sample with a Blood Mini Kit  according to the manufacturer's instructions. Genotypes of the three selected SNPs  and MspA1I for TNFRSF1B (+1663A>G), and the products were separated in 3% MetaPhor agarose gel. After completion of gel electrophoresis, the TNF-α -308 GG genotype produced two bands (127- and 18-bp), AA produced a single band (145-bp), and AG displayed all three bands   , GG produced a single band (248-bp), and TG displayed all three bands     Figure ; TNFRSF1) Figure ; and TNF) Figure Genotyping was performed without knowing the subjects' disease status. Two research assistants independently read the gel pictures and performed the repeated assays, if they did not reach a consensus on the tested genotype. About 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant.2 tests were performed to determine any statistically significant differences in the distributions of the TNF-α and TNFRSF1B genotypes by demographic variables and clinical features. Median survival time (MST) was calculated, and mean survival time was presented when the MST could not be calculated. Kaplan-Meier estimates were used to estimate overall survival (OS) among four genotype groups, and the log-rank test was used to test for equality of the survival distributions. Univariate analysis and multivariate Cox proportional hazards models were conducted to estimate the effect of each genotype on survival with or without the presence of known prognostic factors. Cox stepwise hazards regression model was also conducted to determine independently predictors of NSCLC prognosis, with a significance level of 0.050 for entering and 0.051 for removing the respective explanatory variables. Haplotype frequencies and individual haplotypes based on the observed genotypes were generated by using SAS PROC HAPLOTYPE. Analyses were performed by using SAS statistical package version 9.1 .The two-sided χth edition stage grouping criteria [P = 0.117), ethnicity (P = 0.827), smoking status (P = 0.457), histology (P = 0.092), Karnofsky's performance scores , chemotherapy status (P = 0.407) and radiotherapy dose (P = 0.706). However, sex, tumor stage, and node status were significantly associated with MST of NSCLC . Because these variables may be confounding factors for the effect of the genotypes on OS, they were further adjusted in the multivariable analysis.As shown in Table criteria , 33 caseTNF-α and TNFRSF1B and their associations with OS are summarized in Table TNFRSF1B +676 G>T (rs1061622) were statistically significantly associated with OS , but this association was not observed for the other four SNPs investigated in this study.The genotype distributions of the five SNPs in TNFRSF1B +676 T>G (rs1061622) on NSCLC survival. Three variables  were identified in the regression model with a significance level of 0.050 for entering and 0.051 for removing a variable   genotypes and selected factors including age, ethnicity, tumor stage and node status. The longest survival was observed in subgroups with both rs1061622 GG and age≤62 years (MST = 44.0 months), or white ethnicity (MST = 48.3 months), or T0-2 tumors (MST = 48.3 months), or N2-3 tumors (MST = 44.0 months), compared with the poorest survival in subgroups with both rs1061622 GT/TT and age>62 years (MST = 20.7 months), or non-white (MST = 21.0 months), or T3-4 tumors (MST = 18.2 months), or N0-1 tumors (MST = 29.2 months); however, small sample size in these subgroups might have led to a suboptimal statistical power to detect a statistical difference in the survival time. We also conducted the stratification analysis by histology and different treatments, but the numbers of patients in subgroups were too small, and no significant results were found in every stratum (data not shown).As shown in Figure TNF-α -308G/-1031T (GT) and TNFRSF1B +676T/+1663A/-1709A (TAA) were the most common haplotypes in the patients with the frequencies of 63.3% and 40.2%, respectively. However, we did not found significant associations between other haplotypes and OS of NSCLC patients, compared to the most common haplotypes (data not shown), which is likely due to limited study power.In addition, we also evaluated the combined effect of these polymorphisms on NSCLC survival by using the haplotype analysis. The results showed that TNF-α and TNFRSF1B on survival of NSCLC patients treated with chemoradiotherapy or radiotherapy alone. We found that the TNFRSF1B +676 GG (rs1061622) variant homozygous genotype was associated with a significantly improved survival of NSCLC in this non-Hispanic patient population. Such an effect was not observed for other SPNs under investigation.In the present study, we examined the effect of five selected polymorphisms in TNF-α is mostly regulated at the transcriptional level, and polymorphisms within the TNF-α promoter have been related to TNF-α levels [TNF-α-308 G/A in the promoter of TNF-α has been reported to be associated with higher expression levels of TNF-α [TNF-α-308 G/A and another SNP in the promoter of TNF-α  were significantly associated with prognosis of bladder cancer and breast cancer [TNF-α-308 G/A or TNF-α-1031 T>C and the prognosis of several other cancers, including gastric cancer [Tumor necrosis factor-alpha (TNF-α) executes multiple functions in immunity, inflammation, differentiation, control of cell proliferation, and apoptosis through distinct receptors known as TNF receptor type I (TNFR1) and type II (TNFR2) . Expressα levels ,23. For of TNF-α  and thusof TNF-α , breastsof TNF-α , oral caof TNF-α ,32, bladof TNF-α  and lungof TNF-α . Additiot cancer ,29,36, ct cancer . Howeverc cancer , colorecc cancer , Hodgkinc cancer , and brec cancer . Such a TNFRSF1B rs1061622 GG might be an independent predictor for survival of NSCLC patients treated with chemoradiotherapy or radiotherapy alone. The TNFRSF1B is the principal TNF receptor found on circulating T lymphocytes, which functions both as a TNF antagonist by neutralizing it and as an agonist by facilitating interaction between TNF and TNFR1 at the cell surface [TNFRSF1B (M196R) that causes a functional amino acid change from methionine (M) to arginine (R) [Interestingly, logistic and stepwise regression analysis in our study indicated that  surface . rs10616nine (R) , which inine (R) ,48.TNFRSF1B +676 T>G (rs1061622) with susceptibility and survival of breast cancer patients [TNFRSF1B +676 T>G (rs1061622) and lung cancer survival, and the exact biological mechanism underlying this association in our study remains to be investigated. It is possible that this SNP affects the function of TNFRSF1B and enhances radiotherapy or chemotherapy efficacy through inhibiting tumor therapy resistance. Another possibility is that this SNP may change the expression level of TNFRSF1B and influence the character of immunological response to infection in lung cancer patients, finally resulting in the difference of survival in NSCLC [TNFRSF1B +676 T>G (rs1061622) genotypes and selected factors including age, ethnicity, tumor stage and node status. Although our sample size was relatively small in these subgroups, it provided a clue that effect of this SNP on prognosis of NSCLC may be dependent on patients' characteristics and clinical features as well. However, the exact functional relevance of this SNP in the prognosis of NSCLC patients treated with chemoradiotherapy remains to be explored.However, the results from published studies on associations between rs1061622 and prognosis of cancers were controversial rather than conclusive. For example, a Tunisian study showed significant associations of patients , but anopatients . Up to nin NSCLC . FurtherTNF and TNFRSF1B genes in NSCLC patients, several potential limitations should be taken into consideration. Firstly, our sample size is relatively small with a limited statistical power to evaluate interactions between the studied polymorphisms and clinical factors including tumor stage and node status. For the same reason, the results from our subgroups by TNFRSF1B +676 T>G (rs1061622) genotypes and selected factors need validation by larger studies. Secondly, only five potentially functional SNPs in TNF and TNFRSF1B were included in our study, which is far from comprehensive. Indeed, both genes are highly polymorphic, and it is possible that some important SNPs may have been missed or the observed association may have been due to other polymorphisms in LD with the studied ones. Again, much larger studies would have the statistical power to confirm the associations of more SNPs with OS in such a patient population.Although our study reported some new findings about polymorphisms of TNFRSF1B +676 T>G in the prognosis of NSCLC. However, replication studies with diverse ethnic groups, larger sample sizes and functional characterizations are warranted. Currently, we have ongoing projects that help continue recruiting similar patients for additional analysis in the future.In summary, in this cohort of NSCLC patients, we identified a possible role of TNF-α: Tumor necrosis factor-alpha; TNFR2: tumor necrosis factor receptor super family 2; PCR-RFLP: polymerase chain reaction-restriction fragment length polymorphism; HR: hazard ratio; CI: confidence interval; SNP: single nucleotide polymorphism; NSCLC: non-small cell lung cancer; OS: overall survival.The authors declare that they have no competing interests.XXG, QYW and ZXL designed the study and prepared the manuscript. XXG performed the experiments with the assistance from ZSL and JQ. XXG and HXM conducted the analysis with assistance from LEW. XLY, DG and RK helped to collect and organize the clinical data. HXM, QYW and LEW helped to finalize the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/447/prepub


The large amount of literature published over the last two decades on human papillomavirus (HPV)-DNA testing has definitely demonstrated the association between high-risk viral genotypes (hrHPV) and cervical cancer. Moreover, hrHPV-DNA testing has shown excellent performance in several clinical applications, from screening settings to the follow-up of treated patients, compared to conventional cytology or colposcopy options. On the other hand, when a huge number of reports are published on the same subject in a relatively short period of time, with many variations in settings, study designs and applications, the result is often confusion and decreased comprehension by readers. In daily office practice, several different situations (in symptomatic or asymptomatic women) can be positively managed by the correct use of hrHPV-DNA testing. Validated hrHPV-DNA testing and, specifically, the HC2® assay, due to its excellent sensitivity and negative predictive value together with optimal reproducibility, currently represent a powerful tool in the clinician’s hands to optimally manage several situations related to HPV infection and the potential development of cervical cancer. Despite the large amount of data available on the value of human papillomavirus (HPV)-DNA testing for the detection of cervical cancer precursors, both in the primary cervical screening and in the management of ‘borderline’ or ASCUS  cytology, HPV-DNA tests have not always and correctly been translated into clinical practice by clinicians and within national cervical screening programs. This article is intended as a synthesis of the literature aimed at supplying useful and simplified ‘take-home messages’ to the clinical community. The recommendations have been formulated consistent with the evidence-based literature and rated according to their level of quality and strength, employing a generally recognized and approved rating system, described in Several population-based studies have established that tests for high-risk HPV (hrHPV) DNA have higher clinical sensitivity than cytology in detecting cervical intraepithelial neoplasia (CIN) of grades 2 and above (CIN2+) , 5, and Increased sensitivity has two important clinical outcomes: reduced mortality and an elongation of screening intervals; the latter implies better compliance with screening and lower costs. Recently, two papers confirmed these early assumptions. A publication from rural India, a setting where no population screening is in place, showed that a single round of hrHPV testing by Hybrid Capture 2 (HC2®) was associated with a significant decline in the rate of advanced cervical cancers and associated deaths, as compared with the unscreened control group . In contAnother added value of HPV testing over cytology is its high reproducibility: Carozzi  investigINK4A (P16) to triage positive women initially screened with HC2®; this strategy showed the same positive predictive value of conventional Pap smear screening , while retaining the higher NPV of screening by HPV testing [While the NPV of HPV testing over the Pap smear is well established, the management of HPV positive patients in the screening setting is still controversial. In a recent paper, different strategies have been evaluated: the authors concluded that primary HPV-DNA-based screening with cytology triage and repeat HPV-DNA testing of cytology-negative women appears to be the most feasible strategy . The Ame testing . In the INK4A P6 to tria testing . A scree testing .According to the emerging scenario of the post-HPV vaccination era, it is to mention the particular value of hrHPV-DNA testing as the most suitable screening test for vaccinated women; what is expected from vaccination in the next future, a very low prevalence of HPV-correlated diseases, depicts another added value of this screening option.Conclusions about the use of hrHPV-DNA testing in primary screening are summarized in Pap test reporting classifications have evolved during the last decades, with the current standard being the Bethesda System in its latest 2001 revision , 22. Of In the USA, the vast majority of women diagnosed with an ASCUS are triaged to colposcopy or other follow-up by an hrHPV-DNA test. In contrast, outside the USA there has been a long lasting lack of consensus as to the appropriate management of the millions of women with ASCUS or equivocal cytologic abnormalities. Three major management strategies have been used in Europe. Immediate colposcopy has been assumed to be the safest option but with the disadvantages of high costs and potential overtreatments. Because the sensitivity of a single round of conventional cervical cytology is relatively low, a program of repeated cytology has been used in most countries. Several large studies have evaluated the performance of hrHPV-DNA testing to guide management in the ASCUS population, with sensitivity for the detection of CIN2+ using HC2® reported as 83–100% .The ASCUS/LSIL Triage Study (ALTS) represents a hallmark randomized, multicenter clinical trial sponsored by the National Cancer Institute designed to compare three management options . HPV-DNABased on these studies, the ASCCP, in the 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests, recommends a HPV-DNA testing-based triage for women with ASCUS cytology . CervicaEvidence-based statements about the ASCUS cytology management are summarized in An important setting where hrHPV-DNA testing has shown potential benefit is the follow-up of women treated for CIN, to detect residual or recurrent disease . The lonIn conclusion, for women double negative (both Pap and HC2® tests) at six months retesting at 12 months could be safely skipped, maintaining the 24-month follow-up visit scheduled . In studIt can therefore be concluded that the evidence-based post-treatment follow-up should include both the conventional cytology and HC2® testing to identify patients at increased risk for disease recurrence (AII).Over the past 50 years, the relative proportion, as well as absolute incidence, of invasive and pre-invasive glandular lesions of the uterine cervix has been increasing in western countries. Reports from the 1950s and 1960s indicated that adenocarcinomas accounted for only 5% of cervical cancer cases, while in the 1970s they represented 20–25% of all cervical carcinomas . In receIn conclusion, in the follow-up of AIS patients who desire to preserve fertility, the combination of HC2® and cytology showed a high sensitivity in detecting persistent lesions. In addition, the NPV of 100% seems to be very useful in preventing unnecessary hysterectomies (AIII) .More than 40 different genotypes of HPV can cause infections of the cervix. Based on available cross-sectional and short-term prospective data, the risks of persistence and neoplastic progression markedly differ by HPV type , 44 but Several studies investigated the potential use and limitations of hrHPV genotyping in screening, the triage of borderline smears and post-treatment follow-up clinical situations. At present, only the ASCCP guideline includes genotyping in the management algorithm for use in the screening setting of triage cytology negative and hrHPV-DNA positive patients. HPV 16 and/or 18 positive women are recommended for immediate colposcopy, while HPV 16 and/or 18 negative but other hrHPV positive women are referred to annual follow-up visits. The clinical evidence supporting this clinical guideline is supported by many studies [HPV 16 and/or 18 positive women with an ASCUS or low-grade SIL Pap smear carry an increased risk of CIN2+ lesions. In the ALTS study, women with ASCUS or LSIL who were HPV16 positive were at increased risk for ≥CIN3 compared with women who were HPV negative ; this risk was fivefold greater than the risk in women who were positive for non-HPV 16 carcinogenic HPV types   (CII). THPV-DNA testing has been introduced into clinical practice as a cure test. Some studies investigated the potential use of genotyping in this clinical setting , 47. TheAt present, there are no clear indications of the appropriate use of genotyping in clinical practice; testing without changing the clinical protocol carries the potential to increase costs and harm to the patient, with no added benefit. It is recommended that the performance of the many HPV genotyping tests available on the market should be clinically validated by substantial clinical data and acceptable test inter-laboratory reproducibility confirmed before implementing genotyping into clinical practice. Due to current test limitations, the clinical application of genotyping is not included in most international guidelines.Cytology has reduced the incidence and mortality of cervical cancer in countries with organized screening, but in Europe there are still an estimated 68,000 incident cases while in the USA there are ~12,000 new cases per year . NeverthThe impending broad implementation of cervical cancer screening by hrHPV-DNA testing as a first-line screen should take into account both the substantial impacts of ongoing vaccination programs, with two vaccines showing efficacy in preventing both infections and associated precancerous lesions , 50 due Multiple variables simulating the natural history of cervical cancer, and specific details and costs of delivering clinical interventions, are used in such complex modelling systems. Moreover, models have to be tailored to the specific populations targeted for screening programs, which may significantly differ in HPV infection prevalence, rates of other related cervical cancer risk factors, costs and cost-related variables. The rate of screening coverage and adherence radically impacts each given scenario.posteriori analysis of observed data, as obtained through continued surveillance and post-marketing studies, is mandatory [Although well-designed model-based simulations are considered to be useful and predictive for future clinical applications, real world validity should be further confirmed. As recently stated by the European Guidelines for Quality Assurance in Cervical Cancer Screening, a andatory .The most relevant variables used by mathematical models to evaluate the cost-efficacy of hrHPV-DNA testing-based screening are the longitudinal clinical performance indicators: sensitivity, specificity and positive-NPVs. Regarding clinical sensitivity, a recent meta-analysis reported a pooled sensitivity for HC2® in primary screening of 97.9% (AI) . The expConsidering the epidemiology and natural history of HPV infections, selecting the appropriate age of screening is important. The age of 30 years (concerning the age-related decline of transient infections) has been considered appropriate to start hrHPV-DNA screening in Europe , 57 ;vaccinated girls (vaccination starting at age 12 years): screening starting at 25 years with hrHPV-DNA testing with cytology triage (rounds every five years) .If HPV vaccination is included in the analysis, the following strategies were valued as being more cost-effective than the ongoing cytology screening:The optimal screening strategy for women vaccinated at older ages (18 years or above) has not yet been determined, and until specific guidance data are available, they should be screened as per the algorithm for unvaccinated women.A cost-effectiveness analysis in 2006 concluded that hrHPV-DNA testing is an economically viable strategy for the triage of ASCUS cytology (AI) .The proper education of women and of the various healthcare professionals involved in the process are fundamental aspects in the area of HPV-DNA testing. The fact that cervical carcinoma is caused by a virus which is sexually transmitted introduces additional complexity and high-emotional impact to the doctor–patient relationship. HPV is extremely widespread in healthy populations, and the concepts of temporary infection, persistent infection, and illness are strikingly different, but conceptually difficult to understand. The availability of the HPV test in primary screening implicates the management—clinical and psychological—of a fair number of women with normal cytology and positive viral tests, especially if young women (<30 years old) are included. Resorting to self-obtained information in the area of health is in continuous expansion, and frequently, the sources of the available materials are of debatable quality. HPV and related diseases are certainly newsworthy and of interest to the media.The minimum set of information for women is summarized in Available time, adequacy of the environmental setting, technical competence, the communication skills of the health professional and the receptive level of the patient are the most important variables to consider and possibly to face , 62–64. The whole universe of cervical pathology should, in our minds, be framed in the concept of cervical cancer risk. Considering specifically the persistent positivity of viral tests as one of the most significant indicators in the degree of probability that a woman, over time, will develop a serious cervical pathology—as is similarly done in cases of plasma cholesterol levels or obesity in cardio-vascular risk—may help to correctly position the examination in the imaginary collective and introduce implications of varying follow-up procedures  in relation to risk stratification .In light of this, the positivity of the test does not represent an illness, but simply the presence of a risk factor that, if confirmed one year after the first sampling, will suggest the plain observance of a non-invasive cytological or instrumental triage. As a whole, first and second level cervical screening tests guarantee a degree of protection from the real disease—invasive carcinoma—that has no equivalent in medicine (AI).Human papillomavirus (HPV) is well-recognized as the necessary cause of neoplastic transformation of the uterine cervix epithelia. As regards uterine cervical cancer prevention, a significant body of literature has definitely demonstrated the higher performance of hrHPV-DNA testing over cytology in terms of sensitivity, negative predictive power (NPP) and reproducibility in different clinical settings: primary screening, triage of ‘borderline’ (ASCUS) cytology and follow-up of treated preneoplastic disease (CIN). Compared to cytology, either conventional or liquid based, internationally validated HPV-DNA tests demonstrate a very close to 100% sensitivity and NPV in the identification of cervical preneoplastic conditions. Thus, the new screening algorithm should include hrHPV testing as primary test, with cytological triage only in hrHPV positive women. However, although the validity of hrHPV-DNA testing is already worldwide accepted as triage for ASCUS cytology or as test of cure after CIN therapy, it is not yet translated as front-line test into clinical practice in organized programs (with the exception of the Netherlands) or in opportunistic screening as well. The added value, compared to conventional cytological screening, is concerning not only squamous cervical lesions, but also glandular types , which are less prevented by the national programs of surveillance.In view of the epidemiology and natural history of HPV infections, the age of 30 years appears to be appropriate to start hrHPV-DNA testing as primary screening, avoiding younger women where HPV-infection is highly prevalent, consisting in large amounts of spontaneously regressive lesions.Due to variability in performance , no clear indications of the appropriate use of HPV genotyping in clinical practice can be drawn. Unfortunately, the huge amount of data of literature in the field is not consistent with the clinical practice, and many clinicians still rely on Pap smear or suggest to patients the use of HPV-DNA testing with questionable indications; strong evidence-based recommendations can be made in order to achieve the best results of cervical cancer prevention: primary screening, triage of ASCUS cytology and follow up of conservatively treated intraepithelial cervical lesion are definitely the settings in which HPV-DNA testing is the optimal option.


Objective. To verify the relationship between vaginal pH and human papillomavirus (HPV) infection and to measure cytokine levels in endocervical secretions of women with bacterial vaginosis (BV) or HPV. Methods. 173 women (16–48 years old) were enrolled and divided into groups: BV, HPV, and controls. Microbiological culture and vaginal pH were measured. HPV detect by PCR, and cytokines by ELISA . Results. Of 173 women, 60 were control group (34.7%) and 113 were distributed in HPV , BV , vaginitis  and, BV and HPV-associated groups . Vaginal pH > 4.5 was related with HPV infection. IL-2 and IL-12 were increased in BV and HPV groups, and IL-6 (only BV group), compared to control group. IL-12 and IFN-γ were higher in HPV than BV group. Conclusion. The increase of vaginal pH was associated with HPV infection; BV and HPV groups had a Th1 cytokines immune response. The vagina and cervix are the first lines of physical and immunological defense against sexually transmitted pathogens , 2. The Gardnerella vaginalis, Mobiluncus spp., Bacteroides sp., Prevotella spp., Peptostreptococcus spp., Mycoplasma hominis, or Ureaplasma urealyticum are routinely detected and are accompanied by a subsequent increase in the vaginal pH  andAfter each participant gave written informed consent, biological material was collected from the posterior fornix by using a gynecological brush . The sam2. In those cases, diagnosis was confirmed by tests of carbohydrate fermentation, motility, and rapid hippurate hydrolysis [Diagnosis of bacterial vaginosis was made when three of the four established clinical criteria were met: vaginal discharge, pH > 4.5, positive amine test, and the presence of clue cells [drolysis .μL, which was then aliquoted and stored at −70°C until use for cytokine measurement by ELISA. The cultured plate was used for microbiological analysis after 48–72 hours of incubation at 37.0 ± 0.5°C.The sample that had been collected from the endocervix was used for laboratory measurement of cytokine levels and the presence of high-risk and low-risk HPV. The tube containing the endocervical secretion was centrifuged and saline (0.85%) was added to obtain a final volume of 500 α which was standardized at 100 ng/mL. The ELISAs for the samples and controls were performed in duplicate at the Oncology Research Institute of Federal University of Triângulo Mineiro—UFTM. Standard recombinant cytokines were used as controls. Samples previously diluted in saline were further serially diluted with assay diluent , according to the BD recommendations. The spectrophotometric absorbance at 405 and 490 nm was measured and the difference of the values was multiplied by the dilution factor of the initial dilution of the samples in saline. The concentration of cytokines was determined in pg/mL by comparing the absorbances with those of a standard curve for the respective recombinant cytokine [For cytokine measurement, an aliquot of the endocervical secretion was thawed and subjected to ELISA using pairs of commercially available monoclonal antibodies . All cytokines were standardized in a concentration of 10 ng/mL, except for that for TNF-cytokine .μL of the initial 500 μL sample in TRIzol to extract DNA, according to the manufacturer's instructions . We performed PCR to test for the presence of low-risk HPV 6 and 11 (40 cycles) and high-risk HPV 16, 18, 31, 33, and 35 (35 cycles) using the primers 5′ to 3′ TACACTGCTGGACAACATGC and 5′ to 3′ GTGCGCAGATGGGACACAC, for the low-risk strains, and 5′ to 3′ TTTGTTACTGTGGTAGATACTAC and 5′ to 3′ GAAAAATAAACTGTAAATCATATTC for the high-risk strains, as previously described [μL of sample buffer and electrophoresed through a polyacrylamide gel (10%) at 90 volts for 2 hours and stained with 2% silver nitrate. A standard 50 bp molecular weight marker was used on each gel . The gel was then analyzed for the presence of specific banding patterns [We diluted 100 escribed . The amppatterns .odds ratios. The chi-squared and Mann-Whitney tests were used to determine if differences among the groups reached statistical significance (P < 0.05).Descriptive statistical analyses were carried out, including examination of the frequency distributions of categorical data. The prevalence was calculated to reflect the relative frequency of genital infections using confidence intervals (CIs) and n = 36, 20.8%), BV , vaginitis , and BV and HPV-associated were in . Of the 47 patients infected with HPV, 36 were only by this virus and 11 were associated with BV. Among those infected only with HPV 19 (52.8%) had only high-risk HPV, 12 (33.3%) the low-risk, and 5 (13.9%) had both subtypes. Coincidentally, the same number of patients was diagnosed with BV (47) and 36 (76.6%) had only this disease, 6 (12.8%) were associated with high-risk HPV, 4 (8.5%) associated with HPV low-risk, and only one of them had both BV and viral types. In women with BV, 41 (87.2%) were associated with the presence of Gardnerella vaginalis and 6 with other anaerobic bacteria.The women were divided into three groups according to presence of BV, high- and low-risk HPV infection or noninfected healthy controls see . Of a toThe diagnosis of BV was made in 47 cases when three of the four routine clinical criteria were detected. In particular, pap smear analysis revealed clue cells in 41 of the cases. However, Gram staining was positive in 95.8% of the 47 diagnosed BV patients.Lactobacillus sp. and other bacilli and Gram-positive cocci. The 47 BV-positive cases harbored Gardnerella vaginalis , Prevotella sp. (in 42.5%), and Clostridium sp. (in 14.9%), in addition to other anaerobes. Among the 41 cases of GV diagnosed by culture, 11 also had HPV , and six of these 11 were of high risk (P = 0.850), four were of HPV low-risk (P = 0.616), and one had both low and high-risk strains (P = 0.200). In the group without GV, we observed a similar rate of HPV infection . The same comparisons were made for the group with and without increased anaerobic bacterial flora. Forty-three (27.0%) of the women who presented with the prevalent of anaerobic had HPV, which was similar to those in which we did not identify any prevalence of anaerobes (28.6%) (P = 0.902). There was not observed an association between the presence of HPV and the presence of predominant anaerobic bacteria however, we identified a statistical association between vaginal pH > 4.5 and HPV infection , BV and low-risk HPV (n = 4), BV and high-risk HPV (n = 6), and VB and high-low risks HPV (n = 1) could not be statistically determined due to the relatively low numbers of patients. The other cytokine concentrations and the association between bacterial vaginosis (BV), HPV, high-risk HPV, and low-risk HPV are in Levels of the cytokines IL-2, IL-6, IL-12, IFN-were not . The assIn this study, our cohort exhibited incidences of BV and HPV that were higher than those previously reported . This fiGardnerella vaginalis as the main microorganism associated with the appearance of BV in our study cohort. These particular results are consistent with other previous studies [ Papanicolaou smears are a routine method used to identify the presence of clue cells associated with BV , 22. We  studies , 5, 24. Gardnerella vaginalis (P = 0.890), Prevotella sp. (P = 0.150), or any other species of anaerobic bacteria (P = 0.840) in the vaginal mucosa and conclude that the predominance of anaerobic microorganisms in vaginal flora was not significantly associated with the appearance of HPV infections.Changes in the vaginal flora, such as the appearance of clue cells, suggest that BV occurs with a significantly higher frequency in women with cervical cytological abnormalities. A higher frequency of HPV has also been observed in these patients and is presumed to be supported by the production of nitrosamines by anaerobic bacteria and subsequent stimulation of cytokine production . HoweverConsidering that BV, cervicitis, and CIN have been associated in some studies, it would not be surprising if the patients with BV and CIN presented with similar profiles in their localized immune response . In contP = 0.031) [One of the most common recognized factors among women with BV is the elevation of the vaginal pH . Murta e= 0.031) , 26. Mor= 0.031) . KnowingSampling the endocervical secretion is a reliable, highly reproducible method to detect cytokine concentrations in the genital tract . By thisP = 0.039), but not those who have coinfection with high-risk HPV strains (P = 0.256).The local production of cytokines in cervix is important for the regulation of immunity in the genital tract . ResearcThe levels of IL-2 are not increased in patients with HPV, HIV, or other STDs, probably because this cytokine is produced very early in the immune process and rapidly degraded . HoweverThese cytokines IL-6 and IL-8 are probably associated with the development of cervical cell lesions , 31. Theγ in women with HPV except in low-risk HPV were rather above those of the control group. According to a previous study, the presence of HPV associated with the regression of cervical lesions may be the result of a Th1 response (decreased IL-8 and increased IFN-γ). Although one study reported a decrease of IFN-γ during HPV infections [In this study, the levels of IFN-fections , our stufections .α have not been reported as significantly different between BV-positive and -negative subjects [Gardnerella sp. and Prevotella sp., having a Nugent score >4 when compared to a group with an abundance of Lactobacillus sp. [α and leukocytes in BV-diagnosed women with GV has also been reported [α and the presence of BV or in association with BV and HPV infections. Further studies are needed to confirm the lack of change in TNF-α for cases of HPV infections and BV. Other vaginal infections also are influenced by the concentration of cytokines. In candidiasis, increases in IL-10 and decreases in IFN-γ have been reported [The increases in TNF-subjects . Due to subjects . Previoullus sp. . An incrreported . Trichomreported ; howeverreported .Unfortunately, it wasnot possible to eliminate the natural variations in cytokine concentrations that occur during different phases of the menstrual cycle because the samples were collected at different times of the cycle, and it was difficult to accurately determine the duration of the infection for the individuals included in our study. Stages of infection can also influence the different cytokine levels and the secreted cytokine profile can influence the outcome of an infection. A cohort in order to control these factors could contribute for analyzing those variables.γ were higher in HPV group than only BV. By comparing the levels of cytokines in vaginal secretion among high-risk HPV, low-risk HPV, and BV groups, we found that a Th1 immunological response was occurring.We conclude elevation of vaginal pH (>4.5) was frequent in genital HPV infections. We also found IL-2 and IL-12 was increased in individuals with BV and HPV infections, whereas individuals with only BV had higher cytokine levels of IL-2, IL-12, and IL-6, compared to control group, the cytokines IL-12 and IFN-


Low avidity of antibodies against viral, bacterial and parasitic agents has been used for differential diagnosis of acute versus recent/past infections. The low-avidity antibodies may however, persist for a longer period in some individuals.We studied the association of human papillomavirus (HPV) type 16 antibody avidity with seroprevalence to HPV types 6/11/18/31/33/45. Antibody avidity was analysed for 365 HPV16 seropositive pregnant Finnish and Ugandan women using a modified ELISA.Low avidity of HPV16 antibodies was found in 15% of Finnish and 26% of Ugandan women. Ugandan women with low-avidity HPV16 antibodies had an increased risk estimate for HPV6/11  seropositivity but not to high-risk HPV types 18/31/33/45.Association of the low avidity HPV16 antibody "phenotype" with possible susceptibility to infections with other HPV types warrants investigation. Persistent infections with high-risk human papillomavirus (hrHPV) type 16 cause half of cervical cancer (CxCa) morbidity/mortality . InfectiHPV16 causes genital infections. Following the infection, development of antibody response takes from 6 to 18 months ,13. DuriMaturation of the IgG antibody avidity takes approximately 6 months -16. ThisWe evaluated whether presence of low-avidity of HPV16 antibodies is associated with an increased risk of prevalence for other HPV types.A total of 4748 pregnant Finnish (2784) and Ugandan (1964) women participated in an epidemiological study . In FinlChlamydia trachomatis and HIV antibodies, and serum cotinine (current smoking > 20 ng/ml) were analysed by ELISA as described [escribed . Standarescribed ,24-27, wescribed -19 for Hescribed . Wells Aescribed -20. Cut-escribed -20. The Chlamydia trachomatis and HIV antibodies, and cotinine. Statistical analyses were done using Stata 8 .Logistic regression was used to calculate the relative risk  of being HPV6/11/18/31/33/45 seropositive associated with low avidity HPV16 antibodies. Single HPV16 seropositives were the reference group. Adjustment was stepwise for age, Overall, HPV16 seroprevelances were equal (21%) in both countries . AltogetC. trachomatis, and smoking were 0%, 25% and 23%  among the Finns and 9%, 27% and 2%  among the Ugandans.Overall, the prevalence of low-avidity HPV16 antibodies was 15% (38/248) among the Finns and 26% (30/117) among the Ugandans (p < 0.0001). There was no statistically significant correlation  between HPV16 antibody avidity and age . After stratification by country and adjustment, increased risk for being HPV6/11 seropositive was found only among the Ugandans . Estimates on the association of low-avidity with seropositivity for other hrHPVs were not different from unity  of HPV16 serology  some HPVThe association was seen only in the Ugandans, whose HIV seroprevalence was 9%. Adjusting for HIV, however, had no effect on the low-avidity associated risk, antibody avidity is not affected by HIV infection ,22. SmokPBN, CB, LH, KH, EKM, RB, MM, RK, HMS declare no conflicts of interest. ML has obtained grants from Merck&Co. Inc. and GSK-Biologicals for HPV vaccination studies through his employers.PBN, designed, ran the study, conducted data collection and analysis and wrote the report.ML designed the study, supervised PBN in data analysis, data interpretation and report writing. KH, developed the modified ELISA. KH, LH, HMS supervised PBN in laboratory analysis and contributed to data interpretation and report writing. KD processed the samples. EKM, CB, RB, MM, RK, contributed to study design. All authors read and approved the final manuscript.


Human papillomavirus (HPV) is an oncogenic virus causing oropharyngeal cancers and resulting in a favorable outcome after the treatment. The role of HPV in oral cavity squamous cell carcinoma (OSCC) remains ambiguous.This study aimed to examine the effect of HPV infection on disease control among patients with OSCC following radical surgery with radiation-based adjuvant therapy.We prospectively followed 173 patients with advanced OSCC (96% were stage III/IV) who had undergone radical surgery and adjuvant therapy between 2004 and 2006. They were followed between surgery and death or up to 60 months. Surgical specimens were examined using a PCR-based HPV blot test. The primary endpoints were the risk of relapse and the time to relapse; the secondary endpoints were disease-free survival, disease-specific survival, and overall survival.P = 0.005), disease-free survival (P = 0.037), disease-specific survival (P = 0.006), and overall survival (P = 0.010), whereas HPV-18 infection had no impact on 5-year outcomes. The rate of 5-year distant metastases was significantly higher in the HPV-16 or level IV/V metastasis group compared with both the extracapsular spread or tumor depth ≥11-mm group and patients without risk factors (P<0.001).The prevalence of HPV-positive OSCC was 22%; HPV-16 (9%) and HPV-18 (7%) were the genotypes most commonly encountered. Solitary HPV-16 infection was a poor predictor of 5-year distant metastases (hazard ratio, 3.4; 95% confidence interval, 1.4–8.0; HPV infections in advanced OSCC patients are not uncommon and clinically relevant. Compared with HPV-16-negative advanced OSCC patients, those with a single HPV-16 infection are at higher risk of distant metastases and poor survival despite undergoing radiation-based adjuvant therapy and require a more aggressive adjuvant treatment and a more thorough follow-up. Human papillomavirus (HPV) is a well-known oncogenic virus often observed in patients who have had a favorable outcome after the treatment of oropharyngeal cancers However, the role of HPV in oral cavity squamous cell carcinoma (OSCC) remains ambiguous because of the relatively small number of recorded OSCC patients in comparison with the larger population of oropharyngeal cancer Several questions about the role of HPV in advanced OSCC patients who require adjuvant therapy following radical surgery in southern Asia remain unanswered. For example, what is the incidence of HPV infections among OSCC patients? Are the clinical and biological behaviors of HPV in OSCC the same as those in oropharyngeal SCC? Are different treatment strategies and follow-up protocols appropriate in HPV-positive OSCC patients? Does HPV infection affect the outcomes of postoperative adjuvant therapy? To answer these questions, we studied a large cohort of patients with previously untreated OSCC who underwent radical surgery with or without adjuvant therapy; in particular, we focused on the impact of HPV infections on the outcomes of radiation-based adjuvant therapy for advanced OSCC. Accordingly, this study aimed to test the hypothesis that HPV infections among advanced OSCC patients are associated with a decreased risk of disease relapse, including local recurrence, neck recurrence, and distant metastasis, and therefore improve the rates of survival, including disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS).The Institutional Review Board at Chang Gung Memorial Hospital approved this study, which complied with the Declaration of Helsinki. All participants provided written informed consent. The inclusion criteria were as follows: a histological diagnosis of OSCC, the presence of a previously untreated tumor scheduled for radical surgery with neck dissection (ND), the absence of other suspected distant metastatic lesions detected by imaging, and a willingness to undergo imaging-guided biopsy or exploratory surgery if necessary. The exclusion criteria included a refusal or inability to undergo radical surgery.Between 2004 and 2006, 333 patients were prospectively included in this study. All patients consented to and participated in the long-term outcome survey program of the Head and Neck Oncology Group at the Chang Gung Memorial Hospital. All participants underwent an extensive presurgical evaluation that included a medical history and a complete physical examination, flexible fiberoptic pharyngoscopy, a complete blood count, routine blood biochemistry, CT or MRI scans of the head and neck, chest radiographs, bone scans, and liver ultrasonography. Cancer staging was performed according to the 2002 American Joint Committee on Cancer 6th edition staging criteria 2 cisplatin biweekly plus 800 mg daily oral tegafur and 60 mg leucovorin, or 30 mg/m2 weekly cisplatin All patients underwent radical excision of the primary tumor with ≥1 cm gross safety margins . Classic radical or modified NDs (level I–V) were performed in the patients with clinically positive lymph node disease. Supra-omohyoid NDs (level I–III) were performed in clinically node-negative patients. Most of the uncomplicated patients underwent surgery alone except for those who unexpectedly had close margins ≤4 mm and/or positive lymph nodes as identified by pathological examinations. In this study, the subjects who underwent adjuvant therapy were considered as advanced OSCC patients. The indications for postoperative RT (60–66 Gy) included pathological T4 tumor, a positive lymph node, or a close margin ≤4 mm. ECS or multiple lymph node metastases were the reasons for the administration of CCRT with 50 mg/mPatient data were extracted from medical records and classified according to our previously identified risk factors for OSCC, which were described in detail elsewhere Excised tumor samples were collected during radical surgery. DNA was extracted from paraffin-embedded tumor samples using a Lab Turbo 48 automatic nucleic acid extraction system and a Lab Turbo Virus Mini Kit LVN500 . Finally, 50 µL of DNA solution was eluted, and 1 µL was used as the PCR template. HPV infection was diagnosed in subjects using PCR on the HPV L1 gene. HPV DNA was amplified with MY11/biotinylated GP6+ primers, which targeted the L1 region and produced a 192-bp DNA fragment. The PCR reaction volume was 25 µL, which included a 2-µL aliquot of purified DNA. In the positive cases, the HPV L1 gene was genotyped using an HPV Blot kit  that can differentiate the 39 HPV types . HPV type-specific probes were immobilized on a nylon membrane, which was used for reverse blot hybridization to detect HPV DNA in a single assay. The HPV types were determined using a visual assessment protocol provided by the manufacturer The primary endpoint was time to disease relapse including local recurrence, neck recurrence, and distant metastasis. The secondary endpoints were DFS, DSS, and OS. DFS was calculated as the date of primary surgery to the date of disease relapse. DSS was calculated as the date of primary surgery to the date of death caused by a disease recurrence, and OS was defined as the time period between primary surgery and death caused by any reason.Follow-up visits continued until December 2011. All patients received follow-up examinations for at least 60 months after surgery or until death. The procedure used for selecting the optimal cutoff values for clinicopathological factors has been previously described (20). Five-year local control, neck control, distant metastasis, DFS, DSS, and OS rates were computed using the Kaplan-Meier method (log-rank test). Univariate and multivariate analyses were used to identify independent predictors of 5-year outcomes. Independent prognostic factors were identified using multivariate Cox regression analysis with a forward selection procedure. Statistical analyses were performed using the SPSS software . A two-sided P value <0.05 was considered statistically significant.During the study period, we recruited 333 OSCC patients . A total of 240 patients (72%) reported drinking alcohol, 284 (85%) reported chewing betel quid, and 290 (87%) reported smoking cigarettes. Nineteen patients (6%) underwent primary tumor excision only, and the other 314 (94%) underwent ND in addition to primary tumor excision. The results of the pathological staging were as follows: pT1 (15%); pT2 (41%); pT3 (15%); pT4 (29%); pNx (6%); pN0 (55%); pN1 (12%); pN2b (24%); and pN2c (3%). When pNx (in the absence of ND) was classified as pN0, the pathological stages were as follows: p-stage I (14%), p-stage II (29%), p-stage III (15%), and p-stage IV (43%). The 5-year control and survival rates for all of the OSCC patients were as follows: local control, 89%; neck control, 86%; distant metastases, 12%; DFS, 73%; DSS, 79%; and OS, 66%.Among the 333 patients, 52% had advanced OSCC and underwent radical surgery followed by adjuvant therapy , whereas 48% were the uncomplicated OSCC patients who underwent surgery alone. The tumor aggressiveness that was observed in the advanced OSCC patients was distinctively different from that of the uncomplicated OSCC patients in terms of pathological T-status , pathological N-status , pathological stage , and ECS . The 5-year control/survival rates were significantly worse among the advanced OSCC patients compared with those of the uncomplicated OSCC patients: local control , neck control , distant metastases , DFS , DSS , and OS .The median duration of follow-up for the advanced OSCC patients was 58 months . At the time of the analysis, 81 of the 173 patients (47%) were alive, and 92 (53%) were dead . Twenty-five patients (15%) developed local recurrences, 31 (18%) had neck recurrences, and 35 (20%) experienced distant metastases. A total of 47 patients (27%) exhibited local and/or neck recurrence, salvage therapy was performed in 29 individuals (62%) and 21 (72%) dead at the time of the analysis.The overall prevalence of HPV infection among the OSCC patients was 21.3% (n = 71), and the 3 most common genotypes, including single and multiple infections, were as follows: HPV-16 , HPV-18 , and HPV-52 . The proportion of HPV-positive cases among the advanced OSCC patients was similar to that among the uncomplicated OSCC patients . The distributions of HPV-16 (9.2% vs. 6.3%), HPV-18 (6.9% vs. 6.9%), and HPV-52 (0.6% vs. 3.1%) were similar in both groups .We calculated the 5-year DFS, DSS, and OS rates among the OSCC patients according to HPV status. Compared with the HPV-negative patients, those with HPV-positive tumors had similar rates of DFS . The 5-year outcomes were calculated in both the advanced OSCC group and in the uncomplicated OSCC group. There were no significant differences in time-to-recurrence or time-to-death among the HPV subgroups in the uncomplicated OSCC group . To further comparison, we further divided HPV infection into 3 subgroups according to genotype frequency . Among the advanced OSCC patients, single HPV-16 infection was unrelated to local control or neck recurrence. Solitary HPV-16 infection was associated with a higher rate of distant metastases than the HPV-negative status  and markedly lower DSS and OS rates . The local control and neck control status were similar in both groups . The multivariate analyses of important risk factors, including solitary HPV-16 infection, pN2, level IV/V metastases, ECS, tumor depth ≥11 mm, and lymphatic invasion, are shown in Using level IV/V metastases, solitary HPV-16 infection, ECS, and tumor depth ≥11 mm as independent predictors, we created four subgroups : an “HPV-16 or level IV/V metastases” group (n = 24), a “both ECS and tumor depth ≥11 mm” group (n = 39), an “ECS or tumor depth ≥11 mm” group (n = 71), and a “no risk factor” group (n = 39). The 5-year distant metastatic rate was higher in the HPV-16 or level IV/V group than in the ECS and tumor depth ≥11 mm group (P = 0.075), the ECS or tumor depth ≥11 mm group (P<0.001), and the no risk factor group (P<0.001) .n): the HPV-16 (6/8) or level IV/V (6/7) group, the tumor depth ≥11 mm group (12/39), and the tumor depth <11 mm group (3/24). The 5-year distant metastasis rates of the ECS patients were higher in the HPV-16 or level IV/V group than in the tumor depth ≥11 mm group  and the tumor depth <11 mm group   .The null hypothesis of the present study was rejected, that is, HPV infections did not decrease the risk of disease relapse and was not associated with a better survival among advanced OSCC patients undergoing radical surgery and radiation-based adjuvant therapy  & Fig. 2Our findings are surprisingly different from the recent HPV-related outcome surveys in the field of head and neck cancers that have shown positive clinical impacts of HPV on DSS or OS The evidence regarding the clinical impact of HPV in OSCC patients remains inconclusive n = 12) in advanced OSCC. However, there was no difference in relapse or survival between patients with and without solitary HPV-18 infections. In this context, we further focused our study on HPV-16. Previous studies have reported that HPV-positive oropharyngeal cancers are associated with poorly differentiated histology, T1–T2 disease, N2–N3, and radiosensitivity HPV-18 infection is uncommon  in oropharyngeal cancer In addition to solitary HPV-16 infection, we further showed that level IV/V metastases, ECS, and tumor depth ≥11 mm are independent risk factors for 5-year distant metastases in advanced OSCC. Four subgroups of distant metastases were thus created . We prevSeveral caveats of this study merit comment. First, a potential limitation of our report is the use of specific PCR assays for detecting the HPV L1 gene. Because PCR amplification of HPV DNA is a very sensitive technique, we ruled out the possibility of laboratory artifacts and the presence of environmental virions by performing all amplifications in duplicate and with the use of two different PCR assays. We acknowledge that reverse transcriptase-PCR assays for E6 and E7 transcripts may be more reliable for the detection of oncogenic HPV infections. Moreover, the lack of available tumor marker data  does not allow us to draw any conclusion on the activity of viral oncogenes. To further characterize the possible mechanisms by which HPV-16 infections could be related to the risk of distant metastases and death, the measurement of E6 and E7 expression and HPV-associated biomarkers will be required in future studies.In conclusion, HPV infection does not represent a favorable prognosticator in the OSCC patients who receive radical surgery regardless of radiation-based adjuvant therapy. Notably, the advanced OSCC patients with solitary HPV-16 infection require priority adjuvant treatment and follow-up due to increased risk of early distant metastases and death. Moreover, in particular, patients with level IV/V metastases or HPV-16 infection with ECS require a more intensive therapeutic protocol. Our findings suggest that different types of HPV infections present distinct clinical and biological challenges among advanced OSCC patients, and there is at least one such HPV-16 associated with unfavorable outcomes in individuals who have received conventional adjuvant treatment. The significance of HPV-16 infection should be further studied in future translational and clinical research.


HPV DNA was detected in 1.5% of all samples, and in 3% and 0.4% of samples from females and males, respectively. In approximately 70% of HPV DNA positive adolescents, the infection was due to a single genotype, with 88.9% of genotypes belonging to the HR-clade. The only two HPV-positive boys (14 and 18 years old) had HPV-70 genotype. Only one of the 11 HPV-infected girls was in the 11-14 age-group. HPV prevalence was 4.2% in girls aged 15-18 years and 60% of infections were due to vaccine types HPV-16 or HPV-6/-11. This is one of the few studies, the first conducted in Italy, on HPV infection in adolescents. Urine testing is the easier way of detecting HPV infection in younger populations. Our data revealed a very low HPV prevalence, and no infections were observed in the 12-year-old vaccine target population. The majority of infections were seen in females aged 15-18 years. Overall, more than 50% and 30% of the potentially persistent HPV infections detected in this group could have been prevented by the quadrivalent and the bivalent vaccines, respectively. The introduction of vaccination against Human Papillomavirus (HPV) in adolescent girls in 2006 has focused virological surveillance on this age group. As few studies have evaluated HPV infections in young populations, further data are needed in order to improve and extend prophylactic policy and to monitor epidemiological changes. The present study aimed at evaluating overall and type-specific HPV prevalence in both female and male adolescents in Italy. HPV DNA detection and genotyping was performed on urine samples collected from 870 unvaccinated adolescents  in five cities in Italy. Following DNA extraction by means of a commercial kit (NucliSENS Human Papillomavirus (HPV) is the cause of the most common sexually transmitted infection and is associated with the development of severe diseases, including invasive cervical cancer, anogenital cancers and oral carcinomas, as well as benign manifestations  .HPV vaccination has been introduced as a preventive measure against cervical cancer, with young women as its primary target . EpidemiTo date, two different vaccine formulations are able to protect against persistent infection caused by the most common oncogenic types (HPV-16 and -18) associated with the development of cervical cancer, one of which also protects against genotypes HPV-6 and -11, which are associated with genital warts . These tThe Italian Medicines Agency (AIFA) approved both vaccines in 2007 ,11 and TNoteworthy, recent evidence has shown that genital HPV infections in adolescent females can occur even before the first sexual intercourse, possibly owing to non-coital sexual behaviours ,18, autoThe quadrivalent HPV vaccine was approved by the FDA and the European Medicines Agency (EMA) also for males 9-26 years of age in 2009 and in 2011, respectively ,22. The Although adolescents are the primary target of prophylactic HPV vaccination programs, few epidemiological studies on this age group are available. Additional data are therefore needed in order to ascertain the HPV infection status of the adolescent population. This knowledge would enable us to improve and extend vaccination policy and to monitor changes in overall and type-specific HPV prevalence rates as a result of HPV vaccination. To add new epidemiological data to this issue, we conducted a study aimed at evaluating the prevalence of HPV infection and the distribution of HPV genotypes among unvaccinated 11- to 18-year-old boys and girls in Italy by means of a molecular test suitable for HPV DNA detection and genotyping in urine samples .The study protocol used by all research units was approved by the Ethics Committee of the Genoa Local Healthcare Unit (LHU), Italy. All subjects gave assent to participate in the study. Informed written consent was obtained from each 18 year-old participant, or from parents or legal guardians if subjects were under 18 years of age. As part of a National Research Program of the Italian Ministry of University and Research (MIUR), this study recruited HPV-unvaccinated male and female adolescents at sports medicine centres and at secondary schools located in five cities of five different Regions: Turin (Piedmont), Milan (Lombardy), Genoa (Liguria), Florence (Tuscany) in North/Central Italy, and Sassari (Sardinia), Insular Italy. On the basis of our preliminary studies (unpublished data), the expected prevalence for HPV positivity in urine from adolescents was around 1%, ranging from 0 to 3%. The power and the statistical significance (two sided) were set at 90% and 5% level, respectively. To determine the sample size in order to detect a difference between proportions in a range of 1% (i.e. between 0.5% and 1.5%), at least 865 individuals were needed to be enrolled. Individuals were proportionally sampled in the five cities on the basis of resident population of 11-18 years of age. Recruitment was conducted between September 2009 and July 2010 and each participant was asked to provide an anonymous self-collected urine sample. Urine samples were collected in sterile containers, maintained at room temperature (RT) and processed within 6-8 hours after collection at each participating centre. To obtain the cellular component, at least 15 ml of each sample was pre-treated by means of two different successive steps of centrifugation and washing, as previously described  and stor® miniMAG®  extraction kit. The concentration and purity of extracted DNA was evaluated by using a spectrophotometer . DNA quality was assessed by amplifying a 268 bp (base pair) fragment of the ubiquitous β-globin gene. HPV DNA was detected through PCR amplification of a 450 bp segment of ORF L1 by using the degenerate primer pair ELSI-f and ELSI-r in previously reported amplification reaction conditions [HPV DNA was extracted from urine samples by means of the NucliSENSnditions . Each PCThe amplification products were visualized by means of electrophoresis analysis of 2% agarose gel containing ethidium bromide (0.5 mg/ml). Amplified product bands were compared with molecular weight standards . PCR products of approximately 450 bp were subjected to restriction fragment length polymorphism (RFLP) genotype analysis.Amplified products were digested with 1U of either RsaI, DdeI or HaeIII, diluted in their respective buffers and incubated at 37°C for 1 hour. Digestion products underwent electrophoresis in 3% agarose gels, and restriction patterns were compared with molecular weight standards . The pattern of fragments obtained from the three digestive processes enabled to genotype to be identified . The RFLSample size was calculated using the statistical software Stata 11.0 . HPV prevalence was expressed as a crude proportion with corresponding 95% confidence intervals (95% CI) calculated using the Mid-p exact test assuming a normal distribution. A P-value <0.05 was considered statistically significant (2-tailed test). Statistical analysis was performed using OpenEpi, version 3.01 .A total of 870 adolescents aged 11-18 years  were enrolled: 501 males  and 369 females . The final proportions of recruited individuals in the five cities did not significantly differ from those expected by sample calculation (Chi squared P = 0.35): 34.0% of urine samples were collected from adolescents in Turin, 30.7% in Milan, 21.4% in Genoa, and 8.6% and 5.3% from boys and girls in Florence and Sassari, respectively. The median age and the male/female distribution of the participants was comparable between the cities involved.The β-globin gene was amplified from all 870 urine samples collected, confirming the suitability of the extraction protocol. Overall, 13  subjects were HPV DNA-positive .The geographic distribution of HPV-positive adolescents was as follows: 0.7%  in Turin, 1.5%  in Milan, 2.7%  in Genoa, and 4.3%  in Sassari. HPV DNA was not detected in samples collected from participants in Florence . No significant differences were observed.Overall, 17 different infections were identified. In 69.2%  of HPV DNA positive adolescents, the infection was due to a single genotype and 88.9%  of the genotypes identified belonged to the HR-clade. The remaining 4 subjects  had double infections, two of which were sustained by LR HPV types and two by at least one genotype belonging to the HR-clade.HPV prevalence rates were significantly different between genders (P<0.05): 0.4%  and 3.0%  in males and females, respectively . Among the 11 HPV DNA-positive girls , only one (14 years old) belonged to the 11-14-year age-group . In the female 15-18 age-group, the prevalence of HPV infection was 4.2% , significantly higher than that detected in 11-14 age-group (P<0.05) .In the two HPV DNA-positive boys (14 and 18 years of age) the infection was attributed to HPV-70. Approximately sixty-four percent  of the 11 HPV-positive girls had a single infection and 4  were infected with two genotypes. A total of 8 different genotypes were detected among 15 infections identified in females: HPV-16 , HPV-70 , HPV-6 and HPV-11  and HPV-52, HPV-54, HPV-66 and HPV-87  . The only HPV positive girl among the 11-14-year age-group was infected with HPV-70. Sixty per cent of the positive girls in the 15-18 age-group were infected with the HPV-16 vaccine type or co-infected with the LR- types HPV-6 and -11 included in one of the two available vaccines.To our knowledge, no research has been planned with the aim to evaluate HPV prevalence rates and genotypes distribution in adolescents (11-18 year-old) in Italy prior to our study. An extensive survey carried out in Italy on the prevalence of high-risk HPV infection in women aged 15-73 years found a high prevalence (29.5%) in females between the ages of 15-19 years, but the number of participants was very low and they were all sexually active .Worldwide, several studies have analysed adolescents within a broader age range and by means of different methods and types of sampling, making the results difficult to compare . Only twThe present study identified a very low prevalence rate (1.5%) of HPV infection among Italian adolescents, and found a significantly lower prevalence in boys (0.4%) than in girls (3.0%) (P<0.05). Our findings are consistent with those reported in India by Hussain et al.  that shoIn the present study, no significant differences were observed in HPV infection prevalence rates among adolescents residing in the five cities involved. A slightly, but not significantly, higher percentage of infection was noted among adolescents from Sassari. This may have been due to the low number of samples analysed. However, the Sassari area is known to have a high prevalence of HPV infections, since high rates have been reported in women aged 15-54 years (35.9%) and 15-24 years (53.9%) undergoing Pap-testing at public and private outpatient clinics .In the 11-14-year age-group, only one female, aged 14 years, proved HPV-positive ; this infection was not associated with the vaccine types. No infections were observed in the primary target vaccination group .The majority (57%) of infections found in the 15-18-year-old females were caused by genotypes belonging to the HR-clade, with HPV-16 in 50% of cases. Overall, more than 50% and 30% of the potentially persistent HPV infections detected in this group could have been prevented by the quadrivalent and the bivalent vaccines, respectively. The only two HPV-positive males were infected with HPV-70. This genotype, that is classified by the IARC as "possibly carcinogenic to humans" (group 2B), is not infrequently detected in adolescents. Indeed, it has been shown to be prevalent among males aged 15-18 years in Scotland . FurtherThe method used in the present study appears to be adequately able to identify HPV infections in urine samples, since the pre-treatment required  allowed Several studies have validated urine sampling as an alternative to cervical sampling-based methods for detecting HPV infections in women ,28,32,33In males, the analysis of urine samples for the detection of HPV remains controversial, since there is no ‘gold standard’ sampling method, as there is in females , and few studies have compared the sensitivity of HPV detection in urine with that of detection in other urogenital samples . Furthermore, no studies have compared this detection method with other techniques in male adolescents. Therefore, a consensus regarding which sampling method in males is most effective is still lacking ,35.Although the data presented in this report support the adequacy of urine sampling for the diagnosis of HPV infections in males, the prevalence of HPV infections reported may be underestimated owing to low viral DNA concentrations in urine or to presence of HPV infections in anatomical sites not covered by urine transit ,35.A further limitation of the study was the lack of information on the socio-demographic characteristics, sexual debut and sexual habits of the subjects studied. However, this was not the primary objective of the study, which was designed to evaluate the overall and type-specific HPV prevalence in an unvaccinated adolescent population in Italy.This is one of few studies, and the first conducted in Italy, designed to establish HPV prevalence and genotypes distribution in an adolescent population. The data reported suggest a very low HPV prevalence rate in the age-group studied. The fact that no infections were observed in the primary target vaccination group  indicates that the prevention strategy currently in force in Italy is appropriate. Furthermore, the low prevalence of HPV infections observed in 15-18-year-old females supports the multi-cohort strategy of vaccination adopted by several Italian Regions , also coMolecular testing of urine samples is the non-invasive and easier way to determine the HPV infection status in the youngest age-groups. This is essential to establishing the baseline HPV prevalence, which is a prerequisite to monitoring the impact of HPV vaccination at the population level. Future studies will have to be conducted on a larger population of adolescents representing different geographical areas and socio-cultural features in order to provide a more accurate estimate of HPV infection status in this age group.


It is widely accepted that specific human papillomavirus (HPV) types are the central etiologic agent of cervical carcinogenesis. However, a number of infected women do not develop invasive lesions, suggesting that other environmental and host factors may play decisive roles in the persistence of HPV infection and further malignant conversion of cervical epithelium. Although many previous reports have focused on HPV and environmental factors, the role of host susceptibility to cervical carcinogenesis is largely unknown. Here, we review the findings of genetic association studies in cervical carcinogenesis with special reference to polymorphisms of glutathione-S-transferase (GST) isoforms, p53 codon 72, murine double-minute 2 homolog (MDM2) gene promoter 309, and FAS gene promoter -670 together with HPV types including our recent research results. Cervical cancer is the second most common cancer in women worldwide, and is both a preventable and a curable disease especially if identified at an early stage. It is widely accepted that specific human papillomavirus (HPV) types are the central etiologic agent of cervical carcinogenesis. Other environmental and host factors also play decisive roles in the persistence of HPV infection and further malignant conversion of cervical epithelium . AlthougP < .05 was accepted as statistically significant.A large number of previous studies have suggested the possible correlation between genetic polymorphisms of cancer susceptibility genes and the higher risk of human malignant tumors , 3. GeneThe genes of glutathione-S-transferase (GST) family encode enzymes that appear to be critical in cellular protection against the cytotoxic effects. GSTs play an important role in conjugating glutathione to the products of endogenous lipid peroxidation and inactivating organic hydroperoxides via selenium-independent glutathione peroxidase activity, thus protecting the cell from the deleterious effects of oxidative stress . GST isoWe conducted GST genotype analysis together with HPV typing in a total of 198 cervical smear samples obtained from the patients who received cervical cancer screening. They consist of 54 normal, 102 low-grade squamous intraepithelial lesion (LSIL), and 42 high-grade SIL (HSIL). The protocol of this study was approved by our institutional review board, and all samples were obtained from Japanese women with informed consent. The exfoliated cervical cells were disrupted with lysis buffer, and genomic DNA was extracted with phenol-chloroform and precipitated with ethanol using standard techniques. The GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex PCR techniques according to the method reported by Chen et al.  with somPrevious epidemiological studies of GST and cervical neoplasia found no significant differences in the frequency of GSTM1 or GSTT1 in women with SIL or cancer compared to controls with normal cervical pathology , 9, 10. p53 is a tumor suppressor gene involved in multiple pathways including apoptosis, cellular transcriptional control, and cell cycle regulation , 15. A lWe conducted genotype analysis of p53 codon 72 together with HPV typing in a total of 198 cervical smear samples obtained from the patients who received cervical cancer screening. They consist of 54 normal, 102 LSIL, and 42 HSIL, as described above. PCR restriction fragment length polymorphism (RFLP) analysis of codon 72 of the p53 gene, which was modified from a technique described by Ara et al. , was conAs shown in Our present results revealed that the differences in the polymorphic frequency of p53 Arg, Arg/Pro, and Pro genotypes between SILs and controls were statistically not significant. Moreover, neither Arg nor Pro allele affected the increased risk of SILs with or without high-risk HPVs compared to controls. Some previous studies have reported no correlation between germline polymorphisms of the p53 codon 72 and increased risk of cervical cancer –21. The Murine double-minute 2 homolog (MDM2) is the key negative regulator of p53, and dysfunction of these genes may be associated with an increased rate of accumulation of genetic errors, thereby enhancing the progression of the disease. A single nucleotide polymorphism (SNP) in the MDM2 gene promoter, SNP309 (a T to G change at nucleotide 309 in the first intron), increases the affinity of the promoter for the transcription activator Sp1, resulting in higher level of MDM2 mRNA and MDM2 protein and a subsequent attenuation of the p53 pathway . SNP309 We conducted genotype analysis of MDM2-SNP309 together with HPV typing in a total of 195 cervical smear samples obtained from patients with consent who received cervical cancer screening. They consist of 52 normal, 102 LSIL, and 41 HSIL. Eight human cervical squamous carcinoma cell lines  were also used for genotype analysis of MDM2-SNP309 together with HPV typing. All cell lines were originating from Japanese women, as previously described , were peRecently, Meissner Rde et al.  tested tOur present results using exfoliated cervical cell samples demonstrated that there was an increased OR for TG + GG genotype in HSIL cases compared to controls among the patients with high-risk HPV. We observed that HPV types 16 and 18, the most prevalent and aggressive types worldwide, are predominant in cases with TG + GG genotype and G allele. Moreover, 7 of 8 human cervical squamous carcinoma cell lines that possess high-risk HPV except YUMOTO also showed TG or GG genotype. These observations suggest that MDM2-SNP309 and high-risk HPV infection may be cooperatively associated with cervical carcinogenesis. It would be of interest to further evaluate whether MDM2-SNP309 has the potential to be used in conjunction with HPV-DNA testing and cervical cytology for the management of SIL patients.Apoptosis is a physiological process that regulates normal homeostasis, and alterations of apoptosis-related genes are likely to contribute to the pathogenesis of autoimmune diseases  and maliWe conducted genotype analysis of Fas gene promoter -670 together with HPV typing in a total of 279 cervical smear samples obtained from the patients with consent who received cervical cancer screening. They consist of 63 normal, 167 LSIL, and 49 HSIL. Eight human cervical squamous carcinoma cell lines  were also used for genotype analysis of this SNP together with HPV typing, as described above. PCR-RFLP analysis of the Fas gene promoter -670, modified from a technique described by Lee et al. , was conPolymorphisms in the promoter region or 5′ flanking region of genes can lead to different levels of gene expression and have been also implicated in a number of diseases. Recently, Lai et al.  conducte Previous studies , 43 haveHere, we review the findings of genetic association studies in cervical carcinogenesis with special reference to polymorphisms of GST isoforms, p53 codon 72, MDM2-SNP309, and FAS gene promoter -670 together with HPV types including our recent research results. Our studies using exfoliated cervical cell samples or human cervical squamous carcinoma cell lines have demonstrated that the GSTT1 null genotype, the TG/GG genotype of MDM2-SNP309, and the GA/GG genotype or G allele of Fas promoter -670 are closely associated with cervical carcinogenesis together with high-risk HPV infection. It would be of interest to further evaluate whether these polymorphisms could be used as a disease marker for the natural history of cervical neoplasms in a setting of longitudinal cohort study and for the determination of appropriate screening interval in patients with or without high-risk HPV. HPV are the etiologic agents of cervical and other epithelial cancers. Persistence of infections by high-risk HPV types is the single greatest risk factor for malignant progression. Vaccination against HPV types 16 and 18 has commenced or will soon commence in a number of countries. Our studies have demonstrated that the frequency of G allele increased from LSIL to HSIL and that there was an increased OR for G allele in HSIL cases with high-risk HPV types including 52 and 58. It is known that geographically different oncogenic HPV types 52 and 58 are more prevalent than 16 and 18 in East Asia. HPV prevalence should be considered and treated individually regarding the strategy that best suits the HPV types in a given geographical area. It is likely that novel strategies in combination with vaccination against HPV types 16 and 18 are required in those countries where other types of HPV may be more prevalent. Moreover, further studies on the differential gene expression profiles between normal cervical keratinocytes and cervical cancer cell lines may provide the better understanding for the effect of these polymorphisms in the sequence of cervical carcinogenesis.