High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction. A number of molecular epidemiological studies conducted have accumulated data relating HPV to BC and indicated that HPV DNA is present at a high frequency in BC samples but is rare in normal breast tissues 4 cells) Breast cancer (BC) is one of the major health issues faced by women globally, ranking first in mortality . Epidemiology gave the first hints for an involvement of viral agents in specific human cancers HPV is a DNA virus with a circular double stranded configuration, with more than 100 subtypes found to date in vitro system.Hosts have means such as innate and adaptive immunities to combat against invasion by various pathogens including bacteria and viruses. Innate immunity is a first guardian, which induces interferon resulting in activation of IFN-stimulated genes (ISGs) More than 210 fresh frozen breast tumor specimens were obtained from the National University Health System (NUHS) Tissue Repository (TR) with approval from the Institutional Review Board (IRB) at the National University of Singapore (NUS) and NUHS. The specimens were collected from surgical operations in the National University Hospital (NUS) and banked in the NUHS TR.Genomic DNA (gDNA) was extracted from all above-mentioned BC samples by use of the QIAGEN DNeasy Blood & Tissue Kit  and stored at −20°C before use. After extraction, specimens were subjected to DNA microarray array  for the detection of 13 high-risk HPV genomes, according to manufacturer’s instructions.HPV detection using the TOSHIBA DNA chip was performed according to manufacturer’s instructions 5′-ACGGGGTACCGTCTTATCCTTTGTCCATCTCTTTCTGAC-3′, 5′-ACGGGGTACCGTCTTATCCTTTGTCCATCTCTTTCTGAC-3′, and cloned into pGL3 vector (Promega). pUC-HPV16 and pSP-HPV18 plasmids were constructed as described previously APOBEC3B-promoter (A3B-Pro) was amplified using genomic DNA and specific primers as follows: 293T and MCF10-A cells were obtained from American type tissue culture . 293T cells were maintained in 10% FBS supplemented DMEM. MCF10A cells were cultured with 5% horse serum (Lifetech), 20ng/ml rEGF, 100ng/ml Cholera Toxin, 0.5 µg/ml hydrocortisone  and rInsulin .Stably HPV18 infected MCF10-A (MCF10A-HPV18) cells were established by transfection of linear pSP-HPV18 cut by EcoRI followed by limiting dilution at 7 days after transfection to obtain HPV18 infected cells, in which integration of HPV18 genome was confirmed using Alu-PCR.Anti-HPV18 E7 (8E2) and anti-Flag M2 antibodies were purchased from Abcam  and SIGMA respectively. Anti-γH2AX, γH2AX-Alexa647, β-actin, mouse IgG-HRP and rabbit IgG-HRP antibodies were purchased from Cell Signaling technology.Circular or linear HPV plasmid was transfected into 293T and MCF10-A cells with A3B-Pro and TK-Rluc as control. Cells were harvested at 48–72 hrs post-transfection followed by lysis. Luminescence was measured using Dula-Luciserase kit  and Synergy H1 hybrid Multi-Mode Microplate reader .Patients RNA samples were obtained from the National University Health System (NUHS) Tissue Repository (TR) with approval from the Institutional Review Board (IRB) at the National University of Singapore (NUS) and NUHS. Total RNA from various MCF10-A cells was extracted using RNA easy kit (QIAGEN). Reverse transcription was performed using Goscript reverse transcription system (Promega) after DNase I treatment . Quantification of mRNA was performed using SsoAdvanced SYBR Green supermix  and gene specific primers .MCF10-A and MCF10A-HPV18 cells were fixed by 4% paraformaldehyde/PBS following washing cells with PBS. Cells were treated with PBS containing 4% normal goat serum (Lifetech), 0.5% Triton-X 100 (SIGMA). E7 or γH2AX proteins were stained by anti-HPV18 E7 (8E2) and anti-mouse IgG-Alexa568 Ab or γH2AX-Alexa647 (CST) followed by DAPI staining. After mounting slides using ProLong Gold Antifade Reagent (Lifetech), cells were observed using Olympus X81 immunofluorescent microscope .The pSR-shRNA plasmid was transfected with pVSVG plasmid into Plat-E cells and then supernatants were collected at 60 hrs post-transfection after filtration by 0.45 µM syringe-top filter. Cells were infected with retrovirus vectors for 12hrs in presence of polybrene (SIGMA) followed by puromycin  selection (4 µg/ml) for 48hrs. Cells were cultured in presence of 2 µg/ml of puromycin before use. Knockdown of target genes were checked by qRT-PCR as mentioned above.Microscope slides were coated with 1.5% agarose and dried. Low-melting agarose (0.5% in PBS) was combined 1∶1 with MCF10-A and MCF10A-HPV18 cells. Seven-thousand cells were added to coated slides and the cells were lysed overnight in 10mM Tris, 100mM EDTA, 2.5M NaCl, 10%DMSO and 1% Triton X-100 (pH10). Slides were incubated for 20 min in chilled running buffer  then run at 25V for 25 min at 4°C. Gels were neutralized with 400mM Tris-HCl, pH7.5 followed by propidium iodide staining for 1hr. The microgels were allowed to dry and comets were observed by microscope. The % DNA in tail was calculated by Tail DNA intensity/(Head DNA intensity+Tail DNA intensity) × 100.Data were analyzed using STATA . Associations between HPV status and various breast cancer parameters and risk factors were tested using either Fisher’s exact test or the Chi-squared test for trend. The tests were all two-sided, and statistical significance was defined as p≤0.05.p = 0.0003) while that of IDC was lower than 30%. The HPV positivity was lower when the patients were younger, but it seemed to increase with age (p for trend = 0.1155). Irrespective of ethnicity, all 3 major groups  showed essentially a similar HPV prevalence. The patients with a family history of BC and BC at both sides of the breast seemed to show higher HPV positivity suggesting the infectious origin of some types of BC, especially due to high-risk HPVs (p = 0.0378)  and HPV-18 (36%) followed by HPV-35, -56, -59 and -31 subtypes See . We alsop = 0.047) (HPV-positive tumors showed significantly longer duration period for recurrence after BC resection as compared to virus-negative tumors (= 0.047) . When th= 0.047) . IrrespeTo address whether viral infections could trigger A3B induction and cause mutagenesis of host genomes, we transfected A3B reporter construct together with HPV in either the complete circular form  or fragments , which is widely known as a marker of DNA damage for cancerous phenotype, was significantly increased in MCF10-A persistently infected with HPV18 compared to mock infected cells as revealed by immuno-staining  and WestWe next conducted knockdown experiments for A3B, E6 and E7 using gene specific shRNA to address our hypothesis. Various A3B-specific shRNAs could decrease A3B expression , and theFinally, we checked whether A3B upregulation is associated with HPV infection using BC samples from patients. According to results from To address whether HPV is directly involved in carcinogenesis of BC, we first conducted a molecular epidemiological analysis using patients’ samples. Our results showed the evidence of high-risk HPVs infection in BC patients unambiguously among the different ethnic populations . A possiOur data showed that tumor cells with ER significantly correlated with high HPV infection . The obsWe confirmed several past reports showing that HPV, especially high-risk HPV, was detected from the samples of BC in vivo. Our data is also consistent with those from previous studies by Khan et al. which found that HPV load is very low in BC samples, being far lower than 1 copy/cell We found a positive correlation between A3B expression and HPV infection even in primary BC samples  althoughin vivoIt has been suggested for a long time that inflammation and cancer share some basic mechanisms In present study, we showed that HPV seems to be possible initiator for BCs development through A3B activation leading to genomic instability. However, there are still several pathways for induction of genomic instability and tumor initiation. Among these pathways, oxidative stress (OS) including reactive oxygen species (ROS) is one of the sources to induce DNA damage including DNA double-strand breaks causing cancer initiation In conclusion, we propose here a provocative carcinogenic mechanism of BC that aberrant A3B expression caused by HPV infection might be a mechanism of mutation accumulation in the breast epithelium during HPV-associated breast carcinogenesis which possibly occurs in the early stage of multi-step, multi-factorial carcinogenesis. Recent genome-wide analysis results on broad-spectrum cancer also show the relevance of our contention Figure S1HPV genotypes analysis in BC tissues. Thirteen high-risk HPVs  in derived from fresh BC tissues were detected in gDNAs using the TOSHIBA DNA chip. Percentage of HPV types were analysed with HPV positive samples (n = 217).(TIF)Click here for additional data file.Figure S2Dose-dependent APOBEC3B promoter activation by HPVE6 and E7. (a and b) APOBEC3B (A3B) promoter activity. The indicated dose of E6 (b) and Flag-E7 (c) plasmids were transfected with A3B-Promoter-luciferase and TK-RLuc into 293T followed by detection of luciferase activity at 48hrs post-transfection. Fold A3B-luciferase activity was normalized by TK-Rluc value. Values represent the mean ± SD of three independent experiments. (c) Dose-dependent expression of Flag-E7. Cells were lysed, and then subjected to western blot. The Flag-E7 and β-actin were detected using anti-Flag M2 and β-actin Ab.(TIF)Click here for additional data file.Figure S3Additional γH2AX reduction by shHPV18 E6 or E7 in stable A3B-knockdown MCF10A-HPV18 cells. The γH2AX level in A3B and HPV18 E6 or E7-knockdown MCF10A-HPV18 cells. Stable A3B-knockdown MCF10A-HPV18 cells were transfected with HPV18 E6 or E7 shRNA plasmid. Cells were lysed at 72 hrs after transfection, and then subjected to western blot. The γH2AX and β-actin were detected using anti-γH2AX and β-actin Ab. The number at bottom of panel shows band intensity ratio after normalization by β-actin level.(TIF)Click here for additional data file.Figure S4Possible BC initiation mechanism by HPV. The overall possible scheme of BC initiation mechanism by HPV. 1) HPV is infected to cells, replicates viral proteins and genome and then produces progeny viruses. 2) Estrogen receptor augments HPV replication. 3) A3B production is induced by E6 and E7 proteins derived from HPV episome or integrated genome that results in persistent HPV infection. 4) Induced A3B by E6/E7 and E6/E7 proteins themselves augment genomic instability such as DNA breaks resulting in γH2AX activation. 5) Genomic instability accumulates mutation in host genome and renders cells cancerous. 6) Simultaneously, E6/E7 interfere p53/Rb function respectively to prevent apoptosis and promote cell cycle progression. 7) Those molecular mechanisms cooperatively generate tumor.(TIF)Click here for additional data file.Table S1Primers and Oligonucleotides information. The information of RT-PCR primers and shRNA sequences used in present study.(DOC)Click here for additional data file.Table S2HPV genome copy number in BC. HPV16 and 18 viral DNA in breast cancer (BC) and cervical cancer (CC) tissues were quantified by real-time quantitative polymerase chain reaction (PCR) using a TaqMan probes as mentioned in. Viral DNA was calculated based on the standard curve of control DNA.(DOC)Click here for additional data file.Text S1Supporting information text describes materials and methods for all of supporting figures and tables.(DOC)Click here for additional data file.


Epidemiological studies have examined the association between human papillomavirus (HPV) and breast cancer, but the findings are inconclusive. This study aimed to detect the prevalence of HPV in breast cancer tissue in patients from northeastern China and define the association between HPV and breast cancer using meta-analysis.Polymerase chain reaction (PCR) was used to test cutaneous or mucosal HPV DNA sequence in 77 breast cancer samples and 77 corresponding adjacent normal tissues. The prevalence of HPV in breast cancer was estimated by pooling data from 38 studies. A meta-analysis of 16 case–control studies was conducted to investigate the association between HPV and breast cancer.CI = 25.00–46.10 %). HPV prevalence in breast cancer varied by geographic region, publication period, and PCR detection method. An increased risk of breast cancer was observed in association with exposure to HPV , which was influenced by geographic region, HPV DNA source, PCR primer used, and publication period.We did not find HPV DNA sequence in any of the 154 tissue specimens we tested. However, our meta-analysis revealed a HPV prevalence of 30.30 %  among breast cancer cases; most of these involved high-risk HPV types (35.50 %, 95 % HPV, especially high-risk HPV types, may be associated with an increased risk of breast cancer, and this association varies dramatically among geographic regions. Breast cancer is the leading cause of cancer death in females worldwide, and its incidence and mortality rates have been rising in many Asian countries . AlthougIt is well known that infection with specific types of HPV can cause cervical cancer. Additional evidence indicates a role for these high-risk HPV types in anal cancer, in up to 50 % of other anogenital cancers, and in 25–30 % of head and neck cancers . Later, The aim of our study was to detect any cutaneous or mucosal HPV types in breast cancer using two set of primers: MY09/11 , which tSE = 9.43) selected for our molecular study, 61 had been diagnosed with invasive ductal carcinomas. The clinical characteristics of the breast cancer samples were shown in Table Of the 77 patients . Stratified by region, Oceania yielded the highest HPV prevalence in breast cancer, with 44.30 % (95 % CI = 33.50–55.00 %), followed by Asia with 35.70 % (95 % CI = 20.60–50.80 %), Europe with 32.60 % (95 % CI = 6.50–58.70 %), South America with 14.60 % (95 % CI = 5.80–23.50 %), and North America with 10.70 % (95 % CI = 4.40–17.00 %). When the unconditional logit regression model was introduced to compare HPV prevalence in breast cancer among different geographic regions, the result remained consistent: Oceanian breast cancer patients exhibited the highest HPV prevalence, and the priority of this prevalence was statistically significant (P < 0.001) (Table CI = 20.10–54.50 %) than when HPV DNA was extracted from paraffin-embedded tissues , but this priority of prevalence was statistically insignificant (p = 0.08)  was dramatically lower than that between 1992 and 2006  (p < 0.001)  of the studies we reviewed, while HPV prevalence was 5.30 % (95 % CI = 2.40–8.30 %) using other methods, such as in situ hybridization and Southern blotting (Table CI = 18.30–41.70 %) when broad-spectrum PCR primers were used; it was 31.90 % (95 % CI = 13.00–50.80 %) when type-specific PCR primers were used; and it was 35.30 % (95 % CI = 11.50 –59.00 %) when a combination of these two types of primers was used. Because most studies dealt with invasive ductal cancer as well as non-invasive cancer, the detection rates of HPV in invasive ductal cancer were pooled, yielding HPV prevalence of 32.90 % (95 % CI = 21.50–44.20 %) compared with non-invasive cancer, which showed a prevalence rate of 22.00 % (95 % CI = 12.40–31.50 %) , HPV-33 , HPV-11 , HPV-18 , and HPV-6  (Table CI = 25.00–46.10 %), much higher than the prevalence of low-risk HPV types, which was 11.70 % (95 % CI = 5.80–17.70 %).Although multiple HPV types were determined in breast cancer cases across the 38 studies in our meta-analysis, the five most common HPV types, in decreasing order of prevalence, were the following: HPV-16 , a random model was chosen to calculate the combined OR and its 95 % CI across the 16 studies. When these 16 studies were pooled in the meta-analysis, results showed that HPV infection was associated with higher risk for breast cancer  .A total of 16 case–control studies were included in our evaluation of the association between HPV infection and breast cancer risk  in Oceania and Europe, but pooled OR remained the same . When ORs were grouped by DNA source, however, significantly higher risk for breast cancer was revealed among fresh-tissue methods  without between-study heterogeneity. Similarly, breast cancer risk was greater with HPV infection in studies published between 1992 and 2006  compared with studies published between 2007 and 2012. HPV infection detected using broad-spectrum PCR primers was associated with higher risk for breast cancer , while HPV infection detected using combined PCR primers showed the opposite result .Considering that substantial heterogeneity was observed among the 16 studies for HPV infection and breast cancer, the prevalence of HPV infection in breast cancer was further evaluated by subgroup analysis. The analyzed subgroups were defined according to the main features of pooled studies: geographical region, DNA source, detection method , and publication period Table . In the CI = 22.30–38.40 %). Furthermore, the prevalence estimate varied by geographic region, publication period, and PCR detection method. Our meta-analysis of 16 pooled case–control studies revealed that HPV infection is associated with breast cancer risk.The role of HPV in breast cancer has been controversial; thus, a PCR-based strategy as well as a meta-analysis were conducted to investigate the association between HPV and breast cancer. In present study, 77 breast cancer samples and 77 adjacent normal tissues all tested negative for HPV. However, in our meta-analysis of 38 studies from 20 countries published since 1992, we found the overall HPV prevalence in women with breast cancer to be 30.30 % . Interestingly, the HPV detection rate of studies published between 1992 and 2006 was remarkably higher than that of studies published between 2007 and 2012. This can be explained by the fact that the latter generally included more breast cancer cases and that the specificity of detection advanced over time, thus yielding a conservative result.The prevalence of HPV infection in breast cancer ranged from 10.70 % in North America to 44.30 % in Oceania. The geographic differences of HPV infection rates may be related to the distinctly different cervical cancer burdens across different continents . In 1999In present study, HPV prevalence in 77 breast cancer samples and 77 adjacent normal tissues was zero, which is lower than that estimated by the meta-analysis included in this report. However, this is consistent with the results showed by stratified analysis, i.e. , HPV infection rate varies by geographic region and publication period . All of the patients were women living in northeastern China, and none had a history of cervical carcinoma. Informed consent according to the criteria of Harbin Medical University was obtained from all patients, and this study was approved by the Institutional Research Board of Harbin Medical University (No. HMUIRB20120002).DNA was extracted from fresh frozen breast tissues as previously described, using SDS lysis/proteinase-K digestion, phenol/chloroform extraction, and ethanol precipitation. The DNA quality was confirmed by the amplification of a 268-bp fragment of human β-globin gene using and primers G073 (5′-GAAGAGCCAAG GACAGGTAC-3′) and G074 (5′-CAACTTCATCCACGTTCACC-3′). To avoid contamination between samples, the cutting blade was changed after the sectioning of each sample.2, 200 μM dNTPs, 0.4 μM of each primer and 0.5 U Taq DNA polymerase , and reaction buffer , according to the following conditions: 5 min at 94 °C, then 40 cycles of 5 s at 98 °C, 30s at 50 or 55 °C, and 60 s at 72 °C, followed by a 5-min extension at 72 °C in an automated thermocycler . The full genomes of HPV-16, −11, and −8 were cloned in the pBS322 plasmid (donated by Michelle A. Ozbun), and HPV-18-positive cervical cancer specimens were used as positive controls; sterile water was used as a negative control. The PCR products were separated on 1.5 % agarose gel  and visualized with ethidium bromide staining after electrophoresis.DNA samples were then screened for the presence of HPV by PCR using two sets of primers: MY09/11 and FAP59/64. Consensus primers MY09 (5′-CGTCCMARRGGAWACTGATC-3′) and MY11 (5′-GCMCAGGGWCATAAY AATGG-3′) amplify a 450-bp fragment in the highly conserved region in L1 of mucosal HPV types . Degenerated primers FAP59 (5′-TAACWGTIGGICAYCCWTATT-3′) and FAP64 (5′-CCWATATCWVHCATIT CICCATC-3′) can detect a 478-bp fragment in the L1 region of both mucosal and cutaneous HPV types . PCR was performed in a volume of 20 μL, containing 100 ng of extracted DNA, 1.67 mM MgClEligible articles published from January 1989 to June 2013 were identified by an electronic PubMed search using the MeSH terms “human,” “papillomavirus,” and “breast carcinoma.” Additional articles from the reference lists of retrieved articles were also screened. The inclusion criteria of the studies were as follows: (1) detection of HPV in female breast cancer; (2) full text in English; (3) independent of other studies. The experimental results from the present molecular study were included in the meta-analysis.Two authors (Jinyuan Li and Lanlan Wei) independently extracted data and reached a consensus on all issues. Disagreements were discussed and resolved according to the inclusion criteria and consensus. The following data were extracted from each report: the first author, year of publication, country of origin, ethnicity, sample size (cases and controls), HPV prevalence, DNA source, detection method, PCR primers used, histological subtypes tested, and HPV types detected.An epidemiological review of the overall and type-specific HPV prevalence in breast cancer cases was conducted. Four high-risk HPV types were identified , and two low-risk HPV types were identified (HPV-6 and −11). A meta-analysis was performed to explore the association between HPV infection and breast cancer risk in the form of a case (breast cancer tissues) versus control  comparison. An unconditional logistic regression model was used to compare HPV prevalence according to the influential parameters: HPV DNA source, PCR primers used in detection, and publication period.2-based Q test, and heterogeneity was considered significant if p < 0.05. Subgroup analyses were further performed to explore the source of the existing heterogeneity. Publication bias was evaluated using the linear regression asymmetry tests designed by Egger et al. [A fixed-effect model (Mantel-Haenszel method) and a random-effect model (DerSimonian and Laird method) were used to pool the case–control data. These two models provide similar results when between-study heterogeneity is absent; otherwise, the random-effect model is more appropriate. Between-study heterogeneity was tested using the χr et al. . Analyse


Fifty years ago in the United States, the leading cause of cancer death among women was cervical carcinoma. With the advent of the Papanicolaou (Pap) test in 1945, mortality from this malignancy declined more than 70% . An annual Pap smear was the recommended guideline for many years. New cervical cancer guidelines were released by the United States Preventative Services Task Force (USPSTF) on March 14, 2012.The following points summarize the new USPSTF cervical cancer screening guidelines:Begin screening at age 21 with use of cytology alone every 3 years from ages 21–29. No screening prior to age 21 regardless of age of coitarche.Patients with atypical squamous cells of undetermined significance (ASCUS) on cytology with negative human papillomavirus (HPV) status should be managed the same way as patients with negative cytology.Women ages 30–65 should be screened every 5 years with co-testing (cytology and HPV) or every 3 years with cytology alone if co-testing is not available.Women over age 65 can discontinue screening. Once discontinued, screening should not resume even if a woman has a new sexual partner.No screening is needed after hysterectomy if there is no prior history of cervical intraepithelial neoplasia grade 2 (CIN2) or higher grade lesion.Following spontaneous regression or treatment, women with a history of CIN2 or higher grade lesion should continue screening, even if this extends beyond age 65 .The previous guidelines initiated screening 3 years after beginning sexual activity but no later than age 21. Women younger than 30 were recommended to have a Pap test every 2 years. For women 30 or older, the recommendation was every 3 years if they had had 3 consecutive negative Pap tests. These guidelines issued in 2009 recommended less frequent screening than the prior guidelines from 2003, which recommended women younger than age 30 have an annual exam .In order to understand why the previous screening guidelines, which resulted in such a dramatic decrease in mortality, have now changed, it is essential to understand how the knowledge of cervical carcinogenesis has progressed through the years.Human papillomavirus is the primary causative agent of cervical carcinoma. Persistent infection is necessary for cancer to develop; this is a process that evolves over decades from preinvasive intraepithelial neoplasia to invasive cancer . Human papillomavirus is extremely prevalent; the lifetime risk of infection is 80% . Current estimates are that 50% to 80% of young sexually active women will contract HPV infection within 24 to 36 months of coitarche . The prevalence of HPV infection in the US is 40% among sexually active women ages 14–19, and 49.3% among sexually active women 20–24 years old. The good news is that 90% of women have been found to clear the infection without intervention within 2 years. Human papillomavirus persistence, which is necessary for the development of cervical cancer, affects only a small percentage of women who acquire HPV .The majority of cervical cancers are related to two specific strains of HPV. HPV 16 accounts for 51% of cervical cancers and HPV 18 for 16%. Additional strains of HPV that are related to cervical cancer include HPV 35 (8.7%) and HPV 45 . This equates to a stronger correlation between high-risk HPV and cervical cancer than that between smoking status and lung cancer .Young women are particularly prone to acquiring HPV infection due to the anatomy of the immature cervix. The adult cervix contains protective squamous epithelium, whereas the immature cervix contains columnar epithelium. During the adolescent period, this tissue undergoes metaplasia and transforms to squamous epithelium. During metaplasia, HPV has easier access to the basal cell layer, the site of rapid replication and differentiation . Despite this susceptibility to HPV infection, 90% of young women are able to clear the infection without intervention and hence avoid development of invasive cervical cancer .Women ages 15 through 19 have a very low incidence of invasive cervical cancer, at a rate of 0.1/100,000 . This rate has remained unchanged for 4 decades despite increased screening coverage . Therefore, deferring the onset of cervical screening until age 21 is unlikely to have a significant impact on the overall incidence of cervical cancer. The benefit of delayed onset of screening is the reduction in the number of colposcopies and biopsies for young women with transient HPV changes that will likely resolve on their own in 6 to 36 months . Not only is emotional and physical discomfort avoided, but there is a decrease in health-care costs seen with the elimination of unnecessary colposcopies and biopsies as well. Finally, there are long-term consequences of treatment of dysplasia with cryotherapy or the loop electrosurgical excision procedure (LEEP). These include cervical stenosis, incompetent cervix, increased risk of premature delivery, low birth weight, and premature rupture of membranes .It is important to keep in mind that the new cervical cancer screening guidelines do not apply to women who are immunosuppressed, including patients with HIV or immunosuppression related to organ transplantation. Also excluded from these screening guidelines are women with a history of cervical cancer or in utero exposure to diethylstilbestrol (DES). Additional risk factors for the development of invasive cervical cancer to bear in mind include smoking and having a history of multiple sexual partners .Surveillance, Epidemiology, and End Results (SEER) data demonstrate that cervical cancer is not a disease of youth and HPV acquisition, but rather one that occurs in the middle-aged to older female and is related to the persistence of HPV ; see Table 1. Based on what we have learned through the years about the cause of cervical cancer and the timeline for development from intraepithelial neoplasia to invasive cancer, the new guidelines are medically sound and should continue to adequately protect women from invasive disease.In recent years, we have learned more about the cause and development of invasive cervical cancer and are now poised to move from the practice of secondary prevention to primary prevention. In the past decade, vaccines have been developed to target high-risk strains of HPV. A bivalent vaccine  is targeted toward HPV 16 and 18. A quadravalent vaccine  is targeted to HPV 6, 11, 16, and 18. Both are commercially available and approved for males and females ages 9 through 26. These vaccines are able to decrease the risk of cervical and other HPV-related cancers .The focus of the well-woman examination may now shift from that of the annual Pap test to cancer screening, assessment of cardiovascular risk factors, and evaluation of thyroid health. The clinician will be better able to encourage fitness and promote overall health. As many young women do not regularly see a primary care physician, the annual well-woman visit may be their only health-care contact. Having more time to devote to issues of overall health and well-being will help build stronger relationships between clinicians and patients, resulting in increased mutual benefit.


L1 gene sequencing in cervical smearscollected during gestation and at 12 months after delivery. Outcomes were defined asnonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection ofdifferent types of HPV in the 2 samples), and type-specific HPV persistence (the sameHPV type found in both samples). An unfavourable cytological outcome was consideredwhen the second exam showed progression to squamous intraepithelial lesion or highsquamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistenceoccurred in 50% of the cases composed of type-specific persistence (30%) orre-infection (20%). A low CD4+T-cell count at entry was a risk factor fortype-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almostthree times higher in the type-specific group when compared with the re-infectiongroup . Our findings show thatbonafide (type-specific) HPV persistence is a stronger predictor for the developmentof cytological abnormalities, highlighting the need for HPV typing as opposed to HPVDNA testing in the clinical setting.This study investigated the rate of human papillomavirus (HPV) persistence,associated risk factors, and predictors of cytological alteration outcomes in acohort of human immunodeficiency virus-infected pregnant women over an 18-monthperiod. HPV was typed through  A high prevalence of certain human papillomavirus (HPV) types has been found in cervicalcancer (CC) . The majTP53encodes an arginine (Arg) residue. The Arg/Arg genotype was found to be more prevalent inCC biopsies and in individuals with progression from squamous intraepithelial lesions (SIL)to CC (TP53 genepolymorphisms on the persistence of HPV infection is still controversy (Immunosuppression conditions caused by human immunodeficiency virus (HIV) infection , Riode Janeiro, Brazil, from April 2009 (1st enrollment) to July 2012 (last 18-monthcollection). HIV-positive serological status was confirmed by rapid HIV test or ELISAtests and a subsequent Western blot in distinct samples following recommendation by theBrazilian Ministry of Health. All women who agreed to participate signed an informedconsent form before enrollment. The study was approved by the ethical committees of bothcentres involved in the study: UFRJ  andthe National Cancer Institute (INCA) (protocol: 142/10).This study was conducted in accordance with the ethical standards of the BrazilianNational Ethical Committee as well as the Ethical Committee from the UFRJ and the INCAand with the Helsinki Declaration of 1975, as revised in 1983.Samples and study variables - Cervical smears were collected at twodifferent times from all 90 patients at the second trimester of gestation and 12 monthsafter delivery [average of time interval: 17 (± 4.5) months]. Collection was performedwith two endocervical cytobrushes; one was placed in 100% ethanol for Pap smeardiagnosis, while the other was placed into 1 ml phosphate-buffered saline and sent toINCA for molecular analyses. A structured questionnaire containing demographic data,gynaecologic and obstetric history, and other risk factors for HPV infection wasperformed. Additional variables related to HIV infection have also been collected.Sample processing and molecular analyses - Genomic and viral DNA wasextracted with the QIAamp DNA mini kit  following manufacturer’sspecifications. A genome fragment of the HPV L1 gene was polymerasechain reaction (PCR)-amplified in a nested reaction with standard primers and conditionsas previously described (TP53 gene exon 4 (containing codon 72 of the predicted proteinsequence) was also PCR-amplified as described elsewhere and used to assess DNA qualityand integrity, thus validating the detection of HPV DNA . Sequences were assembled and edited with SeqMan .Sequences suggestive of multiple infections (electropherograms containing multiple peaksat definite positions) were subject to cloning into pMOS  andtransformation into TP53alleles and genotypes were classified by visual inspection of codon 72 of each sequencefor the presence of a cytosine (coding for a proline residue) or a guanine (coding forArg).HPV typing was performed by submitting edited bulk or clonal sequences to the Blastalgorithm (ncbi.nlm.nih.gov/blast) and HPV types were assigned to the best hits of thedatabase for each query sequence. Typing was further confirmed by phylogenetic analysisthrough clustering of query sequences to HPV type references.Definition of HPV oncogenic potential and HPV persistence - The HPVtypes 16, 18, 26, 30, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69,73, and 82 were considered as HR types while HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72,81, 89, 32, 62, 74, 83, 84, 86, 87, and 91 were defined as low-risk (LR) types (R) types .Nonpersistence was defined as the clearance of the HPV DNA in the second samplecollected. Re-infection was defined as the detection of different types of HPV DNA inthe two collected samples. Type-specific HPV persistence required the match of the HPVtype found in both samples, either as major or minor strains (for the multiply-infectedcases). Re-infection cases and type-specific HPV persistence were combined in the groupdenominated HPV DNA persistence. In case of multiple infections, each type was countedseparately for determination of persistence rates.Pap smear cytological classification and outcomes - Cytological resultsof Pap smears were classified according to the 2001 Bethesda reporting guidelines  two-sample tests for those not following a normal distribution.Categorical variables were analysed with chi-square or Fisher’s exact test. Variableswith a p-value ≤ 0.20 in the univariate analyses were included in a logistic regressionanalysis.General cohort characteristics - One hundred and forty patients wereenrolled. Twenty-eight patients (20%) had no detected HPV DNA in their first sample andwere not included in the analysis. In five patients (3.6%), no DNA was recovered in thesecond sample suggesting lack of cells in the sample, and 17 (12.1%) were lost tofollow-up. Ninety patients were analysed.Clearance of HPV infection occurred in 50% (45 patients), type-specific persistence in30% (27 patients), and re-infection in 20% (18 patients). There was no difference in thetime interval between the first and second sample collection among the three groups.+ T-cellcounts at entry were 441 cells/mm3 and almost 57% had counts above 350cells/mm3, the median HIV viral load (VL) was 1,085 copies per mL ofplasma ; this number rose to 44.4% one yearafter delivery. Regarding HIV-1-related variables, 30% of the patients were onpreconceptional combination antiretroviral therapy,the median CD4fplasma .TP53 genotypes at codon 72 were distributed as 38.9% Arg/Arghomozygous, 20% Pro/Pro homozygous, and 41.1% Arg/Pro heterozygous.HPV typing and persistence - Twenty-nine different HPV types wereidentified at baseline , followed by HPV 58 (11 cases), HPV 35 and 53 (5 cases), and HPV 73 and 66 (4cases). HPV 18, consensually recognised as associated to cervical oncogenesis , was onlHPV DNA persistence was found in 45 patients (50%): 27 women (30%) presented atype-specific persistence, while re-infection with another HPV type occurred in 18 women(20%). Risk factors for HPV persistence - We have evaluated factors associatedwith HPV persistence for the three defined groups : 1.00-21.3].sistence  and oddssistence . The totCytological outcomes based on HPV persistence - The proportion ofpatients who showed unfavourable outcomes increased from the nonpersistent (cleared) HPVinfections to re-infections and then to type-specific persistence, being the highest inthe latter group. In the nonpersistent group, there were six LSIL and two HSIL in thefirst sample and all cases improved in the second sample. Four new cases of LSIL wereseen. In the re-infection group, there were eight LSIL at baseline with two improvementsand two new cases of LSIL in the follow-up. In type-specific group, initial sampleshowed 13 LSIL and two HSIL. In the follow-up, three LSIL evolved to HSIL and four newcases of LSIL developed. The OR was almost three times higher in the type-specific groupwhen compared with the re-infection group .In this study, we analysed 90 HIV/HPV co-infected, pregnant women for rate ofpersistence of HPV after delivery and risk factors associated with persistence over 18months. Overall, over 80% of the studied women were infected by HR HPV types atbaseline. HPV DNA persistence occurred in 50% of the women after 18 months, but only in30% showed type-specific persistence. The remaining 18 patients (20%) were ratherre-infected by different HPV types, and do not correspond to bonafide HPV persistence.The use of sequencing methodology to classify the different types of HPV has enable usto determine the true rate of infecting HPV types and avoid overestimation of HPVpersistence.Our HPV typing approach has enabled us to evaluate all cases of persistence byindividual HPV types. Of note, the relative efficiency of HPV persistence by types 16and 58 was possible to infer in view of the higher number of cases in our cohort. HPV 58appears to persist with higher efficiency than HPV 16 , although statistically significance has not been achieved. Finally,despite only four cases of infection were observed, two of HPV 18 and two of HPV 56,both types persisted in the period analysed, highlighting its potential in the fasterdevelopment of cervical abnormalities, as suggested by others , and CC.TP53 genotypes encoding proline at codon72 in our patients (60%) which is in accordance with the genotype among highly mixedpopulations (We find a higher prevalence of ulations . Althougulations .An important issue to be considered in HPV persistence studies is the timespan and thedefinition of persistence. HPV persistence is regularly defined as having two or moreHPV DNA positive tests and test intervals greatly vary between studies, complicatingcomparative analyses .AdditioAs mentioned above, HPV persistence has been associated with development of cytologicalabnormalities and progression to neoplasia. To evaluate the impact of HPV persistence onthe progression of cytological abnormalities, we have compared the cytological outcomesof the women throughout the 18-month analysed period. Our study suggests thattype-specific HPV persistence has a better predictive value to cervical lesionprogression and its use should be reinforced over traditional HPV persistenceassays.The present study has some important limitations. One of them is the lack ofhistopathologic confirmation of the cytologic abnormalities through biopsy. Anotherlimitation was the low HPV persistence rates which precludes us from performing adetailed analyses of the type-specific and re-infection groups. Nevertheless, we showedthe importance of identification of specific HPV types for the discrimination betweenbonafide persistence and clearance followed by re-infection. Measurements of trueincidence of new HPV infections in subjects is a difficult task, because they can beinfected by new viruses and clear them over the time period spanning two collectiontimes, and those transitory infections will not be detected. However, by addressingtype-specific HPV persistence, we can at least get better estimates of new infections,excluding those cases where the HPV types differ between the two timepoints analysed.Additional re-infection cases may also be excluded if the two viruses are geneticallydifferent, even if belonging to the same HPV type, an analysis that requires full-lengthgenome sequencing and phylogenetic estimates, procedures that we did not conduct in thisstudy.HPV persistence is a risk factor for the development of cervical intraepithelialneoplasia and cervical carcinoma, whereas persistence by HR types is a prerequisite forthose conditions . HIV-infWe have found HPV persistence of 50% after delivery in our cohort. Reports ofpersistence after pregnancy are scarce and it decreases with longer time of follow-up.The HPV type 16 was the most prevalent type in this cohort and type 58 was the secondmost common type found. In a meta-analysis of HPV prevalence in five continents, type 58was the third most common type in Latin America and Caribbean. In relation oftype-specific persistence, our results confirm previous evidence found in a recentlongitudinal survey of the Finish Family HPV Study, where HPV 58 showed a higherpersistence time compared to HPV 16 . These r+ T-cell countsare associated with HPV persistence (3 have almosttwo times more chance of HPV persistence when compared with those with a CD4 T-cellcounts > 500 cells/mm3 (Our results corroborate previous report showing that low CD4sistence , and illIn conclusion, the results herein highlight the importance of HPV persistence in thesetting of immunosuppressed patients and their associated risk factors. In addition,efforts to implement true type-specific HPV identification in persistence should be madeto avoid overestimation of persistence and to more accurately predict risk forcytological abnormalities and cervical carcinoma in HPV-infected women.


Reffering to an increase in cervical cancer in the recent years, rapid, sensitive and economical detection of human papillomaviruses (HPVs) as causative agents of cervical cancer is important. The traditional methods for the detection of HPVs in cervical cancer, such as pap smear, suffer from limitation and PCR has a potential to overcome the limitaitons. The purpose of present research work was to identify the five important strains of HPV  simultaneously by Multiplex PCR application.Study was done on 100 cervical lesions of women. DNA was extracted from specimens by a genomic DNA purification kit. A 5-plex PCR was developed for the simultaneous detection of major HPV. Five pair of new primers was designed for detection of HPV 16, 18, 31, 33 and 45 by Multiplex PCR.Among the 100 evaluated samples, 82 were found positive to HPVs. In the meantime the highest rate of infection was for HPV 16. Also 30 of HPV positive samples had infections with two or more HPV types.Multiplex PCR assay used in present study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of vaginal samples. Human Papilloma Viruses (HPVs) are agents causing cervical cancer that have been detected in up to 99.7% of cervical squamous cell carcinoma and 94 to 100% of cervical adenocarcinomas.7Cervical cancer is the second most common cancer that affects more than four hundred thousand women each year and a leading cause of cancer-related death worldwide.10HPV cannot grow in conventional cell culture, and serological assays have only limited accuracy. Cervical cancer screening is currently with the papnicolaou (pap) smear which has had a significant impact on the reduction in the incidence of cervical cancer. However, the sensitivity of cytological test is variable greatly according to experience of the cytologist and other conditions.Those women who had cervical lesion in refferal and teaching hospital affiliated to Isfahan University of Medical Sciences  were included in the study. One hudnred samples were collected (during winter season 2014). All samples were taken using of sterile cotton swab, and were stored at -20ºC until required for DNA extraction. Permision from the ethical committee was taken. The anonymity of participants and data confidentiality was protected. Protocols and approved proposal according to local law and regulations, by the Institutional Review Boards of each participating referral hospital.http://wwwncbi.nlm.nih.gov/BLAST/).The gene bank accession for the DNA sequences used, are shown in The sterile cotton swabs were placed in 1.5 ml sterile tubes and viral DNA was extracted by a genomic DNA purification kit  according to the manufacturer’s recommendation. The extracted DNA was quantified by spectrophotometric measurement at a wavelength of 260 nm and was kept frozen at -20°C until used.A 5-plex PCR was performed for identification of HPV-16, 18, 31, 33 and 45. The Multiplex PCR was run in final reaction volumes of 50 μl containing the following reagents: 100 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.5), 6 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, 50 pmol of each primer and 5 unit of Taq DNA polymerase . The PCR assay was performed at 95ºC for 6 minutes (pre denaturation) and then for 40 cycles of 94ºC for one minutes, 60ºC for one minutes, 72ºC for minutes, with final extension at 72ºC for seven minutes in a thermal cycler , Negative controls were included in each run contain HPV 54.The PCR-amplified products were detected in 1.5% ethidium bromide (EtBr)-stained agarose gel electrophoresis. Aliquots of 15 μL of PCR products were applied to the gel. Voltage of 120 V for two minutes and then 85 V for 25 minutes was used for products separation. The DNA molecular weight marker  was used as a size marker. After electrophoresis, images were obtained in UVIdoc gel documentation systems (UK).P values <0.05 were considered significant.For presence of HPVs  all data were analyzed by MS Excel 2007 and the Chi-square test using the SPSS 17  software. One hundred samples of mucosal cervical lesion from women were examined for presence of HPVs DNA . The primers used were derived from the E6 HPV gene of HPVs. Agarose gel electrophoresis of positive samples revealed 202 bp fragment (HPV 16), 279 bp fragment (HPV 18), 339 bp fragment (HPV 31), 154 bp fragment (HPV 33) and 451 bp fragment (HPV 45). An example of Multiplex PCR amplification of mucosal samples is shown in An increasing number of malignancies are directly or indirectly the result of viral infection. Cervical cancer is a worldwide public health problem among women, especially in emerging nations.This study describes a rapid and sensitive assay for detection of mucosal high-risk HPV types with a high specificity for the detection of several mucosal high-risk HPV types in women at increased risk of cancer and also infected with single and multiple HPV types. We suggest use of this assay for epidemiological studies that aim to determine the distributions of various HPV types in different parts of the world.


Federally funded surveys of human papillomavirus (HPV) vaccine uptake are important for pinpointing geographically based health disparities. Although national and state level data are available, local  data are not due to small sample sizes, confidentiality concerns, and cost. Local level HPV vaccine uptake data may be feasible to obtain by targeting specific geographic areas through social media advertising and recruitment strategies, in combination with online surveys.Our goal was to use Facebook-based recruitment and online surveys to estimate local variation in HPV vaccine uptake among young men and women in Minnesota.From November 2012 to January 2013, men and women were recruited via a targeted Facebook advertisement campaign to complete an online survey about HPV vaccination practices. The Facebook advertisements were targeted to recruit men and women by location , age (18-30 years), and language (English).Of the 2079 men and women who responded to the Facebook advertisements and visited the study website, 1003 (48.2%) enrolled in the study and completed the survey. The average advertising cost per completed survey was US $1.36. Among those who reported their postal code, 90.6% (881/972) of the participants lived within the previously defined geographic study area. Receipt of 1 dose or more of HPV vaccine was reported by 65.6% women (351/535), and 13.0% (45/347) of men. These results differ from previously reported Minnesota state level estimates (53.8% for young women and 20.8% for young men) and from national estimates (34.5% for women and 2.3% for men).This study shows that recruiting a representative sample of young men and women based on county and postal code location to complete a survey on HPV vaccination uptake via the Internet is a cost-effective and feasible strategy. This study also highlights the need for local estimates to assess the variation in HPV vaccine uptake, as these estimates differ considerably from those obtained using survey data that are aggregated to the state or federal level. Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States  and is tTwo vaccinations against HPV infection are currently licensed in the United States. The vaccinations were originally licensed for use in girls, but as of October 2011, the Advisory Committee on Immunization Practices extended their recommendation of the quadrivalent vaccine to include both boys and girls aged 11 or 12 years old ,7. HowevPrevious research on HPV vaccine coverage has used publicly available data from five national health surveys  -15. ThesThe Internet provides a unique point of contact to reach young adults for health research. Several studies have demonstrated that Internet-based research can be used to elicit high response rates at a fraction of the cost of traditional recruitment methods -18. In aThe objective of this study was to estimate HPV vaccination practices among a local population of young adult men and women in the United States using an Internet-based recruitment strategy.Men and women from Minnesota were surveyed about their HPV vaccination practices via the Internet from November 21, 2012, through January 31, 2013. Participants were English-speaking, aged 18-30 years, had a Facebook account, and resided in the greater Twin Cities Metropolitan Area . This age range was used to target men and women who were eligible to receive the HPV vaccine, participate in cervical cancer screening (women), and able to provide informed consent. The Twin Cities Metropolitan Area was selected due to the variation of HPV-related cancer incidence rates exhibited in this area during the past 15 years, the high concentration of colleges and universities, and the large population of 18-30 year olds residing in this area . Al. Al40]. Limitations include the fact that the survey responses were self-reported by persons over the Internet and may be subject to under or overreporting. However, other Internet-based studies have shown increased self-disclosure and reporting with online surveys, which may reduce potential response biases  ,21. AddiTo our knowledge, this is the first study to estimate local level vaccination uptake among young men in the United States. Understanding the local variation and patterns of HPV vaccination of young men could serve to identify areas where HPV infection-related health disparities may continue if neglected. In particular, the online survey also allowed us to collect data on sexual orientation, which in turn would allow us to understand whether men who have sex with men, who are at high risk of HPV-related anal cancer, are receiving the vaccine and to also determine whether reductions in the overall risk of HPV infection will affect transmission to females ,43.The results from this study suggest that more detailed and local assessments of HPV vaccine uptake are necessary as estimates vary greatly from national surveys. In addition, recruiting young adults via the Internet is efficient, cost-effective, and can produce a representative sample of the target population. Future work is needed to understand the pattern of HPV vaccine uptake at local levels in order to identify areas that may be best served by vaccine programs.


The incidence of lung cancer in Koreans is increasing in women and in both men and women with a never-smoking history. Human papillomavirus (HPV) infection has been suggested as a modifiable risk factor of lung cancer in never-smokers and women (LCNSW). This systematic review (SR) aimed to evaluate an association between HPV infection and lung cancer risk in LCNSW.Based on a prior SR and some expert reviews, we identified refereed, cited, or related articles using the PubMed and Scopus databases. All case-control studies that reported the odds ratio of HPV infection in LCNSW were selected. An estimate of the summary odds ratio (SOR) with 95% confidence intervals (CI) was calculated.A total of four case-control studies were included. The fixed-effect model was applied because of homogeneity (I-squared=0.0%). The SORs in women and in never-smokers were 5.32  and 4.78 (2.25 to 10.15) respectively.These results showed a significant effect of HPV infection in LCNSW. It is evident that developing a preventive plan against LCNSW may be necessary. Lung cancer ranks the first in cancer mortality in Korea and is the primary cancer with the heaviest disease burden . AccordiThe increasing incidences of lung cancer among women never-smokers is a global trend ,5, and iHPV deoxynucleic acid (DNA) is detected in approximately 20% of lung cancer tissues -20, and www.ncbi.nlm.nih.gov/pubmed) and the Scopus (www.elsevier.com/solutions/scopus) database.In order to maximize the utility of previously conducted systematic reviews in the literature search, we used a manual search method rather than an automatic method ,27,28. TThe finally selection criterion for the study objective was a case-control study in which HPV DNA was tested on never-smokers and women. Accordingly, for each study in the lists described above, the following exclusion criteria were applied to the abstract or the main body of the article: (1) a study dealing with different hypotheses, (2) an expert’s review or a systematic review study, and (3) a case report study. Of the remaining case-control studies after the first three exclusion criteria were applied, the final set of studies were selected using the following two exclusion criteria: (4) a study in which HPV DNA testing was not done on the pathological tissue, and (5) a study from which information on women or never-smokers cannot be obtained, even if it was a case-control study.Two researchers applied the exclusion criteria and obtained HPV-related information from each study. ORs and 95% CIs were computed for the confirmed patient groups and positive HPV values. Heterogeneity was evaluated using the I-squared values (%), and when homogeneity was confirmed, a meta-analysis was performed to compute summary odds ratios (SORs) and 95% CIs using a fixed-effect model. Statistical significance was determined at the level of 5%, and the statistical program Stata version 14.0  was used to conduct the analyses.The SOR for lung cancer associated with HPV infection was 5.32 for the women and 4.78 for the never-smokers. These are at a level similar to SOR 5.67, which is an odds ratio computed for men and women together . ConsideThe four studies selected in this systematic review -32 were In contrast, via a manual search we were able to identify four studies that met the selection criteria of the systematic review by Zhai et al. , but werLung tissues are known to have the highest sensitivity for HPV DNA detection , and sixIn order to prove that a specific virus causes cancer, a case-control study must be conducted in order to meet necessary standards ,51. Howe


E2F1 increased survival of mice ubiquitously expressing HPV oncogenes. E2F1 similarly functioned as a tumor suppressor in HPV-positive oral tumors as tumors grew faster with homozygous loss of E2F1 compared to tumors with heterozygous loss of E2F1. Re-expression of E2F1 caused decreased clonogenicity in HPV-positive cancer cells. Our results indicate that HPV oncogenes activated the E2F1 pathway to cause lethality in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2F1 pathway, which is activated in HPV tumors, may facilitate tumor regression.The Human Papillomavirus (HPV) is associated with several human cancers, including head and neck squamous cell carcinomas (HNSCCs). HPV expresses the viral oncogene E7 that binds to the retinoblastoma protein (RB1) in order to activate the E2F pathway. RB1 can mediate contradictory pathways—cell growth and cell death via E2F family members. Here, we assessed the extent to which E2F1 mediates lethality of HPV oncogenes. Ubiquitous expression of the HPV oncogenes E6 and E7 caused lethality in mice that was associated with focal necrosis in hepatocytes and pancreatic tissues. Furthermore, all organs expressing HPV oncogenes displayed up-regulation of several E2F1 target genes. The E2F1 pathway mediated lethality in HPV-positive mice because deletion of  The Human Papillomavirus is associated with many epithelial cancers, including those afflicting the oropharynx and anogenital regions ,2,3. HPVRB1 makes mice prone to develop thyroid and pituitary tumors [RB1 results in embryonic lethality due to abnormal proliferation, apoptosis, and cellular differentiation [RB1 loss is mediated, in part, by E2F family members that regulate the transcription of genes involved in the cell proliferation and cell death [RB1 [Paradoxically, the RB-E2F1 pathway has been implicated both in control on proliferation and cell death. Heterozygous loss of y tumors ,8,9. By ntiation ,11. Embrll death . In partll death ,15,16,17ath [RB1 . By contath [RB1 . TherefoRB1 loss caused embryonic lethality, we hypothesized that HPV oncogenes may also cause lethality and inhibit tumor growth via an E7-RB1-E2F pathway [Previous reports have not described the lethality of HPV oncogenes in transgenic mice consistent with the innocuous HPV infections in humans. The majority of these HPV transgenic mouse models used constitutively active tissue-specific promoters including cytokeratin 14 and cytokeratin 10 that limited E6 and/or E7 expression to the skin which may better tolerate oncogene expression ,21,22. H pathway .E2F1 rescued HPV-induced lethality in adult mice. Furthermore, loss of E2F1 increased HPV-positive oral tumor growth that was associated with increased tumor cell proliferation. Together, our results indicated that the E2F1 pathway, which is controlled by the HPV E7 oncogene, inhibits cell proliferation causing lethality in HPV-positive mice and suppressing HPV-positive oral tumor growth.Here, we used a tamoxifen (TAM)-inducible HPV transgenic mouse model  to ubiquRB1 causes embryonic lethality [p < 0.001; Homozygous loss of ethality ,10,11. Sethality . Activatp < 0.001; + tissues caused embryonic lethality. By contrast, breeding iHPV mice to Albumin-Cre mice, GFAP-Cre mice, K14-Cre mice, or PDX1-Cre mice resulted in double-transgenic mice at expected Mendelian ratios. Therefore, different tissues have distinct sensitivity to HPV oncogenes resulting embryonic lethality when expressed ubiquitously or in Nestin+ tissues.Given that HPV infections are not associated overt skin toxicity ,5, the etam mice (RosaHPV) that expressed a TAM-regulated Cre recombinase driven by a ubiquitous Rosa promoter. Tamoxifen treatment of RosaHPV mice caused recombination of the LSL-E6E7 transgenes in the skin, spleen, liver, brain and pancreas . In addition, we bred Rosa-HPV mice to a 2C TCR transgenic mice [+/+, RosaHPV-Rag1+/− and RosaHPV-Rag1−/− mice , minichromosome maintenance complex component 2, 5, and 6 , ribonucleotide reductase M1 (RRM1) and ttk protein kinase (TTK). TAM treatment caused induction of E2F1 target genes in the skin, spleen, liver, and brain  Furthermore, fewer RosaHPV-E2F1−/− mice displayed gait abnormalities or paralysis compared to RosaHPV-E2F1+/− mice or RosaHPV-E2F1+/+ mice NatGt(ROSA)26Sor/J) [All mice were maintained under specific pathogen-free conditions and used according to protocols approved by The University of Chicago  Institutional Animal Care and Use Committee and Institutional Biosafety Committee (Protocol ACUP 72136). iHPV mice containing the Lox-Stop-Lox-HPV-Luc (LSL-E6E7) transgene  and KH mransgene  have beeT)Nat/J) , K14-CreT)Nat/J) , AlbuminT)Nat/J) , PDX1-CrT)Nat/J) , CMV-CreT)Nat/J) , Nestin-T)Nat/J)  and GFAPT)Nat/J)  were obtTamoxifen was purchased from Sigma . DMBA and TPA were dissolved in acetone. Tamoxifen was dissolved in sunflower seed oil at 20 mg/mL and 0.1 mL was injected intraperitoneally daily for five days. All oligonucleotides were synthesized by IDT.All PCR reactions were performed using 2× Go Taq Green Master Mix . For detection of LSL-E6E7 recombination, gDNA was isolated and the LSL-HPV transgene was amplified by PCR using the primers: forward primer: 5ʹ-CAACGTGGTTATTGTGCTG-3ʹ corresponding to the 3ʹ end of the CAG promoter and the reverse primer: 5ʹ-GGTAACTTTCTGGGTCGCTCCT-3ʹ corresponding to the 5ʹ region of the E6E7 gene. The 1629 bp amplicon represents the unexcised HPV vector and the 388 bp amplicon represents the excised iE6E7 gene.+/− and KHR-E2F1−/− oral tumors at the end of the experiment. Total RNA was isolated using TRIzol , and treated with DNase I. The cDNA was generated using ProtoScript® First Strand cDNA Synthesis Kit  and subjected to qPCR using SYBR Green Supermix . The endogenous GAPDH  gene was used as a reference gene. QPCR Primers: E6E7 primers (E6E7-F: 5ʹ-ATTTGCAACCAGAGACAACT and E6E7-R:5ʹ-ACAGGTCTTCCAAAGTACGA), Luc primers (Luc-F: 5ʹ-ACTGGGACGAAGACGAACAC and Luc- R: 5ʹ-GGCGACGTAATCCACGATCT), and GAPDH mouse primers (GAPDH-F: 5ʹ-AATGTGTCCGTCGTGGATCT and GAPDH-R: 5ʹGGTCCTCAGTGTAGCCCAAG). In addition, validated mouse primer pairs for E2F1, E2F2, E2F3, LIG1, Ttk6, MCM2, MCM5 MCM6, CCNE1, CCNE2, RRM2, and B2M were obtained from PrimePCRTM Assays . QPCR was carried out using iQ™ SYBR® Green Supermix with hot-start iTaq DNA polymerase and iQ™ 5 Multicolor Real-time QPCR Detection System Machine (Bio-rad). CT values for individual samples were subtracted from the corresponding CT values for GAPDH or B2m (∆∆CT). Normalized fold expression was obtained using the –log2∆∆CT formula.RNA was extracted from the indicated organs of Rosa-HPV mice 5d after treatment with TAM or vehicle or from KHR, KHR-E2F1The HPV-positive cell line HeLa was obtained from Kenneth Alexander (The University of Chicago) and was validated by IDEXX laboratories nine-loci STR testing. Cells were maintained in complete DMEM (cDMEM) with 10% fetal bovine serum plus L-glutamine and Penn/Strep at 37 °C. HeLa cells were plated in six-well plates and transfected with a bicistronic expression plasmid containing human E2F-1 and EGFP  or the empty plasmid containing EGFP using lipofectamine 2000. After 24 h, a 1000 cells were split into six-well plates and incubated for 13 days. After 13 days, the media was removed and washed with PBS. The cells were then fixed with methanol for 5 min and then stained with 1% crystal violet for 30 min. Colonies were counted and the experiment was performed in triplicate.in utero and in adult mice that is associated with necrosis in normal tissues without the development of neoplastic lesions. HPV oncogene lethality was mediated, in part, by the HPV oncogene E7 targeting E2F1, as loss of E2F1 reversed lethality in adult mice. Furthermore, loss of E2F1 potentiated HPV-positive oral tumor growth and overexpression of E2F1 decreased the clonogenicity of HPV-positive cancer cells. Understanding how to manipulate cell death pathways activated by HPV oncogenes via E2F1 may serve as novel approaches to treat head and neck cancers and other cancers in general.Here, we find that HPV oncogenes caused lethality


Neuropsychological batteries are infrequently used to assess cognitive impairment in multiple sclerosis because they are time-consuming and require trained personnel. The Symbol Digit Modalities Test (SDMT) is suggested to be a useful screening tool to measure cognitive impairment in multiple sclerosis patients and is more valid and reliable over time than the Paced Auditory Serial Addition Test (PASAT). The purpose of this study was to evaluate which of these tests was more sensitive to cognitive impairment at one-year follow-up.A total of 237 patients with relapsing-remitting multiple sclerosis and 57 healthy controls underwent a complete neuropsychological assessment. One year later, we assessed 196 patients using the Brief Repeatable Battery of Neuropsychological Tests. We also administered other executive function and prospective memory tests, together with fatigue and depression questionnaires.A total of 33.8% of patients were classified as cognitively impaired. The SDMT and the PASAT 3 seconds test (PASAT3) had a sensitivity of 0.809 and 0.783, respectively, thereby classifying patients as cognitively impaired. Analysis of 196 patients one year later showed 31.6% had cognitive impairment compared with 27.6% at the first assessment. The sensitivity to detect cognitive impairment after one year was 0.824 for SDMT and 0.796 for PASAT3. When the predictors were removed from the comparative standard battery, SDMT still showed a slightly higher sensitivity. Both SDMT and PASAT3 correlated significantly with all tests, but SDMT showed higher correlation values. Furthermore, SDMT was completed by all subjects while PASAT3 was completed by 86.9% of patients and 94.7% of controls.SDMT is simpler to administer than PASAT3 and may be slightly more sensitive to MS cognitive impairment. It could thus be a suitable test to assess cognitive impairment routinely in people with relapsing-remitting multiple sclerosis. Cognitive impairment (CI) in multiple sclerosis (MS) is common and disabling , affectiNeuropsychological batteries currently available to assess CI in MS patients are time-consuming and therefore are not administered to all patients. The Rao Brief Repeatable Neuropsychological Battery (BRNB) and the Minimal Assessment on Cognitive Function in MS (MACFIMS) are most commonly used and take 25–30 and 90 minutes, respectively, to administer ,5. OtherThe Symbol Digit Modalities Test (SDMT) has been proposed for the assessment of CI in MS. SDMT measures patient attention, concentration and speed of information processing . In a stAnother screening test proposed for use in MS is the Paced Auditory Serial Addition Test (PASAT), an auditory test that also measures working memory. It measures the same cognitive functions as SDMT. PASAT is part of the Multiple Sclerosis Functional Composite (MSFC), developed to measure impairment and disability in MS , and it In a recent study, Sonder et al.  reportedThe aim of our study was to evaluate and compare SDMT and PASAT3 to determine which was more sensitive for the detection of CI when compared with a complete neuropsychological evaluation in a group of patients with relapsing-remitting multiple sclerosis (RRMS) after one year of follow-up.Consecutive patients fulfilling McDonald’s criteria for MS  and atteTwo hundred and thirty-seven patients who met the inclusion criteria and 57 healthy controls recruited from friends and in-law relatives matched by age, gender, and education level agreed to participate in the study. Neuropsychological assessment with parallel forms of the tests was performed in one hundred and ninety-six patients one year later. All participants gave their written informed consent before entering the study. The study was approved by the ethics committee at the Hospital de la Santa Creu i Sant Pau and performed according to the Declaration of Helsinki.Neurological examination of all participants was performed by the same experienced MS neurologist. Relapses were evaluated and disability was assessed to determine the expanded disability status scale (EDSS) score .Neuropsychological examination was always performed by the same clinical neuropsychologist. This examination included the Spanish version of the Brief Repeatable Battery of Neuropsychological Tests (BRB-N) , which cWe used other tests to evaluate executive function: the Stroop Test , the WisIn accordance with previously published classifications ,26, cognWe analyzed differences between the control group and patients regarding demographic variables and cognitive assessment results using a T test for independent samples. The same test was also used to compare demographic characteristics of patients who completed the PASAT3 with those who did not.A Pearson correlation coefficient was used to study the correlation between SDMT and PASAT3 and also between these two tests and other cognitive variables. To avoid the influence of outliers, non-parametric correlations (Spearman correlation coefficient) were used. We applied a multiple linear regression to assess the influence of variables such as age, fatigue, and depression on the correlation between the SDMT and the PASAT3 and the other cognitive tests.A receiver operating characteristic (ROC) curve for SDMT and PASAT3 was also generated to obtain sensitivity and specificity values. The same procedure was followed to identify which test was more sensitive to detect changes in cognitive impairment one year later.Statistical analysis was performed using SPSS 22.0  and a p value <0.05 was considered statistically significant.The group included 237 RRMS patients and 57 healthy controls. Table The control group had significantly higher scores in almost all cognitive variables assessed in the study (p <0.05), except for the STROOP interference score and the perseverative and non-perseverative errors on the WCST. Scores of self-reported inventories of fatigue and depression were significantly higher in the patient group (p <0.001). Results are summarized in Table Thirty-one 13.1%) patients and 3 (5.3%) controls were not willing to perform PASAT3 whereas all participants completed the SDMT. Patients who did not complete the test were older and had fewer years of education. These patients had a longer duration of disease, were treated with disease modifying therapies for longer periods, and had higher EDSS scores. No statistically significant differences were observed regarding time since the last relapse. The Beck Depression Inventory score was higher in the group that did not complete PASAT3. No significant differences were observed in the Fatigue Severity Scale score. Table .1% patieThe Pearson correlation coefficient showed a positive correlation between PASAT3 and SDMT . Both tests showed a significant correlation with other neuropsychological tests. Nevertheless, although differences were not statistically significant, correlation values were higher for SDMT for almost all cognitive tests and for variables such as fatigue, depression, and EDSS.When controlling for age, fatigue, and depression, multiple linear regression showed that coefficients between SDMT and PASAT3 and other cognitive tests were generally lower, but again, most were higher for SDMT. All tests remained significant. Data are shown in Table In this sample, 33.8% of patients were classified as CI. According to the definition of CI, SDMT had a sensitivity of 0.809 and a specificity of 0.662, while PASAT3 had a sensitivity of 0.783 and a specificity of 0.637. The area under the curve was 0.811 for SDMT  and 0.761 for PASAT3 . A cut-off score of 49 for SDMT classified CI patients with an accuracy of 75%, compared with a cut-off score of 35 and an accuracy of 73% for PASAT3. Figure In the previous analysis, both SDMT and PASAT3 were part of the gold standard for detecting CI in MS patients. Therefore, we performed another analysis using the same criteria of failure in at least two tests but excluding SDMT and PASAT3. We found that 27.4% of patients were classified as CI. SDMT had a sensitivity of 0.779 and a specificity of 0.692, while PASAT3 had a sensitivity of 0.727 and a specificity of 0.585. The area under the curve was 0.809 for SDMT  and 0.700 for PASAT3 . A cut-off score of 49 for SDMT classified CI patients with an accuracy of 75%. A cut-off score of 35 for PASAT3 classified CI patients with an accuracy of 68%. Figure One hundred and ninety-six patients were assessed one year later. The proportion of CI patients in the baseline assessment in this group was 27.6%. At one-year follow-up, the proportion of CI subjects increased to 31.6% with the BRNB battery. SDMT had a sensitivity of 0.824 and a specificity of 0.741, while PASAT3 had a sensitivity of 0.796 and a specificity of 0.684. The area under the curve was 0.860 for SDMT  and 0.772 for PASAT3 . A cut-off score of 49 for SDMT classified CI patients with an accuracy of 80% compared with a cut-off score of 35 for PASAT3, which had an accuracy of 73%. Figure The same analysis was performed without SDMT and PASAT3 as part of the gold standard. According to these criteria, 25% of patients in the baseline assessment in this group were CI. At one-year follow-up, this percentage increased to 26%. SDMT had a sensitivity of 0.800 and a specificity of 0.706, while PASAT3 had a sensitivity of 0.738 and a specificity of 0.588. The area under the curve was 0.826 for SDMT  and 0.697 for PASAT3 . A cut-off score of 49 for SDMT classified CI patients with an accuracy of 77%, and a cut-off score of 35 for PASAT3 classified CI patients with an accuracy of 68%. Figure Fatigue and depression are common symptoms in MS that can affect cognitive function; therefore, we assessed the sensitivity and specificity of SDMT and PASAT taking into account these two factors. We found 50% of patients had symptoms of fatigue and 32.5% had symptoms of depression. When we compared the fatigue group (FSS >3) with the non-fatigue group, and the depressed group (BDI >11) with the non-depressed groups, the area under the curve was higher for patients without fatigue or depressive symptoms.A t-test for repeated measures between the first and the second evaluation showed that the mean score of SDMT was 54.5 at the first assessment and 55 at the second assessment. This difference was not significant (p = 0.308). The mean score of PASAT3 was 45.2 at the first assessment and 46.7 at the second. This was statistically significant (p = 0.011), suggesting a higher learning effect in PASAT3 than in SDMT.This study showed that both SDMT and PASAT3 could detect CI in a significant portion of MS patients but suggests that SDMT has a better ability to detect CI after a year of follow-up. We found significant correlations between PASAT3 and SDMT with measurements of memory and executive function. Correlation values were uniformly higher for SDMT, but were not statistically significant. Parmenter et al.  also repPrevious studies suggested the importance of comparing SDMT and PASAT and defining which of the two is more specific and sensitive as a measure for CI . AccordiOur finding that all patients completed SDMT but that 31 (13.1%) patients were not willing to perform PASAT3 is in agreement with a previous study that reported missing values for PASAT3  and anotWhen we compared factors related to the administration of SDMT and PASAT3, we found that SDMT was completed in a shorter time and that the examples and the instructions were more easily understood and followed by patients during the task. Furthermore, previous studies reported that only minimal training was needed to master the administration of SDMT  and thatWhen administering PASAT3, it was sometimes necessary to repeat the instructions more than once, making the test longer. Some patients decided not to continue the test as they complained of difficulties adding the numbers. An additional inconvenience of PASAT is that performance is affected by mathematical ability ,33.Previous studies showed that SDMT is sensitive for the detection of cognitive deterioration over time, has a moderated practice effect  and is aA limitation of our study is that the low conversion rate to CI in MS over a year might have influenced the ability of SDMT and PASAT3 to detect incident cases of CI. However, our study has several strengths. Both depression and fatigue were taken into account, factors that have not been assessed in previous studies but which may influence cognitive performance . FurtherWe found that even though the differences in sensitivity between SDMT and PASAT3 were minor, the differences were uniformly in the direction of SDMT having slightly better validity, both at baseline and 1-year follow-up. In addition, SDMT was easier and faster to administer compared with PASAT3, and had a greater correlation with other cognitive tests in the presence of fatigue or depression.


Human papillomaviruses (HPVs) infect stratified epithelium and are the causative agents of cervical cancer, the second most common cause of cancer-related death in women. A critical aspect that still persists in the HPV field is the selection of very sensitive and specific HPV diagnostic assays. Here, we provide evidence that the crucial small ubiquitin-like modifier (SUMO) E2-conjugating enzyme Ubc9 is strongly upregulated in cervical lesions. Ubc9 detection could thus be used in diagnosing and/or monitoring the progression of an HPV oncogenic infection. Human papillomaviruses (HPVs) are a group of small, double-stranded DNA viruses known to be the primary cause of cervical cancer. Evidence is now emerging on their role in the aetiology of head and neck and other cancers as well in both women and men.HPVs can be classified as low-risk and high-risk viruses. In the cervix, low-risk HPVs are associated with a spectrum of benign warts, whereas infections with high-risk HPVs, such as HPV16, are manifested by intraepithelial malignant progression caused by viral E6 and E7 . High-riRobust and convincing evidence now reinforces the acceptance of cervical cancer prevention approaches. The strategy is to strongly aim at and focus on persistent infection caused by HPV. Besides laboratory tests to detect HPV DNA, there are other promising markers of HPV oncogenic infection. These include the following: (i) markers of increased HPV mRNA and protein expression; (ii) markers of increased cell proliferation, such as Ki-67, MCM2, TOP2a, and p16INK4a; and (iii) markers of chromosomal instability, such as a gain of chromosome arm 3q and HPV DNA integration [Currently, the most promising candidate as a clinical biomarker is detection of over-expressed p16INK4a (p16) . p16 ovesquamous epithelial cell that has undergone a number of structural changes which occur as a result of infection of the cell by HPV [The most used HPV diagnosis is still based on cytology, since the virus clearly alters the infected cells generating a characteristic vacuolisation around nuclei. This hallmark phenomenon is called koilocytosis and indicates the presence of an HPV infection. A koilocyte is a l by HPV . Such mol by HPV . HoweverSumoylation is a post-translational modification where the small ubiquitin-like modifier (SUMO) proteins are reversibly attached to the protein target through an ubiquitin-like pathway, with many different outcomes on protein stability, interaction and localisation, DNA repair and replication, transcriptional regulation, cell cycle control, apoptosis, cell signalling, and viral replication –11. AmonOur laboratory has previously demonstrated that other viral proteins, such as the adenoviral protein Gam1, can dysregulate the SUMO pathway by interacting with cellular proteins –20. ReceWe thus asked whether we could assess differences in the expression of endogenous Ubc9 in HPV-positive cervical lesions from patients treated at the European Institute of Oncology .The present studies show the upregulation of the sumoylation machinery conjugating enzyme Ubc9 in cervical biopsies in a lesion-dependent manner. Our data pinpoint to the possible use of Ubc9 as a biomarker for HPV oncogenic infection. Indeed, Ubc9 allows precise, sensitive, and selective detection of the infection.www.graphpad.com). Values of P < 0.05 were considered significant.One-way analysis of variance (ANOVA) followed by the Bonferroni test was performed using GraphPad Prism version 5.00  according to established procedures. All sections were counterstained with Mayer’s haematoxylin and visualised using a bright field microscope. Images were generated with a BX51 Upright Microscopes from Olympus America Inc. The following primary antibody was used: anti-Ubc9  , 1:3,200, incubation O/N at 4°C. The Envision Kit from DAKO was utilised, and after the chromogenic visualisation step using the 3,3′-diaminobenzidine (DAB) chromogen, slides were counterstained with haematoxylin and coverslipped.We assessed by immunohistochemistry (IHC) analysis whether there was a differential expression of Ubc9 in different grades of cervical intraepithelial neoplasia (CIN), also referred to as HSILs  or LSILs ) from HPV-positive patients. Typically, CIN1 refers to LSILs, whereas CIN2 and CIN3 to HSILs.We analysed a total of approximately 130 patients, among which there were 54 LSILs and 78 HSILs . In someAs demonstrated by the representative IHC reported in Moreover, in some IHC experiments on FFPE cervical samples from the same cohort of patients, we also quantitated the percentage of Ubc9 positivity by computational analysis. As depicted in The concept of using Ubc9 and other components of the SUMO pathway as diagnostic markers has emerged from other studies as well.et al suggested that Ubc9 may play a role in tumorigenesis and tumour progression in head and neck squamous cell carcinoma (HNSCC) and could potentially be used as a molecular marker for head and neck cancer progression [Ronen gression .et al supported the use of promyelocyte protein-containing nuclear bodies and their association to SUMO-1, as a cytodiagnostic marker paralleling cervical cancer progression [Szendefi gression .et al. showed that expression of SENP1  directly correlated with prostate cancer aggressiveness and recurrence [Wang currence . Their ret al evaluated the contribution of Ubc9 to chemoresistance in breast cancer patients. The expression level of Ubc9 was determined by IHC: the proportion of Ubc9-positive cells was higher in invasive ductal carcinoma compared to normal breast tissues. Furthermore, besides poor clinical response to chemotherapy, high Ubc9 expression associated with poor differentiation, larger tumour size, advanced clinical stage, lymph node metastasis, basal-like phenotype and thus overall worse clinical prognosis [Chen rognosis .In the cohort of patients analysed, we also found increased Ubc9 expression in adenocarcinoma . Taken tThe precise diagnosis of LSILs and HSILs is the most important and key determinant to cervical cancer prevention. Today’s strategy implies that both the identification and the eradication of CIN2/3 help to prevent invasive cancer and to monitor LSILs towards clearance or possible progression to HSILs. The trigger to treatment is the histopathological diagnosis of CIN2/3, in which the present data are indicated as very sensitive to staining with an antibody that recognises the sumoylation pathway conjugating enzyme Ubc9. Furthermore, Ubc9 can also detect CIN1, thus making it a valuable marker to avoid overdiagnosis and therefore overtreatment.Cervical cancer prevention is a worldwide problem, especially increasing in developing countries. To increase the screening power, pathologists must be trained to spot CIN2/3 diagnosis, with no doubt. Thus, Ubc9 staining could be very helpful, and there is clearly a market potential to implement this idea. In conclusion, proteins involved in the sumoylation pathway, in particular Ubc9, could be exploited towards cervical cancer prevention.The authors declare that they have no conflict of interest.


Acquisition of human papillomavirus (HPV) in women occurs predominantly through vaginal sex. However, HPV has been detected in girls reporting no previous sex. We aimed to determine incidence and risk factors for HPV acquisition in girls who report no previous sex in Tanzania, a country with high HPV prevalence and cervical cancer incidence.We followed 503 adolescent girls aged 15–16 years in Mwanza, Tanzania, with face-to-face interviews and self-administered vaginal swabs every 3 months for 18 months; 397 girls reported no sex before enrollment or during follow-up; of whom, 120 were randomly selected. Samples from enrollment, 6-, 12-, and 18-month visits were tested for 37 HPV genotypes. Incidence, clearance, point prevalence, and duration of any HPV and genotype-specific infections were calculated and associated factors were evaluated.Of 120 girls who reported no previous sex, 119 were included, contributing 438 samples. HPV was detected in 51 (11.6%) samples. The overall incidence of new HPV infections was 29.4/100 person-years . The point prevalence of vaccine types HPV-6,-11,-16, and -18 was .9%, .9%, 2.0%, and 0%, respectively. Spending a night away from home and using the Internet were associated with incident HPV, and reporting having seen a pornographic movie was inversely associated with HPV incidence.Incident HPV infections were detected frequently in adolescent girls who reported no previous sex over 18 months. This is likely to reflect under-reporting of sex. A low-point prevalence of HPV genotypes in licensed vaccines was seen, indicating that vaccination of these girls might still be effective. This study demonstrated that some girls who reported never having had sex had incident vaginal HPV, likely due to under-reporting of sex. However, 98% had no evidence of HPV-16 or -18, suggesting that catch-up vaccination campaigns in girls older than current WHO-recommended target population (9–13 years) may be effective at reducing cervical cancer.Human papillomavirus (HPV) infects the mucosal surfaces of the female and male anogenital tract Tanzania has one of the highest rates of cervical cancer in the world and, to date, no national HPV vaccination program The cohort was enrolled between January and August 2012, as described previously During the study, samples were collected every 3 months for 18 months as described previously The Medical Research Coordinating Committee, Tanzania (Ref: NIMR/HQ/R.8a/Vol. IX/1249) and the London School of Hygiene and Tropical Medicine Ethics Committee (Ref: 6040) approved the study protocol. Because participants were younger than the age of legal consent, written informed consent was required from a parent/guardian before written participant assent. Girls with persistent HR HPV at study completion (defined as the same HR HPV genotype detected at two consecutive 6-month visits) were referred for follow-up with the National Cervical Screening Program.Dry vaginal swabs were stored in cryotubes immediately after collection and transported in cold boxes with ice packs before submission to the Mwanza Intervention Trials Unit Laboratory in Mwanza, where they were stored at −20°C until they were shipped to the Catalan Institute of Oncology in Barcelona. Samples from enrollment, month-6, -12, and -18 visits for girls included in this substudy were tested using the Linear Array HPV genotyping assay  which detects 13 HR and 24 low-risk (LR) genotypes Data were double entered into OpenClinica LLC , and analysis was performed using Stata version 10.3 .Reported sexual behaviors were tabulated in all girls who reported not having had sex and in those included in the present study. Genotype-specific incidence was calculated using person-time at risk from the date of enrollment. A new (incident) infection was defined as a first positive test for the specific HPV type among those not infected with that genotype at enrollment. Date of infection was assumed to be midway between the last negative and first positive sample. Girls who had missed visits or missing HPV results, which led to a gap in the observations, were censored at the last available result before the gap.Duration of infection was calculated from the date of infection to the date of clearance (assumed to be midway between the last positive and first negative sample for that genotype). Clearance of a genotype-specific infection was defined as a single negative sample for that genotype. Infections that were not cleared were censored at the date of the last positive sample. If there were two positive samples with one intervening missing sample , duration was considered unknown and censored at the date of the first positive sample.The overall incidence of all HPV infections was calculated as the number of new infections  over person-years at risk. Gaps (explained previously) where a visit was missed were removed, but girls were assumed at risk again after a gap. The overall incidence rate was estimated using Poisson regression with random effects to account for clustering of infections within the same girl. The HPV genotype–specific point prevalence was estimated as the number of visits where the genotype was detected, divided by the total number of visits, including enrollment.p < .10. Time-varying sociodemographic factors were added to this core model sequentially and retained if associated at p < .10. Time-varying behavioral factors were then added sequentially and retained if they remained associated at p < .10. This strategy allowed us to assess the effects of variables at each level of the framework, adjusted for more distal variables.Poisson regression with random effects was used to estimate rate ratios for factors associated with HPV incidence. A conceptual framework with three levels was used to build an adjusted model; age was considered an a priori confounder. Age-adjusted sociodemographic factors at enrollment were retained in a core model if associated with HPV infection at Study enrollment has been described previously At enrollment, 61 of the 119 (51.3%) girls were aged 16 years, and the remainder was aged 15 years. Approximately half (52.9%) lived in urban areas, 92.4% were Christian, 79.0% were in school, and 19.3% were neither working nor in school. The final study visit was attended by 90.8% of girls. At this visit, 108 girls were asked if they were circumcised , and none reported this. Girls contributed 438 valid specimens.Overall, 10 of the 119 girls (8.5%) who never reported having had sex had HPV detected at enrollment. The 119 girls contributed a total of 152.8 person-years (pys) of follow-up; during which, 44 new HPV infections were detected. The overall incidence of new HPV infections in girls who reported never having had sex was 29.4 per 100 pys  and for new HR HPV infections was 11.3/100 pys , followed by HPV59 (2.6/100 pys), and HPV-16, -51, -52, and -6  or having used the Internet  was associated with new HPV infection . There wA total of 119 Tanzanian adolescent girls who reported never having sex were followed for 18 months. Tanzania has a very high HPV prevalence and one of the world's highest incidences of cervical cancer A number of cross-sectional studies have found that cervical or vaginal HPV was detected in 0%–1.5% of self-reported virgins in Europe, Australia, and United States of America Intravaginal cleansing was associated with prevalent HPV in girls who reported never having had sex at the time of cohort enrollment We also observed a strong association between spending a night away from home in the past 6 months and HPV acquisition. Traveling or short-time migration has been associated with HIV infection in previous studies in sub-Saharan Africa One of the limitations of this study was that we only tested samples from girls every 6 months. Therefore, it is likely that HPV-incidence was underestimated because some short-lived infections may have cleared quickly between visits. This may have been partially mitigated by using the highly sensitive HPV Roche Linear Array, reducing the risk of false-negative tests. A further limitation of the study is the relatively small number of events in the risk factor analysis, which resulted in large standard errors and reduced power to detect associations, and made it difficult to adequately adjust for all potential confounders. The major strength of this study was the face-to-face interviews. These included current colloquial terms for sexual behaviors. The interviewers were experienced in sexual behavior research with adolescents and attempted to meet the same participant at every visit, establishing a trusting relationship and potentially reducing under-reporting. A nested study of alternative interview methods found no increase in reporting of sex. Unfortunately, interviews did not include questions about masturbation, which has previously been reported by adolescent girls in Tanzania Our results demonstrate that some Tanzanian adolescent girls who report never having had sex have a relatively high incidence of vaginal HPV. HPV-16 and -18 are responsible for >70% of cervical cancers, and currently available HPV vaccines offer a high level of protection from these genotypes