The Journal of the National Cancer Institute, vol. 103, no. 19, pp. 1444–1451, 2011). In countries with screening programs, adenocarcinoma is the most difficult to detect and treat with later-stage presentation and higher mortality Smith et al.  and Gunnell et al. . With additional cross-protection to HPV 31, 33, and 45 and protection against HPV 16 and 18 lasting at least 9.4 years, Cervarix may reduce adenocarcinomas in screened populations by more than 90%. This paper will detail the evidence about the efficacy, immunogenicity, and safety of Cervarix in the studied populations contrasting public health goals with individual health options.Nearly a half a million women throughout the world develop cervical cancer every year Parkin and Bray ; 80% of these women are in countries without a quality-assured cytology screening program. It is in this setting that Cervarix could reduce the incidence of cervical cancer to about 9.5/100,000 women. New evidence indicates that this might be able to be accomplished with a single dose of Cervarix, a great advantage to public health implementation programs Kreimer et al. (“Proof-of-principle evaluation of the efficacy of fewer than three doses of a bivalent HPV16/18 vaccine,  Cervical cancer is unequivocally initiated by a human papillomavirus (HPV) infection, but much less than 1% of HPV infections progress to cervical cancer  and MeisThe carcinogenic risk potential of the mucosal alpha genus HPV types is categorized as low or high-risk. Low- and high-risk types were so classified based on their propensity to be associated with cervical cancer. High risk types had odds ratios of association with cervical cancer in the hundreds, much higher than the odds ratios found linking tobacco use and lung cancer ; low-risHPV presents in a variety of clinical appearances classified in two categories: benign and oncogenic. One of the most common differentiating factors is the expression of the E2 protein. If E2, an early expressed HPV protein, is not disrupted, then HPV infection most often remains episomal within the epithelial basal cell. As an episomal infection, benign forms such as condyloma and cervical intraepithelial neoplasia grade 1 (CIN 1) are clinically seen, despite the type of HPV that is causing the infection . If the The validity of CIN 2 being a cancer precursor is uncertain due to high misclassification rates , and pooThe natural history of HPV infections in the cervical epithelium in vivo cannot be detected unless the infection is actively replicating. Most actively replicating HPV infections in postpubertal females must reach a significant viral threshold to produce a cytologically detectable lesion , 16. HenThe natural immunologic response of HPV infections is poorly understood, and its meaning is vigorously debated. Most of the data are from studies of young sexually active women. About half of the young women with oncogenic HPV infections seroconvert. Of those who do, seroconversion takes months to detect, usually at low antibody titers –29 whichHPV does not cause a viremia, and hence, as a natural infection, it has very little chance of initiating an anamnestic response of prior primed memory B cells. Immunologically, HPV does not cause cell lysis in either the episomal or integrated infection format. HPV does not incite an inflammatory response at the time of infection or reinfection; there are no proinflammatory cytokines released, and there is poor exposure and activation of the epithelial antigen-presenting cells and Langerhans cells . This laHPV infection is documented by viral DNA detection in the cervicovaginal epithelium or by serologic evidence. Both DNA detection and serologic assays have detected high-risk HPV infections in all ages of females with the incidence as high as 10% in those under 11 years of age –39. PeakMost research on DNA detection of HPV infections has focused on the highest prevalence group of females 16–25 years old attributing the peak rates to sexual activity. In sexually active women older than 25 years, the incidence of oncogenic HPV infections is 5 to 15% –42 with Data available to date indicate that there is a real incidence of new high-risk HPV infections that exceeds 10% in women older than 42 years. Over a 7-year study period these incident infections cause new CIN 3+ lesions in over half of the HPV-infected women . An equaWhile we have no longitudinal data on the progression of incident CIN 3 in women older than 40 years to cervical cancer, the public registries of the Nordic countries where screening is well organized show bimodal distributions of incident CIN 3 and cervical cancer that are separated by the expected 5–10-year time lag . LikewisThe role of past infections as a source of latent viral reactivation for the older woman has been seriously challenged by a recent analysis of a ten-year cohort of nearly 2500 women . In thisAmong women with a high-risk HPV infection that progresses to a CIN 3 lesion there is an increased risk of other anogenital cancers beyond the cervix. Women have a 3–12-fold increased chance of developing another anogenital cancer  within 10 years with increasing risk in the second decade posttreatment for their CIN 3 lesion –52. In aThe distribution of high-risk HPV types varies among increasingly severe cytologic changes and cancer. The prevalence of HPV 16 and 18, the HPV types of most oncogenic potential, is very low among the general population. In the USA population, HPV 16 is the sixth most prevalent infection at 1.5% with HPV 18 occurring in 0.8% of the population . GloballIn contrast, the prevalence of HPV 16 and 18 increases to 27% in women with low-grade cytologic abnormalities ) which by definition means infection with HPV. In women with high-grade squamous intraepithelial lesions (HSILs), the prevalence of 16 and 18 increases to about 50%. Finally, 67% of the squamous cell carcinomas and 75% of adenocarcinomas are caused by HPV 16 and 18  58–62].–62.58–62Prophylactic HPV vaccination will have little cancer-reducing effect in the general screened population, but may prevent cervical cancer among those with no screening opportunities. Prophylactic vaccination may help decrease the numbers of women who develop abnormal cytology screens among those who are screened. Modeling indicates that HPV vaccination will prevent potentially 17% of the abnormal Pap tests based on current knowledge of HPV type distribution  73] and and73] aCervical cancer is the most prevalent in absolute numbers and absolute costs of all the HPV-associated diseases. It comprises 88% of the worldwide burden of HPV-associated cancers and consumes 92% of the monies spent on HPV-associated diseases –82. CervFour times as many cervical cancers occur in countries without quality-assured screening programs as in those countries with ongoing organized screening programs . The incCervical cancer incidence in countries with well-established cytology screening programs ranges from 4 to 10/100,000 women , mostly Cervarix will be most effective in countries without quality-assured cervical cytology screening programs. In these countries, Cervarix may reduce the incidence of cervical cancer to 9.5/100,000 women if the duration of efficacy exceeds at least 15 years . ModelerWhere cervical cancer screening programs are routinely followed, the option to vaccinate with Cervarix may result in a reduction of abnormal cytologies: in the USA estimates are a reduction to 3400/100,000 women per year and CIN 3 lesions to 11/100,000 women per year. This outcome is clinically quite valuable as it reduces the psychological and reproductive morbidity associated with the screening process . The comcervical cancer in industrialized countries, though, not vaccination . T. T112]. Extrapolating from HPV distribution studies and the cross-protection efficacies, Cervarix is expected to protect against more than 90% of adenocarcinomas, those cancers that occur in younger women, are most difficult to detect by screening programs, present at later-stages, and thus have higher mortality , 66, 113Among women who received Cervarix when seronegative and DNA-negative for HPV 16/18 and who participated in repeated cytology screening, Cervarix prevented more than 20% of all abnormal cytology in the first 4 years after vaccination  57]. Ce. Ce57]. Cervarix has excellent efficacy against CIN 2+ caused by HPV 16/18 in women who are already seropositive for the same HPV type, but DNA-negative at the time of initial vaccination  67]. As. As67]. Vaccine efficacy is currently defined after three doses are administered. USA and European data show that less than 20% of 13-year-old girls have received all three doses of vaccine , 118 witEarly data show that Cervarix remains 100% efficacious with just one-dose  68]. Wh. Wh68]. Cervarix studies in women through 55 years have been ongoing for more than four years with immunogenicity results very similar to the 10–15-year-old and the 15–26-year-old population thus bridging to a similar efficacy for the older aged cohort  69–72, , 72, 120While Cervarix provides very high efficacy regardless of serostatus, Cervarix does not cause regression of HPV DNA 16/18 infections already present, nor does Cervarix accelerate progression to CIN 3+ from the current infection .As discussed, cost-effectiveness models indicate no cancer prevention occurs if duration of efficacy is too short. The minimum time analyzed is 10 years of efficacy, most use 15- or 20-year durations; if less than this, the cancers are merely postponed, not prevented , 79–81. T helper cells are important for several immune functions. These include B-cell differentiation, sustained memory, activation of recall reactions, and thus anamnestic responses . T helpeAt 18 months, Cervarix-induced a robust memory B cell response for HPV 16 in 87% of subjects and for HPV 18 in 75% of subjects, both significantly superior to Gardasil , 123.Systemic antibody responses induced by Cervarix, measured by the pseudovirion-based neutralisation assay (PBNA), were robust, despite protective serologic antibody titers having not been defined. Cervarix maintained a 100% seropositivity for both HPV types with substantial titers above natural infection for the full 9.4 years Figures  125]. W. W125]. Local antibody titers at the cervical squamocolumnar junction within the cervicovaginal mucous may play an important adjuvant role in the prevention of HPV 16/18 infection . The valCervarix induces equally high anti-HPV 16 and anti-HPV-18 titers in HPV-DNA-negative women regardless of their serostatus Figures  and 8 1, 130. ThIn 9–25-year-old females Cervarix induces the same peak antibody titers for HPV-16 and 18, measured at month 7 after first injection for three doses  as for two-doses (given at 0 and 6-months). The two dose regimen contained double the antigen concentration at the 6-month dose . With the opportunity to prevent other HPV-associated cancers in males, Cervarix has been trialed in males to document its induced immunologic responses. Among males 10–18 years old, Cervarix induces higher peak antibody titers than in females of similar ages. Specifically, after three doses in males 10–14 years of age anti-HPV 16 and anti-HPV-18 titers are higher than those induced in 10–14-year-old females  132]. T. T132]. The adolescent platform of vaccination was designed around the revised recommendations of HPV, pertussis, and meningococcal vaccination at 11-12 years of age . CoadminSafety has been discussed in detail in previous publications , 138. InCo-administered vaccines did not produce worse local injection side effects and did not increase the reporting of serious adverse events; there was detailed follow-up for 30 days after vaccination and for those subjects who became pregnant .Post-marketing surveillance continues to follow specific pregnancy related side effects. In addition the FDA sponsors the general Vaccine Adverse Events Reporting System (VAERS) to periodically reassess vaccine safety, making changes in recommendations only if the incidence of adverse events is more than twice the very rare incidence of 1/10,000 women . To dateCervarix was approved by the USA Food and Drug Administration (FDA) in October 2009. It is currently licensed in over 100 countries worldwide with over 60 countries approving Cervarix for women older than 25 years. The vaccine is approved for use in the USA in females aged 10 years through 25 years for the prevention of HPV 16/18 attributed cervical cancer, CIN 2+, adenocarcinoma in situ, and CIN 1+ disease . The regThe USA based Advisory Committee on Immunization Practices (ACIP) recommends routine vaccination of females aged 11 or 12 years with 3 doses; the vaccination series can be started as early as 9 years. Likewise, vaccination is recommended for females aged 13 through 26 years who have not been vaccinated previously or who have not completed a 3-dose series of HPV vaccination. If a female reaches the age of 26 years before the vaccination series is complete, remaining doses can be administered after the age of 26 years. The second dose should be administered one to two months after the first dose. The minimum interval between the first and second dose of vaccine is 4 weeks and between the second and third dose is 12 weeks. The minimum interval between the first and third dose is 24 weeks . The product insert recommends Cervarix to be administered intramuscularly in three separate shots, with the initial 0.5 mL dose being followed by two additional shots at one and six months . Cervarix has the potential to reduce the burden of cervical cancer in either one or three doses in those females who are seronegative or seropositive for HPV 16/18. In populations without organized cervical cancer screening, Cervarix can lower the incidence of cervical cancer from the current 50–80/100,000 women to 9.5/100,000 women. In Europe, 18 of the 27 member states of the European Union have cervical cancer incidences above 9.5/100,000  147]. . 147]. In countries with cervical cancer screening programs, Cervarix has the potential to reduce the population incidence of cervical cancer in those subpopulations who do not participate regularly in the screening programs. In the USA this would be the Hispanic and Black populations with current cervical cancer incidences of 12.8 and 11.1 per 100,000, respectively . The largest population benefit of Cervarix in the screened population, though, is the reduction of abnormal Pap test results, colposcopies, and excisional treatments. reduction in CIN 3+ regardless of HPV causation is shown to be 93% for those vaccinated between 16–26 years . In addiCervarix induces anti-HPV 16 and anti-HPV-18 titers in males that exceed titers in similarly aged females, and Cervarix has a proven duration of efficacy for at least 9.4 years.Cervical cancer screening programs have been very successful. Evidence now confirms that decreases in population participation in screening programs do cause an increased overall population incidence of cervical cancer , 103. ThShould vaccination be an option that women choose for their cervical cancer protection, Cervarix is an excellent choice for both screened and unscreened populations due to its long-lasting protection, its broad protection for at least five oncogenic HPV types, the potential to use only one-dose for the same level of protection, and its safety. The most efficient use of Cervarix is in countries without any cervical cancer screening programs where the incidence of cervical cancer can be reduced to 9.5/100,000 women by vaccinating young females. This is the public health target of the World Health Organization (WHO) and most public health authorities. In addition, Cervarix offers the best chance of preventing adenocarcinomas of the cervix. Other options, not public health targets at this time, but supported by trial data, include vaccinating women at any age who are not currently infected with HPV 16/18; this is about 98% of women , 55. A mAnother important individual health benefit, not supported by public health authorities at this time, is the vaccination of older women who are in screening programs , 151. ThCervarix is particularly pertinent for the prevention of CIN 2+ in women older than 45 years because 70% of these CIN 2+ are caused by high-risk HPV types other than HPV 16 . The addCervarix prevents anal HPV infection in women for HPV 16, 18, 31, 33, and 45 , more poFinally, while male uses of Cervarix are not recognized by regulatory authorities at this time, should HPV vaccination for male HPV-associated cancers become a health priority, Cervarix has excellent immunogenicity and safety in males.Within five years Cervarix will have completed its extension trial designed to determine long-term immunogenicity, efficacy, and safety past 15 years. Finland has enrolled over 20,000 16-17-year-old women in the PATRICIA trial who are continuing to be followed. Their data will be linked with the Finnish cancer registry and provide CIN 3/cancer incidence rates between the vaccinated and unvaccinated women at 10 years after initial PATRICIA enrollment providing evidence of efficacy past the 15-year threshold cost-effectiveness models have identified .While vaginal and vulvar lesions are a very small proportion of HPV-associated anogenital diseases, and the PATRICIA studies did not include vulvar or vaginal endpoints, the ad hoc collection of VAIN 2+ and VIN 2+ data show vaccine efficacy of 54% in the TVC-N population .Because of the novel adjuvant formulation of Cervarix, this HPV vaccine may have a unique role in protecting those with autoimmune disorders. Trials enrolling women with HIV, juvenile idiopathic arthritis, systemic lupus erythematosus, and juvenile dermatomyositis (NCT00815282) are ongoing and expected to have reportable results within the next five years. The development of second-generation vaccines continues as solid global evidence that the high-risk types of most importance include the five types already covered by Cervarix and three additional types  . Of moreCervical cancer prevention, the main purpose of HPV vaccination, is best served by continued participation in Pap screening programs with optional vaccination. Cervarix's pertinent strengths areits ability to prevent CIN 2+caused by five oncogenic HPV types for at least 9.4 years ,its high and sustained T- and B-cell immunologic responses, including serum antibody titers, for HPV-16 and 18 that last at least 8.4 years,its ability to prevent CIN 2+ caused by HPV 16/18 in women DNA-negative for HPV 16/18 but seropositive  equally well as in seronegative females for at least 8.4 years,its ability to prevent CIN 2+ in one-dose equally well as in three doses for at least 4 years,its ability to immunobridge CIN 2+ efficacy in women through 55 years its ability to prevent about one in five abnormal Pap tests and to prevent nearly 70% of the excisional treatments in women with access to screening.


We studied anal specimens to determine the distribution of human papillomavirus (HPV) genotypes and co-infection occurrence. This information will contribute to the knowledge of HPV genotype distributions and provide an estimate of the prevalence of different oncogenic HPV genotypes found in patients in Madrid (Spain).We studied a total of 82 anal biopsies from the Hospital General Universitario Gregorio Marañón of Madrid. These included 4 specimens with benign lesions, 52 specimens with low-grade anal squamous intraepithelial lesion, 24 specimens with high-grade anal squamous intraepithelial lesions and 2 specimens with invasive anal carcinoma. HPV genotyping was performed with PCR amplification and reverse dot blot hybridization.We detected 33 different HPV genotypes, including 16 HPVs associated with a high risk of carcinogenesis, 3 HPVs associated with a highly likely risk of carcinogenesis and 14 HPVs associated with a low-risk of carcinogenesis. In two specimens, an uncharacterized HPV genotype was detected. The most frequent HPV genotypes found were HPV-16 , HPV-52  and HPV-43/44 . HPV-18 was only detected in 0.9% (95% CI: 0.1%-3.2%) of the total viruses detected in all lesions. HPV co-infections were found in 83.9% of all types of lesions. The majority of cases (90.2%) were concomitantly infected with the human immunodeficiency virus (HIV).The prevalence of high-risk carcinogenic genotypes in anal pathological samples was remarkable. Therefore, further studies that include a greater number of samples, particularly invasive carcinoma cases are needed to evaluate the potential influence of these HPV genotypes in the appearance of anal carcinomas. Also, the influence of other accompanying infections should be evaluated clarify the appearance of this type of carcinoma.http://www.diagnosticpathology.diagnomx.eu/vs/2075238024106058.The virtual slide(s) for this article can be found here:  Anal squamous cell carcinoma is an uncommon tumour. However, it is the most common type of cancer malignancy that arises from the anal margin. Less frequent anal malignancies include neoplasms from the anal canal, which have been poorly differentiated and are typically non-keratinizing, and malignancies that arise around the dentate line which have been defined as transitional carcinomas ,2.In Spain, anal carcinoma is estimated to represent 1.8% of all tumours of the digestive tract. However, in the last 25 years, its incidence has increased in Spain, as in other Western countries. This increased frequency has been attributed to different factors, including increases in human papillomavirus (HPV) infections and other sexually-transmitted infections, the pandemic of human immunodeficiency virus (HIV) and the increasingly common practice of anal intercourse, in both homosexual and heterosexual settings -6.condyloma acuminatum to severe changes like epithelial dysplasia and invasive carcinoma. The presentation varies with the different specific HPV genotypes that can infect the anogenital or oral mucous membrane. Of 100 recognized HPV subtypes, over 80 have a completely sequenced genome to date; 35 are specific for the cervical and anal epithelium. Each subtype carries a variable capacity for causing dysplastic changes. A recent systematic review showed that 72% of anal invasive carcinomas were associated with the oncogenic subtypes, HPV-16 and HPV-18. Currently, the relationships between the different HPV subtypes and their corresponding effects on the anogenital epithelium are well known [HPV is the most common sexually-transmitted infection world-wide. Different lesions range from simple warts or Patients in immunosuppressed conditions are at increased risk for developing multiple HPV-related benign and malignant anogenital tumours. Patients that are HIV-positive fail to clear HPV-related diseases because they have insufficient cell-mediated immunity to control latent HPV infections. As HIV/AIDS is increasingly managed as a chronic disease, greater attention should be focused on cancer screening and prevention, particularly for non-AIDS-defining cancers in the anus and liver, and for Hodgkin lymphoma, which appears to have increased incidence . A recenPremalignant changes have been classified as low-grade and high-grade anal squamous intraepithelial lesions , based on criteria for evaluating similar lesions in the cervix; however, the classifications, follow the lower anogenital squamous terminology (LAST). This two-tiered scale defines low-grade lesions as those generally related to self-limiting HPV infections and the high-grade lesions as those with potential for progression to invasive carcinoma (IC) ,13. ASILTo date, no study has investigated the relationship between HPV and ASILs in Spain; furthermore the prevalence of HPV is largely unknown. In other European countries, few studies have been published that estimated the HPV genotype distribution in anal lesions, in populations that were either HIV-negative and HIV-positive; thus, further studies in this field are needed -20.The present study aimed to determine the HPV genotype distribution and co-infection rate in anal specimens from individuals in Madrid (Spain). The findings presented in this paper will contribute to the knowledge of HPV genotype distribution and provide an estimate of the prevalence of oncogenic HPV types in anal lesions among Spanish patients.This study had a cross-sectional and retrospective design. We obtained samples from 226 individuals with anal lesions diagnosed in the Hospital General Universitario “Gregorio Marañón” of Madrid, between February 2012 and March 2013. The hospital provided healthcare to a population of about 750,000 individuals .All patients were enrolled in a screening program introduced in the hospital to detect early sings of anal carcinoma. These patients were routinely subjected to anal examinations, followed by cytology. Suspected cases underwent a proctoscopy and were examined under anaesthesia. Suspicious lesions, identified by acetic acid staining, were mapped and biopsied for histological confirmation, according to the recommendations of the European Society for Medical Oncology Clinical Practical Guidelines .The present study includes samples of anal lesions (specimens of fixed tissue sections from biopsies) collected from different departments of the Hospital General Universitario “Gregorio Marañón” of Madrid and submitted to the anatomical pathology laboratory for study.The study included 82 samples from patients with an anatomic-pathological diagnosis of anal HPV-related lesions. Of these 82 patients, 74 with HIV infections were selected to investigate the presence of HPV in corresponding anal lesions. In 6 of these cases, the presence of HPV was negative, and in 6 other cases, the anal specimens were inadequate for HPV testing; in 62 HIV-positive cases, HPV was detected in anal lesions (HIV&HPV). The latter 62 cases were studied further. The average age of these patients was 45.7 years, and the majority  was male.condyloma acuminatum), 40 LASILs , 20 HASILs  and 1 IC.The HIV&HPV samples included 1 benign lesion .DNA for each case was extracted from an anal, liquid-based, cytology sample. The isolated DNA was used as a template for PCR amplification to detect the presence of HPV DNA. We used primers specific for the GP5-6 L1 consensus region. PCR amplification was targeted to a fragment of 450 bp in the L1 consensus region. We performed reverse dot blot hybridization with probes specific for each genotype (based on “flow-through” technology). The amplification products were separated with agarose gel electrophoresis and stained with ethidium bromide, which allowed detection of the products with a UV transilluminator. A sample was considered positive when a band of 150 bp was detected .This diagnostic kit was intended for simultaneous screening and genotyping of 33 HPV types. These HPV types included 15 associated with a high-risk of carcinogenesis , 3 associated with a highly-probable-risk of carcinogenesis  and 15 associated with a low-risk of carcinogenesis .This kit was not able to detect some genotypes separately; thus, some were detected as couples .Two methods were used to estimate the frequency of HPV positivity. First, the percentage of lesions infected by one or several genotypes; and second, the percentage of a given HPV in the total number of viruses detected in all lesions and each group of lesions.Statistical analysis was performed with Stata version 11.1/SE . Relative frequencies of HPV genotypes were estimated as percentages with 95% confidence intervals (95% CI) assessed with Clopper-Pearson method and based on the exact binomial distribution of the tail areas.Treponema pallidum (40.3%) and Hepatitis B and/or C virus (30.6%).At least 83.8% of the entire group of HIV-positive specimens studied was also HPV-positive . Concomitant infections were found in a large proportion of HIV&HPV samples. The most frequent concomitant infections were from As shown in Table Based on all the data, we detected 33 different HPV genotypes, including 16 HR-HPVs, 3 PHR-HPVs and 14 LR-HPVs. As shown in Table In the majority of lesions, at least one HR-HPV was detected. Among all the lesions, HPV-16 was present in 37.1% (95% CI: 25.2%-50.3%), HPV-52 in 30.6% (95% CI: 19.6%-43.7%), HPV-51 in 22.6% (95% CI: 12.9%-35%), HPV-31 in 21% (95% CI: 11.7%-33.2%), and HPV-58 in 21.0% (95% CI: 11.7%-33.2%). Two cases (0.9%) were classified as an uncharacterized HPV type , but we suspect that this classification was most likely related to the missed detection of known HPV types; i.e., it was probably not associated with an undiscovered HPV type ; this was followed by, in order of decreasing frequency, HPV-16 , HPV-43/44 , HPV-51  and HPV-11 . HPV-18 was not detected in this group. Among the LASILs, HR-HPVs were detected more frequently than LR-HPVs (approximately 58.2% vs. 36.9%) and LR-HPV types were rarely identified as single infections (approximately 12.5%).However, among the HASIL samples, HPV-16 was the most common genotype , followed by HPV-58  and the group of HPV-31, HPV-52, HPV-6 and HPV-43/44 . HPV-18 was only detected in 2.5% (95% CI: 0.3%-8.8%). Also among the HASILs, HR-HPVs were detected more frequently than LR-HPVs (approximately 67.1% vs. 29.1%) a greater proportion of HR-HPVs were found in HASIL than in LASIL samples.The only case of IC was found in a 67 year old male with a long history of Candidiasis and Cystoporidium infections. The sample from this individual also exhibited a double infection with HPV-16 and HPV-81.The distribution of HPV genotypes and the analysis between pathology groups vs. HPV risk types is shown in Table Figure The percentages of multiple infections were very high: 80% in LASIL cases, 95% in HASIL cases, and 100% in IC cases. Multiple infections were not detected in benign lesions.As shown in Table Among the LASIL cases with multiple HPV infections, HPV-16 was the most common type found  followed by HPV-43/44, HPV-51 and HPV-52 . HPV-11 occurred in 6.8% (CI: 3.1%-12.5%). Among the HASIL cases with multiple infections, the HPV genotype distribution was similar to that found in all the entire group of HASIL cases.HPV co-infection distributions are shown in Table This study included individuals with visible, HPV-related lesions in the anogenital area, assessed by careful inspection of the external genitalia and perianal area. Also, an intra-anal examination was performed with a digital rectal exam and high-resolution, flexible anoscopy, prior to an acetic acid stain of the mucosa. The population included was nearly universally HIV-infected; they were identified in an anal screening program conducted to prevent anal cancer.In all the cases included in this study, a HPV genotyping assay was routinely performed on cytologic specimens. Some authors hold that the analysis of anal cytology for HPV may lead to an overestimation of genotype-specific associations with dysplasia ; howeverTo our knowledge, this is the first prospective data publically available that estimated the type-specific prevalence of HPV in preinvasive anal lesions among patients that were HIV-seropositive in Spain. Among the results from this study, the main finding was the unexpectedly high presence of high-risk HPV genotypes in patients that were HIV-seropositive with anus pathology. The most frequent genotype found in all lesions was HPV-16 followed (in descending order) by HPV-52, HPV-43/44, HPV-6, HPV-51, HPV-31 and HPV-58. Four of these seven viruses belong to the species, group 9, of the genus, Alpha-papillomavirus, and five are known to be high-risk, oncogenic viruses ,24. ThisThe above suspicion was also noted by Machalek et al. in a recent meta-analysis. They calculated a theoretical rate of progression from HASIL to anal cancer of about one in 600 per year in men that had sex with another man (MSM) and were HIV-positive, in contrast, the rate was only one in 4000 per year in MSM that were HIV-negative . SimilarIn this context, it is currently well known that individuals that are HIV-seropositive have an increased rate of anal epithelial pathology related to HPV infections compared to individuals that are HIV-seronegative -27. In tOf note, it may be important that in our series of patients mostly belonged to a population born between 1958 and 1992. This age group may be related to the probability of survival with AIDS and the possible loss of HIV-infected patients in previous years; thus, relatively younger men were more highly represented compared to those born between 1938 and 1957. This fact can be explained by the undoubted benefit of HAART, which was introduced in 1996.The results of this paper were consistent with those reported recently, which found the most frequent genotype was the HPV-16, and that HIV-seropositive patients exhibited the highest proportion of multiple HPV co-infections ,19,22,32The frequency of cases with the HPV-18 genotype in our study was not as high as that published in other international series ,33. We hAs stated above, the patients with HIV-infections were more likely to have multiple infections and more frequently had oncogenic HPV types than those without HIV-infections. In our series, 83.9% of lesions had multiple infections, which was a higher rate than those reported by Palesfsky and Hesson ,27. HessThe most frequent co-infection pattern was multiple infections with four different HPV types (25%). Although we sought to find a dominant pattern of HPV types in concurrent infection, we only observed random patterns.Finally, to date, there is no established consensus regarding the best treatment for ASILs. The treatments reported for HASILs, both medical and surgical, have not been standardized. Studies that investigated the preventive and therapeutic effects of existing HPV vaccines have reported that vaccines can reduce persistent anal HPV infections and HASILs among immunized MSM. However, the use of vaccines in preventing these lesions in immunocompromised individuals, as expected, has been ineffective or yielded ambiguous results . TherefoIn summary, this study showed that HPV-16 was the prevalent HPV subtype in a Spanish population. HPV-16 was associated with all grades of anal dysplasia and anal squamous carcinomas. Also, the prevalence of high-risk carcinogenic HPV genotypes in patients with anal pathology and at risk for HIV infection was remarkable. Therefore, further studies that include a larger number of samples, particularly samples with IC are needed to evaluate the potential influence of these HPV genotypes in the appearance of anal carcinomas.The present study had some limitations. First, the study had a retrospective design, which relied on medical records. This study design limited the available data to the information contained in the medical charts. The individual’s sexual habits and race/ethnicity were not noted in the chart. This cohort included a population of mostly Caucasian, urban, HIV-seropositive men that were treated in a public hospital; therefore, our findings may not be generalize to other populations.Second, the samples were collected from only one hospital; thus, it may be difficult to generalize the results to the entire population of Spain. However, this hospital was a reference centre that served patients from many districts and municipalities of the Region of Madrid.Third, although more than 40 anogenital HPV types exist, only 33 were investigated in this study. However, these 33 included all the HPV genotypes that are typically implicated in the initiation of anal cancer.Fourth, the modest sample size limited the statistical power for our analyses. However, it is difficult to acquire this kind of samples in Spain; therefore, these results provide valuable information. In the future, it would be of interest to obtain more samples for further studies.ASIL: Anal squamous intraepithelial lesion; HPV: Human papillomavirus; HR-HPV: HPV type associated with a high risk of carcinogenesis; PHR-HPV: HPV type associated with a high probability of risk of carcinogenesis; LR-HPV: HPV type associated with a low risk of carcinogenesis; BL: Benign lesion; LASIL: Low grade anal squamous intraepithelial lesion; HASIL: High grade anal squamous intraepithelial lesion; IC: Invasive carcinoma.The authors declare that they have non-financial competing interests.BGE participated in the design of the study, global data acquisition, analysis and preparation of the first draft. EMR participated in writing the first draft, critically revised the first and final drafts, participated in the data acquisition and participated in planning the study design. EAF conceived the design of the study and participated in the surgical pathological data acquisition and analysis. All authors read and approved the final manuscript.


Findings are inconsistent about whether tobacco, alcohol, and human papillomavirus (HPV) are two independent HNC risk factor groups that distinguish an infection-associated cancer from a tobacco/alcohol-associated HNC. We found that cancer in the oral cavity risk was greater in HPV-E6/E7 seropositive/heavy tobacco users (adjusted OR = 3.5) than in HPV-seronegative/heavy tobacco users (adjusted OR = 1.4); and HPV-seropositive/heavy alcohol users (adjusted OR = 9.8) had greater risk than HPV-seronegative/heavy alcohol users (adjusted OR = 3.1). In contrast, the risk of oropharyngeal cancer was greater in the HPV-seronegative/heavy tobacco (adjusted OR = 11.0) than in HPV-seropositive/heavy tobacco (adjusted OR = 4.7) users and greater in HPV-seronegative/heavy alcohol users (adjusted OR = 24.3) compared to HPV-seropositive/heavy alcohol users (adjusted OR = 8.5). Disease-specific and recurrence-free adjusted survival were significantly worse in oropharyngeal HPV-seronegative cases with no survival differences by HPV status seen in oral cavity cases. The association between tobacco/alcohol, HPV, and tumor site is complex. There appear to be distinct tumor site differences in the combined exposure risks, suggesting that different molecular pathways are involved. Human papillomavirus (HPV) is now an established risk factor for head and neck cancers (HNC) , 2. WhetN = 170) and oropharyngeal (N = 74) sites were included. There were 244 eligible cases enrolled after exclusions. Gender and 5-year age group frequency matched controls (n = 428) were recruited from clinics in Family Medicine and in Internal Medicine at these hospitals who were seeking routine medical care.Patient characteristics and exclusions have been described previously . Cases wPatients signed an institutionally approved informed consent form prior to completing a self-administered risk factor questionnaire that included information about sociodemographics, medical history, tobacco and alcohol use, sexual practices, and history of HPV-related diseases and oral lesions. Prior cancer history, head and neck tumor site, treatments, and staging were collected from medical records and pathology reports. All tobacco and alcoholic products were included and equivalents were used .A blood specimen was drawn from participants to detect HPV at the time of diagnosis prior to cancer treatment for cases or during the interview for controls. Detection of HPV-specific antibodies has been described previously –8. The Lβ-globin gene [Paraffin-embedded tumor tissue was available from 204 cases to evaluate for HPV. Sample preparation, PCR analyses, DNA hybridization, and HPV typing procedures for assessment of tumor tissue were based on a standard protocol . Each PCbin gene  and veribin gene  to detecbin gene . Positivbin gene . DNA seqbin gene . High-riP values for risk factors, and pathologic characteristics were adjusted for continuous age, gender, tobacco pack-years, and average drinks per week. Categorical variables included age, education, number of sexual partners, tobacco pack-years, and average number of alcoholic drinks per week. Cut points for moderate and heavy tobacco or alcohol users were based on the overall distributions or median values among HPV-negative cases and controls. ORs and CIs were calculated using multivariate logistic regression analyses. For variables with zero cells, ORs and CIs were generated using logit methods, adjusting for categorical age, tobacco, and alcohol except when limited by small cell sizes. Odds ratios (OR), 95% confidence intervals (CI) and N = 204 cases). Sensitivity was defined as the percent of HPV DNA positive who were E6/E7 positive; specificity as the percent of HPV DNA negative who were E6/E7 negative; and, concordance as the percent positive for both tests and negative for both tests. Magnitude of agreement was measure by the Kappa statistic. All P values were two sided, and values ≤0.05 were considered statistically significant. Statistical analyses were performed using SAS version 9.2. Individual and joint effects of tobacco and alcohol stratified by HPV status were examined by tumor site and never users of tobacco or alcohol (reference groups) were compared to moderate or heavy tobacco or alcohol users among cases and controls. Additive joints effects and 95% CIs were assessed using the synergy index  while muThe prevalence of HPV-16/18/33 E6 and/or E7 was greater in HNC cases than in controls: 26% versus 7%; it was higher in oropharyngeal cases (64%) compared to oral cavity cases (9%) or controls 7%; . HPV-16 We next examined the relationship between tobacco, alcohol, and HPV E6/E7 status by tumor site . CompareBased on the distinct findings by tumor site for tobacco and alcohol risk, data were next stratified by HPV E6/E7 status . CompareWhen both tobacco and alcohol were examined in stratified analyses associated with HPV status, only the heavy tobacco/alcohol (>30/>21) group was elevated for cancer of the oral cavity . ConsistK = 0.9, 0.7–0.99) for the oropharynx and 84%  in the oral cavity. The sensitivity of HPV E6/E7 antibodies to DNA HPV-positive tumors in the oral cavity was 26% and 100% in the oropharynx. There were 145 oral cavity and 59 oropharynx cases with both serology and tumor tissue assessed for HPV status. The prevalence of HPV-seropositive in the oral cavity was 10% and 64% in the oropharynx, and 13%/58% for oral cavity/oropharynx tumor HPV DNA-positive status . Type-spModerate smokers (compared to never or to heavy smokers) had elevated ORs for both HPV-seropositive and tumor HPV DNA-positive compared to HPV-negative status. Both moderate and heavy alcohol users had elevated ORs compared to never drinkers for either HPV assessment. For the joint effects of alcohol and tobacco, moderate smokers/drinkers (≤30/≤21) and moderate smokers/heavy drinkers (≤30/≥21) had the greatest odds of being HPV-positive; other risks were similar for serology and tumor tissue. Sample sizes were insufficient to evaluate risk factors for HPV serology and DNA by tumor site.N = 157). Among those who did not survive, 56% died from HNC, 28% from other causes and 16% had an unknown cause. A first recurrence occurred in 30% of all patients. Median follow-up time for DSS was 5.3 years (range: 1 month–10.4 years). Among those who had a DS death, 76% died within 2 years of diagnosis. The median follow-up time for RFS was 5.6 years (range: 2 months–10.4 years). For patients with a first recurrence, 76% occurred within 2 years of diagnosis. Time to recurrence did not significantly vary by HPV E6/E7 status for either site.  HPV status was not significantly related to DSS or RFS for oral cavity cancers . In contK = 0.7 both). An examination of sensitivity and specificity for HPV E6/E7 antibodies for HPV DNA status and survival outcomes showed that HPV E6/E7 seronegative status was a strong indicator of DSS and RFS in tumor DNA HPV-negative tumors of the oral cavity . However, there was weaker sensitivity between HPV-seropositive and HPV DNA-positive outcomes . In the oropharynx, serology was a much stronger overall indicator and demonstrated strong sensitivity and specificity for tumor tissue status and survival . The higher percentages in our study associated with the oral cavity may be due to our use of laser microdissection which is more critical for tumors at this subsite.  This also is one of the first studies to examine concordance and sensitivity between E6/E7 serology and tumor DNA for both tumor subsites. We found that they were higher in the oropharynx than in the oral cavity . Previously, Herrero et al.  reported It is unclear why site-specific HPV E6/E7 serology/DNA tumor concordance was consistently high for survival outcomes with the exception of the HPV E6/E7-seropositive/DNA-positive cancers in the oral cavity. Other studies of HPV status in association with HNC survival have focused on oropharyngeal cancer and none has reported on the sensitivity of HPV serology associated with tumor DNA and clinical outcomes . This as This investigation suggests that while risk of HNC by tumor site is both different between oral cavity and oropharynx, both sites are nonetheless associated with independent effects for each of the three major HNC risk factors. The combined effects alter HNC risk differently in a comparison of HPV-negative versus HPV-positive cases in the oral cavity and the oropharynx. Further, patients with oropharyngeal tumors are different from oral cavity cases not only for the three risk factors but also because they are younger in age, have higher prevalence of heavy tobacco and alcohol use, and are diagnosed at a later stage and with nodal involvement. It is not clear how these differences explain the distinctly different survival and recurrence outcomes found. Additional studies are need that examine HPV in the presence or absence of tobacco and alcohol to evaluate differences in chromosomal alterations and molecular pathways to clarify these risk and survival differences by tumor site.


The aim of this study is to describe the prevalence of HPV types in invasive cervical cancers in Italy from 1996 to 2008.A pooled analysis of the three largest case series typed to date was performed. HPV typing was performed on paraffin-embedded slices. Molecular analyses were performed in four laboratories. Multivariate analyses were performed to test the associations between calendar time, age, and geographical area and the proportion of types 16/18.Out of 574 cancers, 24 (4.2%) were HPV negative. HPV 16 and 18 were responsible for 74.4% (378/508) and 80.3% (49/61) of the squamous cancers and adenocarcinomas, respectively. Other frequent types were 31 (9.5%), 45 (6.4%), and 58 (3.3%) for squamous cancers and 45 (13.3%), 31, 35, and 58 (5.0%) for adenocarcinomas. The proportion of HPV 16 and/or 18 decreased with age , while it increased in cancers diagnosed in more recent years .The impact of HPV 16/18 vaccine on cervical cancer will be greater for early onset cancers. In vaccinated women, screening could be started at an older age without reducing protection. Cervical cancer is still the second leading cancer in women worldwide, with 585,278 new cases estimated in 2010. More than 80% of these cases occurred in low-medium income countries .It has been demonstrated that HPV infection is a necessary cause for cervical cancer . In partThe prevalence of HPV types infecting the lower genital tract in the healthy population shows geographical variations . InsteadNevertheless, some differences among continents have been consistently observed: In Africa and Eastern Asia, the proportion of cancers due to HPV types 16 and 18 is slightly lower than in Western Europe and North America ,8. FurthMany hypotheses have been formulated to explain these differences. The one most commonly accepted to explain the higher proportion of type 16 in early onset cancers is that this type has a greater potential to transform the cell compared to other high-risk HPV types and consequently there is a higher probability of progression from infection to cancer in a relatively short time. There is evidence from prospective cohort studies of shorter time to progression from HPV16 infection to high-grade Cervical Intraepithelia Neoplasia (CIN 3) -12. HoweData from cohort studies on time to progression for types 18 and 45 are less consistent ,11 becauThe prevalence of cytological screening uptake in the population strongly influences the epidemiology of cervical cancer. In highly screened populations, Pap test’s greater efficacy in detecting squamous precancerous lesions than glandular lesions leads to marked reduction in the incidence in the former ,14 and sThe aim of this work is to describe the prevalence of HPV types in Italian invasive cervical cancers from 1996 to 2008. The study is based on the pooling of three cases large series [In particular, we describe the associations between ca lendar time, age, and geographical area and the proportion of vaccine-targeted types (16 and 18) and early onset types (types 16/18/45).ad hoc morphological and molecular analyses on archival paraffin-embedded invasive cervical cancer (ICC) specimens.Histologically confirmed cervical cancer diagnoses from three large series were included in the study. All three studies performed Central and Southern Italy Study : 193 casRome Study : 134 casMilan Study : 268 casParaffin block selection and microtome sectioning, performed under strict conditions to avoid potential contamination, employed a sandwich technique to obtain 5 or 10-μm tissue section from each paraffin block. First and last microtome sections were stained for histopathological evaluation, while the internal sections were processed for DNA extraction. In one centre (Naples), morphologic check showed that the mean percentage of tumor cell staining was 64% (range: 5-100%), with a relevant proportion of sample slices containing a limited quantity of neoplastic tissue within a healthy tissue context.No central histologic review was planned for this study.Molecular analyses were performed in four centers: the Milan Study sent specimens to the International Agency for Research on Cancer  , the RomFor each sample, DNA concentration was evaluated by spectrophotometry and expressed in ng/μL.HPV genotyping was done using different PCR-based strategies in a well-defined algorithm.A PCR assay based on GP5+/GP6+ primers was used . All samples and controls were subjected to the reverse line blot (RLB) for detection of 12 high-risk HPV types , 1 probably carcinogenic HPV type (68), and 5 possibly carcinogenic HPV types  [For genotyping HPV DNA, 10 μl of the PCR amplimers of the HPV DNA-positive samples, as identified by DEIA, were analyzed by Line Probe Assay-LiPA25 version 1 , which can detect 25 different high-risk and low-risk HPV types  .Multiplex PCR was performed using the Qiagen Multiplex PCR Kit according to the manufacturer’s instructions, with a mixture containing 19 pairs of HPV-type specific primers  following the procedure described by Gheit et al. . To incrOur tables show the type distribution found in squamous and cervical cancers. We also present the proportion of types 16 and 18 or 16, 18, and 45 by age, calendar time at diagnosis, and geographic area. Proportions with relative 95% confidence intervals were computed. Multivariate models were constructed to identify determinants of the proportion of vaccine-covered types (16 and 18) and early onset types . All the analyses were performed using STATA 11.0.After histological evaluation, 595 ICC specimens from the original studies were included. Of these, 4 cases from the Rome Study and 17 from the Milan Study were excluded from the present study because the information about typing was missing. Thus, 574 cases were available for the analyses.The age range of the women included in this study was 23–85 years (mean age 52.4 years).Squamous cell carcinoma was the most represented histotype (508 cases: 88.5%), followed by adenocarcinoma (55 cases: 9.6%), then adenosquamous cell carcinoma (6 cases: 1.0%), and other minor histotypes (5 cases: 0.9%). For all the following analyses, adenosquamous cell carcinomas were grouped with adenocarcinomas.Adenocarcinoma was not associated with age at diagnosis (p = 0.57), but a strong time trend, with increasing proportion of adenocarcinomas in the recent years, was observed .Twenty-four (4.2%) cases were HPV DNA-negative. Among the HPV-positive cases, 74 (13.4%) were multiple infections, and 7 cases were non-typed.HPV 16 was the most frequent type detected, alone or in coinfection with other types: 69.3% and 58.3% in squamous and adenocarcinomas, respectively (Table HPV 18 was the second most frequent in adenocarcinomas (23.3%), and third in squamous . The cumulative proportion of HPV 16 and/or HPV 18 was slightly higher in adenocarcinomas than in squamous cell carcinomas, though not significantly . HPV 45 was also more frequent in adenocarcinomas than in squamous , while HPV 31 was more frequent in squamous cell carcinomas, though not significantly so (9.5% vs 5.0%). The fifth most frequent type was HPV 58 . HPV 33 ranked sixth (2.9% in squamous and null in adenocarcinomas), and HPV 35 ranked seventh .There was no significant difference in the geographical distribution of HPV 16 and 18, whereas we observed a significant , higher proportion of HPV 31 in northern Italy (13.1%) than in central and southern Italy  Table .There was a significant decreasing trend of HPV 16 and 18 in proportion with age, dropping from 94% to about 75% after the age of 35 . The trend was even stronger when we included HPV 45   Table , with noThe multivariate model shows that the effects of age and calendar time are independent and that they are not due to any bias in center recruitment and laboratory techniques adopted for typing. The same results are obtained if we limit the analyses to squamous cell cancers Table ; stratifThese three large Italian case series confirm some previously published results ,8. The pAgain, in line with de Sanjose’s study, HPV 16 and 18 were detected in about 77% of cervical cancers ,23, whicIt is interesting that HPV 33 did not rank in the top five, in contrast with the worldwide distribution. Other studies have already reported higher prevalence of HPV 31 than 33 in southern Europe .In our series, more than 1 out of 9 cases were adenocarcinomas, which is typical of high-moderate screening coverage , but theThe main objective of this paper was to describe changes in the proportion of vaccine-covered HPV types 16 and 18 and in early onset HPV types 16, 18, and 45, according to the geographical area, age, and the calendar time.Only few differences among geographic areas emerged and, even when statistically significant, they may have been due to chance, given the enormous number of statistical tests that can be performed comparing these distributions.The most remarkable results in our analyses were the trends in HPV 16, 18, and 45 with age and calendar time. The multivariate analysis clearly shows that the two trends are independent and not due to reciprocal confounding, to the increase in adenocarcinomas proportion in recent years, or to different PCR techniques adopted for typing in the three studies.The relation with age was expected since these are known to be early onset types ,9,20,29,On the contrary, the association with calendar time was not observed in other large series ,31 and wThe possibility of a more rapid progression associated with HPV 16 and its variants is supported by the Guanacaste Cohort Study, a 7-year nested case–control study conducted on 10,049 women . The stuAnother hypothesis is that HPV 16/18/45-related cancer precursors occur at a younger age, are far superior in number in comparison with other high-risk HPV-related cancer precursors, are not easily detected by cytology screening, and the transit time from precursor to cancer is not necessarily quicker. Recent population-based studies have shown that by using very sensitive screening tests such as HPV DNA testing, the number of precancerous lesions missed by cytology proves to be considerable . This laBoth these hypotheses consider the three HPV types related to early onset cancers as a unique group, but the evidence available about speed of progression from infection to CIN3 and cancer and about screening sensitivity are different for HPV 16 and HPV 18, and are almost absent for HPV 45.In conclusion, the proportion of HPV 16 and 18 types may be higher in highly screened populations because these types more frequently escape Pap test control. Consequently, the dramatic decrease in cancer incidence after screening introduction may be slightly less relevant for HPV 16- and 18-related cancers. Unfortunately, the effectiveness of Pap test as prevention cannot easily be increased by shortening screening intervals; a large body of evidence shows that reducing the interval from 3 years to 1 has a small effect on cancer incidence and a strong effect on the number of screening-induced treatments ,36. On tAs the present study has several limits, we can only confirm previously observed data or formulate hypotheses to be confirmed in other studies.The first limitation is that we did not perform a central pathology review, even if all the cases were specifically reviewed for the original study in each center ,19,20.As typing was performed on archival paraffin-embedded samples, some trends could be due to reduced analytical accuracy of typing methods on older samples, but this is not very likely to change the proportion of types.Our archives do not report some of the information that might have been crucial to testing our hypothesis. In particular, we do not know if the cases had had a previous negative Pap test. Furthermore, we do not know the stage at diagnosis. It could be extremely interesting to identify the micro-invasive cancers (FIGO IA1), since these cancers are virtually all asymptomatic and are thus mostly screen-detected .Finally, our series do not come from population-based registries and thus may not represent the universe of cancers in Italy. Furthermore, in some of the areas with higher screening coverage, i.e., northern Italy and Rome, the participating centers are not reference centre for screening programs, while in the areas with lower screening coverage, i.e. southern Italy, they are. It is likely that the proportion of screening-detected cancers in our archives does not represent the proportion in the cancer population in each geographic area. This may explain why we did not observe an effect of the geographic area on the proportion of HPV 16, 18, or 45, despite the broad differences in screening coverage among the areas.The impact of HPV 16/18 vaccine on cervical cancer will be proportionally more relevant for early onset cancers. Considering the very low overall risk in women under age 35 and that the proportion of vaccine-targeted cancers is over 90% in that age group, it may be necessary to postpone starting age for screening in vaccinated cohorts in order to reduce the risk of over-treatment and to contain costs.Furthermore, we suggest that screening is less effective in preventing the incidence of HPV 16 and 18 invasive cancers, given their shorter sojourn time in the precancerous stages. If this is true, then the screening interval in vaccinated women should take into account the longer sojourn time of CIN2 and CIN3 lesions due to non-16/18 HPV types.This is a pooled analysis of already published studies. All the three studies were approved by Ethic review board initally. This pooled analysis has been financed by the Ministry of Health and it was planned in the protocol of the most recent of the three studies .Abruzzo: Angeloni Claudio, Lattanzi Amedeo, Maccallini Vincenzo, Caraceni Donatella, Fortunato C.Cagliari: Macis Rosalba, Pilia Massimo, Caredda Valeria.Campania: Carillo Giuseppe, Di Iasi Angela, Santarsiere Aldo, Casto Loredana, Manno Maria, Santangelo Claudia, Pini Maria Teresa, Gallicchio Giuseppina, Scherillo Isabella, Barretta Elena, De Santis Vincenzo, Ercole Filomena.Catania: Scalisi Aurora, Spampinato Pina, Cantarella Maria Antonietta, Miano Maria Giuseppina.Lazio: Giorgi Rossi Paolo, Chini Francesco, Guasticchi Gabriella, Capparucci Paola, Marsili Laila Maria, Tufi Maria Concetta, Gomez Vito, Verrico Giovanna, Schiboni Maria Luisa, Pellegrini Antonella, Bove Emilia, D’Addetta Albina, Placidi Antonio.National Cancer Institute Fond Pascale, Naples: Buonaguro Franco Maria, Tornesello Maria Lina, Loquercio Giovanna, Losito Simona, Botti Gerardo, Vecchione Aldo.European Institute of Oncology, Milan: Sideri Mario, Boveri SaraTuscany: Carozzi Francesca M., Confortini Massimo, Bisanzi Simonetta, Sani Cristina, Venturini Giulia, Burroni Elena, Tinacci Galliano.National Cancer Institute Regina Elena, Rome: Mariani Luciano, Vocaturo Amina, Benevolo Maria, Marandino Fernando.Ministry of Health: Antonio Federici.F.M. Carozzi is an occasional advisor to Gen-Probe, Abbott, Sanofi, and GlaxoSmithKline.PGR, MS, FMC, MLT, FMB and LM conceived the study and planned the analyses. PGR, FMC, FMB, MS, and AV coordinated the sample collection. MLT, MLT, AV, and EB coordinated and performed the laboratory tests. FC planned the data collection and performed the statistical analyses. PGR drafted the paper.0020. All authors read and approved the final manuscript.


MicroRNA (miRNA)-related single nucleotide polymorphisms (SNPs) may compromise miRNA binding affinity and modify mRNA expression levels of the target genes, thus leading to cancer susceptibility. However, few studies have investigated roles of miRNA-related SNPs in the etiology of cervical carcinoma.miR-218-related SNPs involved in the LAMB3-miR-218 pathway and the risk of cervical carcinoma in Eastern Chinese women.In this case–control study of 1,584 cervical cancer cases and 1,394 cancer-free female controls, we investigated associations between two pri-miR-218 rs11134527 variant GG genotype was significantly associated with a decreased risk of cervical carcinoma compared with AA/AG genotypes . However, this association was not observed for the miR-218 binding site SNP (rs2566) on LAMB3. Using the multifactor dimensionality reduction analysis, we observed some evidence of interactions of these two SNPs with other risk factors, especially age at primiparity and menopausal status, in the risk of cervical carcinoma.We found that the pri-miR-218 rs11134527 SNP was significantly associated with the risk of cervical carcinoma in Eastern Chinese women. Larger, independent studies are warranted to validate our findings.The  Severs (UTRs) ,6.Cervical carcinoma is the third most commonly diagnosed cancer and the fourth leading cause of cancer deaths in women worldwide, accounting for 9% 529,800) of the new cancer cases and 8% 275,100) of the cancer deaths among women in 2008 [5,100 of 00 of themiR-218 were associated with infection of high-risk HPV involved in the pathogenesis of cervical cancer [miR-218 expression; in contrast, the RNA interference of E6 oncogene increased the miR-218 expression [Laminin 5 β3 (LAMB3) gene has been found to be one of transcriptional targets of miR-218[LAMB3 was expressed in many epithelial tissues and was involved in tumor microenvironment by increasing carcinoma cell migration [LAMB3 might upregulate the expression levels of the HPV16 E6 oncoprotein though miR-218[LAMB3-miR-218 pathway may be involved in the process of high-risk HPV infection and thus contribute to cervical carcinogenesis. However, its intrinsic mechanisms are still unclear. It is likely that miRNAs and related genetic variations may have effects on cancer development [pri-miR-218 rs11134527 and LAMB3 rs2566) are found to be associated with altered risk of cervical cancer in a Chinese Han population [LAMB3-miR-218 pathway contribute to cervical cancer risk, we performed a case–control study with a much larger sample size to validate the reported associations with cervical cancer risk in Eastern Chinese women.Recent studies have found that the expression levels of l cancer . Specifipression . Moreovef miR-218. LAMB3 wigration . Others h miR-218. Therefoelopment , two of The study population consisted of 1,584 cervical carcinoma patients, who had been operated between February 2008 and March 2011 in Fudan University Shanghai Cancer Center (FUSCC). The tumors were histopathologically confirmed independently as primary cervical carcinoma by two gynecologic pathologists as routine diagnosis at FUSCC. An additional 1,394 cancer-free female controls were enrolled from women who had come to the Outpatient Department of Breast Surgery at FUSCC for breast cancer screening and agreed to participate in this study. These female controls, with the selection criteria including no individual history of cancer, were genetically unrelated and frequency matched to the cases on age (± 5 years) and residential areas in Eastern China.During an in-person survey, all potential subjects were interviewed to identify their willingness to participate in this study. As a result, a response rate for the cases and controls was of approximate 95% and 95%, respectively. Because the vast majority of Chinese women are non-smokers and non-drinkers, our study populations were restricted to women who did not smoke cigarettes or drink alcohol. For the cases, detailed clinico-pathologic information was extracted from the patients′ electronic database of FUSCC, including tumor histology , FIGO sthttp://www.ncbi.nlm.nih.gov/projects/SNP) and the International HapMap Project database (http://hapmap.ncbi.nlm.nih.gov/) based on four criteria: 1) located at the pri-miR-218 gene region or 3′-UTR of the LAMB3 gene, 2) minor allele frequency (MAF) ≥ 5% in Chinese Han populations, 3) with low linkage disequilibrium by using an r2 threshold of < 0.8 for each other, and 4) predicted as potentially functional SNPs by SNP function prediction (FuncPred) software from National Institute of Environmental Health Sciences (http://snpinfo.niehs.nih.gov/snpfunc.htm). As a result, only two reported SNPs  were selected, because pri-miR-218 rs11134527 was predicted to be functional and LAMB3 rs2566 was the only one SNP residing in the miR-218 binding site. Genomic DNA was obtained from the whole blood, and the Taqman assay was performed for genotyping, as described previously [The SNPs were selected from the NCBI dbSNP database .To further explore high-order gene-environment interactions that were individually involved in cervical cancer risk, we performed the MDR analysis, as described previously ,18. This2-test. The associations of genotypes with the risk of cervical carcinoma were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate logistic regression models, with or without adjustment for age, age at primiparity, menopausal status and body mass index (BMI) [P value [P values were two-sided with a significance level of P < 0.05.The differences in selected variables between cervical carcinoma cases and female controls were evaluated by the Pearson's χex (BMI) . The assex (BMI) . A FPRP [P value . All staP=0.226). The cases were more likely to be premenopausal (72.5% vs. 60.5%), thinner  and younger at primiparity  than the controls. Because the differences in age at primiparity, menopausal status and BMI were significant between cases and controls , these variables were further adjusted for any residual confounding effect in later multivariate logistic regression analyses.Among all studied subjects, 19 cases and three controls failed to be genotyped after repeated assays. Thus, the final analysis included 1,565 cases and 1,391 controls. As showed in Additional file pri-miR-218 rs11134527 and LAMB3 rs2566 SNPs as well as their associations with the risk of cervical carcinoma are summarized in Table P=0.083 and 0.094 for rs11134527 and rs2566, respectively). In the recessive genetic model, the pri-miR-218 rs11134527 variant GG genotype was significantly associated with a decreased risk of cervical carcinoma compared with the AA and AA/AG genotypes . However, this association was not observed for the LAMB3 rs2566 SNP.The genotype frequencies of the pri-miR-218 rs11134527 GG genotype was more evident in women who were younger at primiparity  or premenopausal , which was also observed for subgroups of SCC, FIGO stage I, stage II, positive pelvic LN, positive LVSI, deep cervical stromal invasion (> 1/2) and negative expression of ER and PR . However, homogeneity tests suggested that there was no difference in risk estimates between the strata  was still noteworthy in subgroups of premenopausal, SCC, FIGO stage I and positive pelvic LN   and found that the MFE changed from −182.5 kcal/mol to −126.0 kcal/mol when the nucleotide at the pri-miR-218 rs11134527 locus changed from A to G  and the lowest prediction error 43.2%) among all five discrete factors. Intriguingly, the five-factor model had a maximum CVC (100%) and a minimum prediction error (38.6%), which showed a better prediction than one factor , our data did not have statistical evidence to support its association with cervical cancer risk. Our sample size had 100% statistical power to detect an OR of 1.57 that was reported by Zhou et al. [LAMB3 rs2566 SNP between Zhou’s study and ours may be caused by differences in selection of subjects, different catchments of the hospitals and residential regions as well as different sample sizes.In this relatively large hospital-based case–control study of 1,584 cervical cancer cases and 1,394 cancer-free female controls, we validated two previously reported significant miRNA-related SNPs involved in the ulations . We founRecent studies have demonstrated that miRNAs may function as tumor suppressors and/or oncogenes in human cancers ,22, becaIt is well known that genetic variants may modify cancer risk associated with environmental factors. Although there were no two-factor interactions between genotypes and environmental factors, using the MDR analysis , we furtMiR-218, is encoded by an intron of the SLIT2 tumor suppressor gene [miR-218 expression has been observed in cancers of the breast, ovary, lung and stomach [miR-218 (> 2 fold) in HPV-16 or 18 positive cervical cancer cell lines  as well as in cervical tumor tissues [miR-218 as a specific cellular target of high-risk HPV types [miR-218 is likely linked to the process of HPV-associated tumorgenesis. Based on the Microcosm Targets tool software (http://www.ebi.ac.uk/enright-srv/microcosm/), the mature miR-218 was found to have an effect on the mRNA expression regulation through more than 900 target genes, including LAMB3[RICTOR[ROBO1[BIRC5[LAMB3 that expressed in many epithelial tissues could induce carcinogenesis by increasing carcinoma cell migration and disturbing tumor microenvironment [LAMB3 increased expression levels of the HPV16 E6 oncoprotein in cervical cancer cells and this process might be mediated by miR-218[LAMB3-miR-218 pathway involved in the development of cervical carcinoma.sor gene , is knowsor gene . The dec stomach ,26,27, a tissues . They alPV types , suggesting LAMB3, RICTOR[B3[RICTOR, ROBO1[2BO1[BIRC5, that maBO1[BIRC5. LamininBO1[BIRC5. LAMB3 tironment . Moreovey miR-218, which ipri-miR-218 rs11134527 SNP could alter the local second structure of the pri-miR-218 mRNA, we performed the RNAfold prediction analysis and found an obviously changed mRNA structure from rs11134527 allele A to G. These findings further suggest that germline genetic variations of pri-miR-218, such as rs11134527, may lead to an alteration of miR-218 expression and affect the miRNA binding process and thus are associated with cervical cancer susceptibility.It is known that the mRNA secondary structure is critical for mRNA-miRNA interactions and gene functions . To invemiR-218-related SNPs involved in the LAMB3-miR-218 pathway  were investigated in this study. Cancer is a complex and multifactorial disease, and any single SNP may not be sufficient for the prediction of the overall risk [Several limitations of our study need to be addressed. Firstly, this hospital-based case–control study may have selection bias and information bias, which may be minimized by frequency-matching cases and controls as well as the adjustment for potential confounding factors in the final analyses. Secondly, only two all risk . Future all risk . Therefopri-miR-218 rs11134527 SNP was associated with the risk of cervical carcinoma in Eastern Chinese women. Our findings suggest some possible interactions between genetic variations involved in the LAMB3-miR-218 pathway and other risk factors for cervical carcinoma. However, well-designed prospective studies with larger sample sizes are required to validate our findings.In summary, in the current case–control study of 1,584 cases and 1,394 controls, we found that the miRNA: microRNA;nt: Nucleotide;UTR: Untranslated region;pri-miRNA: Primary miRNA;pre-miRNA: Precursor miRNA;SNP: Single nucleotide polymorphism;SCC: Squamous cell carcinoma;HPV: Human papillomavirus;LAMB3: Laminin 5 β3;FUSCC: Fudan University Shanghai Cancer Center;FIGO: International Federation of Gynecology and Obstetrics;LN: Lymph node;LVSI: Lympho-vascular space invasion;ER: Estrogen receptor;PR: Progesterone receptor;MAF: Minor allele frequency;MDR: Multifactor dimensionality reduction;CVC: Cross-validation consistency;OR: Odds ratio;CI: Confidence interval;BMI: Body mass index;FPRP: False-positive report probability;HWE: Hardy-Weinberg equilibrium;MFE: Minimum free energyThe authors declare that they have no competing interests.Conception and design: QW, XW. In-person survey and data collection: XJC, XC, KDY, ZMS, MHS. Genotyping and Provision of study materials: TYS, MLZ, MYW, JH, XYZ. Data analysis and interpretation: TYS, QW. Manuscript writing: TYS, QW, XW. Final approval of manuscript: TYS, XJC, MLZ, MYW, JH, KDY, ZMS, MHS, XYZ, XC, XW, QW. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/19/prepubTable S2. Interactions between genotypes of the LAMB3-miR-218 pathway and environmental factors on cervical cancer risk. Table S3. False-positive report probability values for associations between genotypes of the LAMB3-miR-218 pathway and cervical cancer risk.Distributions of selected variables in cervical cancer cases and cancer-free female controls. Click here for file


Leukoplakia is the most common potentially malignant lesion of the oral cavity and can be categorised according to its clinical appearance as homogeneous or nonhomogenous. Tobacco and areca nut use, either alone or in combination are the most common risk factors for oral leukoplakia, but some oral leukoplakias are idiopathic. Some leukoplakias arise within fields of precancerized oral epithelium in which the keratinocytes may be at different stages of cytogenetic transformation. Leukoplakias may unpredictably regress, may remain stable, or may progress to carcinoma. There is a greater risk of carcinomatous transformation of idiopathic leukoplakia, of non-homogenous leukoplakia, of leukoplakia affecting the floor of the mouth; the ventrolateral surface of the tongue and the maxillary retromolar and adjoining soft palate , of leukoplakia with high-grade epithelial dysplasia, and of leukoplakia in which the keratinocytes carry cytogenetic alterations associated with carcinomatous transformation. Although there appears to be some link between human papillomavirus (HPV) and oral leukoplakia, there is little evidence to support a causal relationship either between HPV infection and oral leukoplakia or between HPV-infected leukoplakic keratinocytes and their carcinomatous transformation. Leukoplakia is the most common potentially malignant lesion of the oral cavity , 2. LeukAbout 70–90% of oral leukoplakias are related to smoking and areca nut use, either alone or in combination, and there is a direct relationship between the frequency and the duration of cigarette, pipe, or cigar smoking and the prevalence of oral leukoplakia , 9. The However it is possible that infection of the oral epithelium with human papillomavirus (HPV) and excessive consumption of alcoholic beverages may be associated with oral leukoplakia, but there is little evidence of a causal relationship between either HPV infection or alcohol, and oral leukoplakia .It has been suggested that a definitive diagnosis of oral leukoplakia must be established by histopathological exclusion of other keratotic oral lesions that are recognised as specific entities, and by exclusion of any aetiological agents other than tobacco/areca nut use .It is the opinion of the authors that these criteria are unrealistically limiting, ignoring as they do the possible roles of HPV, alcohol, chronic inflammation, and low-grade chronic frictional trauma in the pathogenesis of oral leukoplakia. Referring to the WHO definition of 2005 above, it is difficult to understand how such a definition could gain any useful currency since it is so “exclusive” that it leaves no rational guidance for the everyday diagnosis of oral leukoplakia.According to its clinical appearance, oral leukoplakia can be categorised into two main clinical types: homogeneous and non-homogeneous. Either type may occur as an isolated lesion or as multiple lesions. The leukoplakic lesion can vary in size from a few millimetres to several centimeters , 2, 5–12Homogeneous leukoplakia is a uniformly white flat plaque with a smooth or relatively smooth surface; non-homogeneous leukoplakia may be nodular or verrucous having a wrinkled or corrugated surface or may be a mingling of white and red areas termed erythroleukoplakia , 10, 11.The clinical appearance of oral leukoplakia may change over time. Some homogeneous lesions may become larger, or non-homogeneous, but most oral leukoplakias will remain stable or will regress, while some few will undergo carcinomatous transformation –15.Oral erythroplakia, of all precancerous oral lesions, carries the greatest threat of malignant transformation. It has a velvety-red appearance, and about 50% of all cases of erythroplakia are already squamous cell carcinomata at the time of diagnosis. The erythroplakic component of oral erythroleukoplakia is identical to erythroplakia .Proliferative verrucous leukoplakia, considered to be either a clinical subtype of non-homogeneous oral leukoplakia or to be a distinct clinical entity, is not strongly associated with smoking, is characterized by multiple leukoplakic lesions that affect wide areas of the oral epithelium, and may progress either to verrucous carcinoma or to squamous cell carcinoma –20. In mThe data from epidemiological studies on oral leukoplakia is inconsistent, most probably owing to differences in case selection criteria  and in methodology  , 10, 11.Estimates of the global prevalence of oral leukoplakia range from 0.5% to 3.46%, and of the rates of carcinomatous transformation of oral leukoplakia from 0.7% to 2.9% . Oral leOral leukoplakia is usually diagnosed in middle age, and its prevalence increases with age. About 10% of oral leukoplakias are idiopathic and the greater part of the remaining 90% is associated with the use of tobacco/areca nut . Males aThe literature on the relationship between race and oral leukoplakia is sparse. In a South African study of archived histopathological material, 86% of oral leukoplakias were from whites, 9% from blacks, and 5% from Asians, despite the fact that the vast majority of South Africans are black . This isThe reported prevalence of epithelial dysplasia in oral leukoplakia ranges from 5% to 25% . DysplasThe presence of epithelial dysplasia is a marker of the malignant potential of oral leukoplakia, and the risk of an individual leukoplakic lesion to progress to carcinoma increases with the increase of the grade of the epithelial dysplasia , 14, 30.However, some dysplastic oral leukoplakias can remain stable or even regress, while some oral leukoplakias without epithelial dysplasia will indeed progress to carcinoma , 11, 12.Treatment of dysplastic oral leukoplakia by excision, by laser or by cryosurgery, or by topical or systemic chemotherapy does not eliminate either the risk of relapse or recurrence, or the risk of carcinomatous transformation , 5, 6. TIt is evident from this data that some cases of treated oral leukoplakia are unpredictably destined to recur or to undergo carcinomatous transformation, and that there are not yet any diagnostic methods available , to confidently identify these cases .Epithelial dysplasia in oral leukoplakia is a useful marker of the risk of carcinomatous transformation and is an important guide to clinical management , 11, 28.The progression of oral leukoplakia to carcinoma is unpredictable but is relatively infrequent with an estimated overall risk of less than 2% per year , 13, 26;de novo in normal-looking epithelium [de novo usually runs a more aggressive course and has a less favourable prognosis than squamous cell carcinoma arising from preexisting leukoplakia [de novo in close proximity to oral leukoplakias [In populations where smoking, the use of smokeless tobacco, reverse smoking, and the use of areca nut are very prevalent, most squamous cell carcinomata arise from preexisting leukoplakias; while in populations with a lower prevalence of these habits, most squamous cell carcinomata arise ithelium . It has koplakia , 26, butkoplakia . Sometimoplakias .Non-homogeneous leukoplakia has a greater risk of carcinomatous transformation (20–25%) than homogeneous leukoplakia (0.6–5%) , 13. MosThe rates of progression of large oral leukoplakias (>5 mm) and of leukoplakias at sites in the mouth known to be at most risk of developing carcinoma  are greater than for smaller leukoplakias or for leukoplakias at other sites in the mouth , 33, 38.There is evidence that clearly suggests that some leukoplakias arise from cytogenetically altered transformed keratinocytes within fields of precancerized oral epithelium. Keratinocytes of oral leukoplakia show cytogenetic changes including alterations in the p53 tumour suppressor gene, aberrations in their DNA content, and loss of heterozygosity (LoH) at chromosomal regions of candidate tumour suppressor genes , 39–45. However, some precancerous oral leukoplakias in which cytogenetic alterations in the keratinocytes cannot be demonstrated, nevertheless undergo carcinomatous transformation , 42. TheHuman papilloma viruses (HPVs) are strictly epitheliotropic and infect either cutaneous or mucosal squamous epithelium, depending upon their genotype , 49. ThoLow-risk HPV genotypes have been implicated in the pathogenesis of the benign oral proliferative epithelial lesions, squamous cell papilloma, common wart (verrucous vulgaris), condyloma acuminatum, and focal epithelial hyperplasia (Heck disease); while high-risk types have been associated with precancerous and cancerous oral and oropharyngeal epithelial lesions , 51–56.There is an extreme variation in the reported prevalence of HPV infection in oral precancerous and cancerous lesions ranging from 0% to 100% , 58. ThiMany studies investigating the association of HPV and squamous cell carcinoma of the mucosa of the upper aerodigestive tract used PCR techniques for detection of HPV DNA without also quantifying the DNA viral load. PCR can detect extremely small fragments of DNA that may represent either contamination of the sample or biologically insignificant HPV infection , 57, 58.Whether these are legitimate findings or are the results of inconsistencies and errors in methodology, several HPV genotypes have been detected in precancerous oral lesions. High-risk HPV genotypes, in particular HPV-16, have been reported to be the most prevalent in oral leukoplakias, including proliferative verrucous leukoplakia , 65. OthThis suggests that there may be some link between HPV infection and oral precancerous and cancerous lesions. As E6 and E7 oncoproteins of high-risk HPV genotypes have the capacity to mediate carcinomatous transformation of infected keratinocytes by inactivating cellular p53 and Rb tumour suppressor pathways , 52, HPVIn fact, HPV-16 has been found to be causally associated primarily with squamous cell carcinoma of the palatal tonsils –70 in a Recent meta-analyses and comprehensive studies , 67, 75 With oropharyngeal carcinoma as the model, a causal association between HPV and cancerization in oral epithelium is likely if the cells of the lesion contain HPV DNA expressing E6 and/or E7 mRNA , if therIt is well established that non-homogeneous leukoplakias more frequently undergo malignant transformation than homogeneous leukoplakias, yet it appears that HPV is found more commonly in homogeneous than in non-homogeneous leukoplakia , 78. NevLittle is known about the E6 and E7 proteins of low-risk HPV either with regard to their role in the pathogenesis of HPV-infected oral leukoplakias, or with regard to their role in the carcinomatous transformation of some leukoplakias. It is possible that as in other HPV-associated benign proliferative oral epithelial lesions, E6 and E7 proteins of low-risk HPV types found in oral leukoplakia may stimulate suprabasal postmitotic infected keratinocytes to reenter the S-phase of the cell cycle resulting in epithelial proliferation and disturbed maturation, without causing the genomic instability possibly associated with subsequent cell transformation. This mechanism may be a co-determinant of the development of the leukoplakia, but there is no concrete evidence to support this.As oral leukoplakia is potentially malignant, and as some leukoplakias will unpredictably progress to carcinoma, ideally all oral leukoplakias should be treated. When dealing with two or three accessible circumscribed lesions, the treatment of choice is surgical excision. For multiple or for large leukoplakias where surgical treatment would be impractical because it would result in unacceptable deformities or in functional disabilities, treatment can be by cryosurgery, laser surgery, or by the use of topical bleomycin. However, regardless of the extent of the lesion or of the modality of treatment, in as many as 30% of treated cases, leukoplakias will recur and treatment will not prevent the progression of some leukoplakias to squamous cell carcinoma , 21, 32.Idiopathic leukoplakia, non-homogeneous leukoplakia, leukoplakia affecting high-risk oral sites, and leukoplakia showing moderate or severe grades of epithelial dysplasia and particularly leukoplakias, in which a combination of these factors affect the risk of carcinomatous transformation, should be treated aggressively. Any changes in colour, texture or size, and appearance of additional leukoplakias at new oral sites are advance warning of the possibility of carcinomatous transformation.Leukoplakia is the most common potentially malignant lesion of the mouth. It may unpredictably regress, may remain stable, or may undergo carcinomatous transformation. Those leukoplakias that are committed to a cancerous pathway most probably arise within a field of precancerized epithelium consisting of keratinocytes at different stages of cytogenetic transformation. This may explain the high rate of recurrence of oral leukoplakia despite treatment, and why some leukoplakias progress to carcinoma.Many studies have reported the presence of HPV DNA in oral leukoplakias. However, there is not enough evidence to prove any casual association either between HPV and the development of oral leukoplakia, or between HPV and the progression of oral leukoplakia to carcinoma. The nature of the link between HPV infection and oral leukoplakia is as yet unknown.


Increasingly, countries have introduced female vaccination against human papillomavirus (HPV), causally linked to several cancers and genital warts, but few have recommended vaccination of boys. Declining vaccine prices and strong evidence of vaccine impact on reducing HPV-related conditions in both women and men prompt countries to reevaluate whether HPV vaccination of boys is warranted.A previously-published dynamic model of HPV transmission was empirically calibrated to Norway. Reductions in the incidence of HPV, including both direct and indirect benefits, were applied to a natural history model of cervical cancer, and to incidence-based models for other non-cervical HPV-related diseases. We calculated the health outcomes and costs of the different HPV-related conditions under a gender-neutral vaccination program compared to a female-only program.Vaccine price had a decisive impact on results. For example, assuming 71% coverage, high vaccine efficacy and a reasonable vaccine tender price of $75 per dose, we found vaccinating both girls and boys fell below a commonly cited cost-effectiveness threshold in Norway  gained) when including vaccine benefit for all HPV-related diseases. However, at the current market price, including boys would not be considered ‘good value for money.’ For settings with a lower cost-effectiveness threshold , it would not be considered cost-effective to expand the current program to include boys, unless the vaccine price was less than $36/dose. Increasing vaccination coverage to 90% among girls was more effective and less costly than the benefits achieved by vaccinating both genders with 71% coverage.At the anticipated tender price, expanding the HPV vaccination program to boys may be cost-effective and may warrant a change in the current female-only vaccination policy in Norway. However, increasing coverage in girls is uniformly more effective and cost-effective than expanding vaccination coverage to boys and should be considered a priority. Persistent infection with human papillomavirus (HPV), a known causal agent for cervical cancer, is emerging as an important risk factor for several diseases in both women and men. High-risk, oncogenic HPV infections, most importantly HPV-16 and to a lesser extent HPV-18, are responsible for a proportion of vulva, vaginal, anal, penile and oropharygeal cancers Figure 1In Norway, a 3-dose schedule of the quadrivalent HPV vaccine, shown to have high efficacy against HPV-16, -18, -6, and -11, has been offered to pre-adolescent girls through school-based delivery in the 7th grade since 2009. The most recent cohort of girls (born in 1999) has achieved 2- and 3-dose coverage rates of 79% and 71%, respectively In light of declining vaccine prices and the growing evidence of vaccine impact on reducing multiple HPV-related conditions in both women and men 15 and a range of threshold values cited in other settings.We adapted a dynamic model of HPV sexual transmission and multiple disease simulation models to reflect the health and economic burden of HPV-related conditions in Norway across multiple birth cohorts of men and women We refined a previously-developed dynamic model of HPV-16 and -18 transmission The dynamic model is age-structured in yearly intervals and simulates multiple birth cohorts over their lifetimes. Individuals are designated into one of four sexual activity groups , which governs the rate of partner change per year and varies by age and gender, based on data from two Norwegian sexual behavior surveys The individual-based stochastic model, previously adapted to the Norwegian context Initial parameters for both models were based on data from epidemiological and demographic studies Baseline costs associated with HPV vaccination included costs for all three vaccine doses using the estimated tender price of $75 per dose Health state utility weights for cervical cancer varied according to stage Table 1 We synthesized available data from Norway, or from surrounding countries when Norwegian-specific data were not available, to inform parameter inputs, such as disease incidence, survival and cases attributable to vaccine-targeted HPV types Table 1.We compared a scenario of routine HPV vaccination of 12-year-old girls only at the current Norwegian 3-dose coverage level (71%) to a scenario that assumes similar coverage is achieved by 12-year-old boys. We calculated the health outcomes and costs of the different HPV-related conditions and explored the impact of different vaccine prices ranging from $20–$160 per dose. We evaluated the impact of model assumptions on cost-effectiveness using one- and multi-way sensitivity analysis. For one-way sensitivity analysis, we varied vaccine efficacy, duration, incidence of oropharyngeal cancer and considered an alternate vaccine dose schedule (assuming two doses confer the same vaccine protection as three doses). To provide an approximate estimate of the impact of the men who have sex with men (MSM) population on results, we systematically reduced the herd immunity benefits conferred to the male-population in the female-only vaccination strategy. For the multi-way sensitivity analysis, we simultaneously varied treatment costs and the attributable fraction of HPV-16 and -18 in each HPV-related condition to determine “optimistic” and “pessimistic” results. We also varied analytic assumptions, such as the discount rate (0% and 3%) and consideration of direct costs only, consistent with Norwegian guidelines Assuming the current 3-dose vaccination coverage rate among pre-adolescent girls remains constant at 71% with 100% lifelong efficacy, the girls-only vaccination program was projected to substantially reduce future cancer incidence Table 2.At the assumed tender price of $75 per dose, the cost per QALY gained from routine vaccination of girls only (compared to no vaccination) was $20,600 when including only benefits related to cervical outcomes and $5,000 when including benefits associated with all female and male HPV-related conditions Table 3.The impact of model assumptions on the incremental cost-effectiveness ratio of a gender-neutral vaccine program compared to a girls-only program  for three vaccine prices is shown in Apart from vaccine price, the discount rate and increasing vaccination coverage in pre-adolescent girls had the most influence on results. For example, increasing vaccination coverage of girls alone to 90% was more effective and less costly, and therefore dominated, a scenario of vaccinating both genders with 71% coverage. We calculated that more than twice the amount per vaccinated girl – or six times the amount, if the funds were targeted specifically to those who did not previously uptake – could be spent before adding boys to the vaccination program would be equally cost-effective as increasing participation among girls only. Although extending HPV vaccination to boys provides benefits to both genders, increasing coverage within a girls-only program prevents more HPV-16,-18 related female cancers than a gender-neutral program that achieves 71% coverage. Through additional herd immunity benefits, increasing female-only vaccine uptake can prevent nearly as many HPV-related cancers among men as by vaccinating boys directly Figure 3Our findings confirm that vaccine price is one of the most influential parameters when determining cost-effectiveness of extending the current female-only HPV vaccination program to include boys. In order to aid policy decisions in settings where stakeholders are privy to the national tenders procured at a lower price per dose than the publicly available price, we express the incremental cost-effectiveness ratios across a range of plausible vaccine prices. Our analysis suggests that there may be some combinations of vaccine price and willingness-to-pay thresholds where adding boys is cost-effective, even when current 3-dose coverage rates are already high .In our base case scenario, which considers a realistic Norwegian vaccine tender price of $75 per dose, we found that adding 12-year-old boys to the current HPV vaccination program may be considered ‘good value for money’ at a willingness-to-pay threshold of $83,000 per QALY gained. However, the most recent Norwegian guidelines for economic evaluation emphasize that consensus surrounding a single Norwegian threshold value has not been established To our knowledge, there are only three other studies that have assessed the potential value of adding boys to the pre-adolescent HPV vaccination program that simultaneously account for HPV transmission dynamics, consider all HPV-related outcomes, and report results in terms of cost-effectiveness For a specified vaccine price, our findings were generally stable to variations in critical parameters, with the notable exception of considering a scenario in which we compared expanding vaccination to boys versus increasing the coverage rate among girls, consistent with another study Finally, as both genders are responsible for HPV transmission, one may argue on equity grounds that both genders should get vaccinated to share the burden in reducing the risk of HPV-related disease, as well as have equal access to direct vaccine benefits. Equity versus efficiency arguments should be considered along-side the decision-making process and are particularly relevant in Norway where guidelines explicitly emphasize this trade-off Limitations of our modeling approach have been previously discussed We did not account for any level of vaccine cross-protection related to non-vaccine types observed in clinical trials The quality and completeness of the Norwegian Cancer Registry have been documented At Norway's assumed HPV vaccine tender price, vaccinating boys seems attractive and may warrant a change in the current female-only vaccination policy. However, increasing coverage in girls is uniformly more effective and cost-effective than expanding vaccination coverage to boys and should be considered a priority.File S1Supplementary appendix providing additional information on model inputs, the Norwegian-specific calibration process, and additional results.(DOCX)Click here for additional data file.