Today, most oropharyngeal squamous cell carcinoma (OSCC) is human papillomavirus (HPV) positive and HPV alone or in combination with p16 is reported to be a favorable prognostic factor for OSCC. Patients with tumors at other OSCC sites (OOSCC) are often included in the same treatment and study protocols as patients with tonsillar- and base of tongue SCC, even though the prevalence and clinical significance of HPV infection in OOSCC is unknown. Since tonsillar and base of tongue SSC cover roughly 90% of all OSCC, there is an obvious risk that there may be a misinterpretation of the results for OOSCC. Herein, we therefore study the prevalence of HPV and p16 and their impact on survival in OOSCC. A total of 69 patients were included in the study, and 61 were included in the survival analysis. HPV and p16 were present in only 17% (12/69) and 25% (17/69) of the OOSCC cases, respectively, while the majority 69% (48/69) was both HPV and p16 negative. Neither HPV nor p16 had predictive value for clinical outcome in OOSCC in this study. In conclusion, the prevalence of HPV and/or p16 is much lower in OOSCC compared to earlier reports including all OSCC, or tonsillar- and base of tongue cancer alone and HPV and p16 had no impact on clinical outcome in OSCC in this study. Our data highlight the diversity of head neck cancer sub-sites and the importance of taking OSCC sub-sites in consideration in future clinical trials and treatment. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide . DespiteINK4 (p16) is strongly associated to HPV-positive OSCC [Overexpression of the cyclin dependent kinase inhibitor 16, p16ive OSCC –17, and ive OSCC . p16 hasive OSCC . Even thive OSCC . It has ive OSCC .Nevertheless, studies of OSCC do not always recognize the complexity of the region. Tonsillar and base of tongue SSC, both originating of the Waldener's ring, cover roughly 90% of all OSCC . HoweverFor this purpose, we herein study the prevalence of HPV and p16 in the OOSCC sites separately, as well as their impact on patient survival.All patients diagnosed between 2000 and 2008, in the County of Stockholm, Sweden, with OSCC outside the tonsils and base of tongue (OOSCC), where pretreatment biopsies were available were included in this study. The International Classification of Diseases (ICD) system 10, where the oropharynx is subdivided into the anterior wall, that is, the base of tongue and vallecula; the lateral wall, that is, the tonsillar fossa; the posterior wall; the superior wall; and the soft palate, was used for diagnosis. OSCC C10.0–C10.9 and C50.1–C50.8 were included in the analysis. Sub-site C50.8 was analyzed to assess tumors from the soft palate; however, one patient was excluded due to the origin of the tumor in the hard palate. All patients' charts were analyzed, and age, gender, TNM-stage, treatment, and survival were recorded. Treatment was categorized as surgery, radiotherapy, including both external and interstitial (brachytherapy) radiation sources, with doses up to 68 Gray, or chemoradiotherapy. Patients were evaluated for disease progression every 3 months the first 3 years, then every 6 months for a total of 5 years. All patients were evaluated for a minimum of 3 years, and the follow-up period ranged from 1 to 122 months. For the survival analysis, age was categorized into two groups, either under or over the age of 67 (mean age for the whole group). Patients categorized as Stages I or II at time of diagnosis were pooled into one group and compared to patients categorized as Stages III and IV. The study was conducted according to ethical permissions 2005/431-31/4 and 2005/1330-32 and 2009/1278-31/4 from the Ethical Committee at Karolinska Institutet, Stockholm, Sweden.HPV-DNA was detected as previously , 6, 23. INK4a primary monoclonal mouse anti-human p16INK4a antibody . In brief, 4-μm sections of formalin-fixed and paraffin-embedded tissue were deparaffinized in xylene and re-hydrated in graded alcohol. Epitope retrieval in citrate buffer (pH 6) in micro-oven for 20 min and cooled on bench followed by peroxidase blocking (3% H2O2) for 10 min. Slides were then blocked with BSA followed by incubation with the primary monoclonal antibody over night (8°C). Secondary biotynilated anti-mouse antibody was added and was followed by a standard avidin-biotin-complex-PO technique using the VECTASTATIN® Elite® ABC kit . Slides were developed in DAB and counterstained in hematoxylin. p16 staining was graded on a four-tier scale . Samples with >75% p16-positive tumor cells were considered as p16 positive and notably for all tumors either >90% or <10% of the tumor cells were p16 positive.Immunohistochemistry (IHC) was performed with the p16http://faculty.vassar.edu/lowry/VassarStats.html).The Fischer's exact test was used to compare differences in tumor stage, gender, and treatment between HPV-positive, p16-positive, and negative samples. Kruskal–Wallis test was used to compare age in the different HPV and p16 groups. Disease-free survival was calculated from the end of treatment until any recurrence. Deaths from other causes or patients lost to follow-up were censored. Overall survival was calculated from date of diagnosis to death of any cause. The HPV-positive cases were compared to the HPV-negative cases and analyzed using the Kaplan–Meier method, and survival curves were compared by use of the log-rank test in the univariate analysis. A similar analysis was made comparing HPV-positive and p16-positive cases to the rest. In the multivariate analysis, a Cox proportional hazards model was used to adjust for all covariates in the univariate analysis. STATA was used for the survival analysis, whereas Fischer's exact test and Kruskal–Wallis test was calculated using VassarStat website  of the 75 patients with OOSCC, with correct diagnosis and available pretreatment biopsies were included in the HPV and p16 analysis. Patients and tumor characteristics are summarized in Of all tumors, only 17% (12/69) were HPV positive , and only 25% (17/69) were p16-positive . Of the There were no significant differences for the presence or absence of HPV or p16 between the different sub-sites . There wn = 11) and those where biopsies were not available for HPV analysis (n = 6). Of the six patients, where biopsies (four with C05 and two with C10) could not be obtained, three were treated with palliative intent. All patients with HPV-positive and p16-positive tumors were included in the analysis. The mean age in the group not available for HPV analysis was 64 years. Further characteristics of the patients and their tumors are listed in For the survival analysis, 61 OOSCC patients were available of the original 75 patients, after excluding those treated with palliative intent (The mean follow-up time was 34 months (range 1–122). In all, 49 patients died during follow-up, seven patients with HPV-positive cancer, and 42 with HPV-negative cancer. However, of these, three patients with HPV-positive cancer and five patients with HPV-negative cancer died free of disease. Four patients with HPV-positive tumors (33%) and 27 patients with HPV-negative tumors (55%) had recurrence.There was no prognostic significance for HPV  or HPV iIn this study, we analyzed OSCC outside the tonsillar fossa and base of tongue (OOSCC) for presence of HPV-DNA and expression of p16 and only found a minority (16%) to be HPV positive and 25% to be p16 positive. In addition, there was no complete correlation between HPV status and p16. Furthermore, in contrast to a clear positive impact of HPV and p16 on clinical outcome in tonsillar and base of tongue cancer, this was not the case for OOSCC.The low prevalence of HPV in OOSCC obtained in this study (16%) could be due to the fact that the epithelial tissue of OOSCC differs from that in tonsillar and base of tongue cancer. In addition, an even lower prevalence of HPV was observed when assessing tumors positive for both HPV and p16 (12%). Our data were well within the range of HPV prevalence in OOSCC reported earlier (0%–60%) –28 howevNotably, in this study, in the survival analysis including 61 patients, it was not obvious that the presence of HPV or p16 showed a clear-cut benefit for prognosis in OOSCC. This is in contrast to several previous studies that have shown that prognosis for OSCC patients with HPV-positive cancer is clearly better than for those with HPV-negative cancer independent of nodal status, age, stage, tumor differentiation, or gender , 21, 30.Also, in contrast to earlier data showing a very high correlation between presence of p16 and HPV in OSCC , we founThere are some limitations in our study and first of all this is a retrospective study with its potential bias involved. However, since OOSCC is a rare disease, it is difficult to study prospectively, and to our knowledge, this is the largest presented data on non-tonsillar, non-base of tongue OSCC. Nevertheless, there was still a limitation in the study sample size, which may explain the lack of significance in the survival analysis in the age and stage group. However, there was a clear trend toward a worse outcome for patients with a higher stage and age. Moreover, the quality of the data on smoking status and comorbidity was poor and inconsistent; thus, we were not able to adjust for these factors. Even though there are limitations in this analysis, there is still a marked difference in survival when compared to similar studies with similar sample sizes .The fact that the prevalence of HPV was lower than that in tonsillar and base of tongue cancer was not so controversial, since it was similar to that in other reports . Still, In summary, the prevalence of HPV and p16 is relatively low in OOSCC, and HPV and p16 could not be used here independently or in combination to show an impact on survival. In this limited cohort the correlation between HPV status and/or p16 in the tumors was not as strong as shown in previous studies including all OSCC or tonsillar SCC , 31–33.


ND4), mutated in most cases of the blinding disease Leber hereditary optic neuropathy (LHON), on the host mouse mitochondrial genome.To determine the effects of mitochondrial targeting sequence (MTS) modified AAV gene delivery of wild-type human NADH dehydrogenase subunit 4  directly to the mitochondria of the mouse retina. Control viruses consisting of scAAV lacking the COX8 targeting sequence and containing human ND4, or scAAV containing GFP, were also vitreally injected. Using next-generation sequencing of mitochondrial DNA extracted from the pooled mouse retinas of experimental and control eyes, we tested for the presence of the transferred human ND4, and any potential recombination of the transferred human ND4 gene with the endogenous host mitochondrial genome.We injected a modified self-complementary (sc) AAV vector, to which we appended the cytochrome oxidase subunit 8 (COX8) leader to one of the three capsid proteins (VP2) comprising the protein shell of the AAV virion, into the mouse vitreous to deliver the human ND4 DNA reads in mitochondrial samples of MTS AAV-ND4-injected eyes, a few human ND4 reads with AAV-ND4 lacking the MTS, and none with AAV-GFP injection. Putative chimeric read pairs at the 5′ or 3′ ends of human ND4 showed only vector sequences without the flanking mouse sequences expected with homologous recombination of human ND4 with the murine ND4. Examination of mouse mitochondrial ND4 sequences for evidence of intra-molecular small-scale homologous recombination events yielded no significant stretches greater than three to four nucleotides attributable to human ND4. Furthermore, in no instance did human ND4 insert into other non-homologous sites of the 16 kb host mtDNA.We found hundreds of human ND4 remains episomal in host mitochondria and is not disruptive to any of the endogenous mitochondrial genes of the host genome. Therefore, mitochondrial gene transfer with an MTS-AAV is non-mutagenic and likely to be safe if used to treat LHON patients with mutated ND4.Our findings suggest that human  The clinical features of Leber hereditary optic neuropathy (LHON), a degenerative vision disorder, were first described in 1871 . It is nND1 gene, or the T14484C mutation, which affects the ND6 gene. These three mutations are considered the primary causes of LHON, and each presents a significant risk of permanent visual loss. All are associated with focal degeneration of retinal ganglion cells (RGCs). Two of the primary LHON mutations, G3460A and T14484C, lead to an 80% reduction in NADH dehydrogenase activity in mitochondria isolated from platelets of patients [Most LHON cases are associated with mutations in one of three mitochondrial genes for subunits of NADH ubiquinone oxidoreductase, which is complex I of the mitochondrial respiratory chain ,4. This patients . Howeverpatients . With thpatients , there rpatients -13.ND4 DNA and the degraded capsid protein with GFP attached to the MTS enter the organelle through the import channels [ND4 gene in the mitochondrial genetic code into the organelle rescued the defective energy production of G11778A LHON hybrid cells.Therapies for LHON in common with all disorders caused by mutated mtDNA are inadequate, in large part because of the barrier in delivering DNA into the organelle. We have broken through this barrier . Our piochannels , resultichannels . This deND4 gene in the adult rodent visual system that prevented visual loss and atrophy of the optic nerve induced by coadministration of a second AAV containing the allotopic human mutant ND4 allele; i.e., the arginine to histidine transition at amino acid 340 (R340H). Allotopic R340H ND4 AAV injected into the vitreous of rodents was previously demonstrated by us to cause optic nerve swelling, followed later by atrophy of the nerve along with loss of RGCs [ND4 gene that, when exchanged for the wild-type ND4 in the scAAV construct and subsequently injected into the mouse vitreous, induced visual loss and optic atrophy [We then applied our MTS AAV technology to animals by testing for the presence and expression of the human  of RGCs . The sam of RGCs . In prio atrophy . These a atrophy .ND4-injected eyes suggested possible homologous recombination of human ND4 with the host mouse ND4 mtDNA. Here we used next-generation sequencing to test for the safety of MTS-AAV gene transfer by looking for any adverse recombination of the MTS AAV-transferred human ND4 with the host mitochondrial genome of the mouse.In our prior work with the MTS AAV , PCR of ND4 was fused in frame with FLAG and cloned into a self-complementary (sc) AAV backbone under the control of the mitochondrial heavy strand promoter (HSP) to give sc-HSP-ND4FLAG. VP2COX8GFP was generated by first linking the 23 amino acid MTS presequence of cytochrome oxidase subunit 8 (COX 8) to GFP and then inserting the whole cassette into the VP2 capsid of AAV at the Eag I site at residue 138. Self-complementary AAV2/sc-HSP-ND4FLAG or a control scAAV2-GFP was produced by the plasmid cotransfection method. The plasmids were amplified and purified by cesium chloride gradient centrifugation and then packaged with the VP2COX8GFP or standard VP2 plus VP1, VP3, and the helper plasmid PXX6 (threefold excess) into AAV2 recombinant virus by transfection into 293 cells as described previously [Human wild-type eviously . In brieND4FLAG , scAAV2/VP2ND4FLAG, (n=3 mice), or scAAV-GFP  was injected into the vitreous body of both eyes. Animals were euthanized nine days post-injection . Mitochondrial-enriched fractions were isolated from retinal tissues and used to extract DNA using the DNeasy blood and tissue kit  according to the manufacturer’s instructions as previously described [All animal procedures were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and the ARVO Statement for the use of Animals in Ophthalmic and Vision Research and were approved by the University of Miami IACUC. For the intraocular injection of recombinant AAV, three month old DBA/1J mice were sedated by inhalation with 1.5% to 2% isofluorane. A local anesthetic (proparacaine HCl) was applied topically to the cornea, and then a 32-gauge needle attached to a Hamilton syringe was inserted through the pars plana. One microliter of scAAV2/COX8VP2escribed . RetinalND4 AAV constructs. Human and mouse mitochondrial alignments were subsequently extracted from their respective BAM  files. To identify potential mitochondrial insertions of the construct, we first searched for evidence of paired-end reads, wherein one of the paired ends mapped to the construct sequence (human ND4 or elsewhere in construct) and the other pair was aligned within the mitochondrial genome of the mouse. Using BWA mapping results from both the human and mouse alignments, along with custom PERL scripting, we identified all putative instances of chimeric mapping events for additional manual evaluation of possible microscale homologous recombination events between the transfected human construct and mouse mitochondria. For these analyses, it was only possible to assess microscale recombination events at regions of homology between the construct and the mouse mitochondria, such as the ND4 gene. As described above, BWA alignment was first performed targeting the mouse mm9 genome. All mitochondrial alignments were subsequently extracted from the resulting BAM files. At each homologous position where the human and mouse bases differed , we examined the Illumina sequence data for instances where the mouse mitochondrial sequence matched the human. Since, by chance alone, any sequencing error or mutation on the mouse mitochondria had a 1/3 chance of matching the corresponding human base, only stretches of multiple human bases were taken as evidence for putative homologous recombination events and selected for further manual examination.Library construction was performed according to the Illumina ® TruSeq™ DNA Sample Preparation Guide . The final median insert size was 250–300 bp. Samples were bar-coded to allow for multiplexing. Cluster generation took place on the Illumina cBot according to the manufacturer’s recommendations. Sequencing occurred on the Illumina HiSeq2000 using the reagents provided in the Illumina TruSeq PE Cluster Kit v3 and the TruSeq SBS Kit – HS (200 cycle) kit. Sequence alignment was performed with the Burroughs-Wheeler Aligner  with default parameter settings. Two 100 bp paired-end reads from the Illumina Hiseq2000 were separately aligned to the human (hg19) and mouse (mm9) genomes, as well as the ND4 to mitochondria, then we should be able to detect human ND4 DNA in infected mouse retinas. Indeed, we found 579 sequencing reads (100 bp long) spanning the entire 1377 nucleotides of human ND4 in the mouse retinal samples injected with the MTS AAV containing human ND4 , which typically delivers its DNA to the nucleus . Looking at the ratio of the entire mitochondrial genome reads to the nuclear genome reads of the mouse, ratios were 0.00963 for the MTS AAV-ND4 relative to 0.01274 for the untargeted AAV-ND4; However, the ratio for the AAV-GFP injected eyes was 0.005665. Relative to the AAV-GFP control, mitochondrial/nuclear read ratios increased 170% for MTS AAV-ND4 and 225% for untargeted AAV-ND4. Taken together, we found no evidence for mitochondrial DNA depletion at 9 days induced by the presence of episomal human AAV-ND4 in retinal mitochondria.As our findings indicated human ND4 remains episomal in mitochondria of the mouse retina. Despite the mixing of human and mouse mitochondrial genes in RGCs, we found no evidence for homologous recombination of transferred human ND4 with the host ND4 using highly sensitive next-generation sequencing technology. Our previous PCR data had shown sequences of the histidine tRNA at the 3′ end of human ND4 that suggested homologous recombination [ND4, we found only the adjacent FLAG epitope and vector sequences and not the histidine tRNA that is adjacent to the 3′ end of murine ND4 in the host mitochondrial genome. Similarly, at the other end adjacent to the start methionine of human ND4, we found only vector sequences and not the ND4L gene adjacent to the start methionine of mouse ND4. Therefore, human ND4 did not replace mouse ND4 of the host mitochondrial genome. Replication of the human ND4 DNA delivered by our MTS-AAV may have occurred by the GC rich region of the inverted terminal repeat (iTR) of the AAV vector that is highly homologous to the GC rich control sequence box 2 (CSB2) in the origin of replication region of the host mitochondrial genome.Our results here suggest that the MTS AAV-transferred human bination . HoweverND4 were sequenced in the mouse ND4 samples obtained from MTS AAV-ND4-injected eyes, but not with standard untargeted AAV-ND4 or AAV-GFP. Such events, if frequent, could potentially result in mutated host ND4 proteins. Fortunately, this is unlikely, as these small-scale recombinations were seen in only a few reads out of several thousand. Still, AAV serotype 2 (to which we attached the MTS and injected into the vitreous cavity) is directed primarily to RGCs, the cell type affected in LHON [ND4 reads relative to total murine ND4 reads. In that context, small-scale homologous recombinations could exert a deleterious effect as demonstrated by Fan et al. [ND4 nucleotides into the mouse ND4, finding only non-synonymous amino acid changes that are of no known pathogenicity.As exchanges of mitochondrial DNA between different plant species, some animals, and (very rarely) in humans have been reported -26, we l in LHON . It shou in LHON . Therefon et al. , who fouND4 into non-homologous sites that could have disrupted mitochondrial protein synthesis and oxidative phosphorylation. In our study, we used 3 month old mice that showed no evidence of mitochondrial deletions characteristically seen in cells and tissues of older mammals including humans [ND4 into the mouse mitochondrial genome at non-homologous sites [ND4 is nonpathogenic to host mitochondria, and supports our previous studies that indicate an MTS AAV-ND4 can be used to rescue visual loss and optic neuropathy induced by the mutant ND4 allele responsible for most cases of LHON [Finally, sequencing of the entire mouse mitochondrial genome failed to reveal insertions of the human g humans -31. Suchus sites . Taken tus sites . Moreove of LHON . It rema of LHON , occurs  of LHON  or perha of LHON , then a


HPV16 variants correlate with geographic origin and ethnicity. The association between infection with a specific variant and the cervical disease risk remains unclear. We studied the prevalence, persistence and association with cervical intraepithelial neoplasia (CIN) of different HPV16 variants, using cervical swabs and whole tissue sections (WTS) of biopsies from 548 women in the placebo group of a HPV16/18 vaccine trial. In HPV16-positive samples, HPV16 variants were identified by a reverse hybridization assay (RHA). Laser-capture micro-dissection (LCM) was performed for localized detection of HPV. HPV16 variants were determined in 47 women. Frequency of mixed HPV16 variant infections was lower (8.5%) than for multiple HPV genotypes (39.1%). Among 35 women having consecutive HPV16 variant-positive swabs, 32 (91.4%) had the same variant while in three (8.6%) women a change in variant(s) was observed. HPV16-positive WTS were obtained from 12 women having consecutive HPV16 variant-positive swabs. The same variant was present in WTS of 10 women, while two were negative. WTS of five women were histologically normal. A single HPV16 variant was detected in four women having CIN1-3, while additional HPV genotypes were found in three other women having CIN2 and CIN3. In the WTS of one woman with mixed genotypes, the HPV16 variant was assigned to a CIN2 lesion by LCM. HPV16 variant infections can be effectively studied in cervical swabs and tissue specimens by the HPV16 variant RHA. Multiple HPV16 variants in one woman are rare. The HPV16 genotype consistently detected in follow-up samples usually involves a persistent infection with the same variant. Human papillomavirus (HPV) is a DNA virus that infects cutaneous and mucosal epithelium and induces epithelial proliferation. More than 40 HPV genotypes have been detected in the anogenital region, and clinically most important are the oncogenic (high-risk) HPV genotypes  which are involved in the development of high grade cervical intraepithelial neoplasias (CIN) and cervical cancer , and perDiagnosis of HPV infections is based on detection of its genomic DNA in cervical cell samples or cervical biopsy specimens by molecular methods, such as liquid hybridization  ,11 or thHPV comprises at least 189 distinct genotypes . By defiHPV16 has been studied more extensively than other genotypes, and most information about viral variants is available from the E6, L1 and LCR regions of the viral genome . The avaHPV16 subtypes and variants have been found to correlate with geographic origin and ethnicity of the infected patients. Sequence variation in the E6 region permitted discrimination between several subtypes, which have been designated as European prototype (E-P), African (with Af1 and Af2 as variants), Asian (As), North American (NA) and Asian American (AA) ,20,22. TSeveral studies have investigated the clinical and epidemiological relevance of HPV subtypes and variants. The association between infection with a specific subtype or variant of HPV (mainly HPV16) and the risk of cervical disease remains unclear. Some studies have reported a higher risk for specific variants, whereas other studies did not find any significant association -37. In sThe aim of the present study was to identify the different variants of HPV16 in a large group of women participating in a HPV16/18 vaccine efficacy study who were assigned to the placebo group. The prevalence, persistence and association with cervical intraepithelial neoplasia (CIN) by different HPV16 variants were investigated in follow-up cervical swab and tissue material from the placebo group.Cervarix®, GlaxoSmithKline Vaccines, Rixensart, Belgium) [HPV16-positive cervical swab and tissue specimens were selected for HPV16 variant analysis. These materials were available from the placebo group of women participating in a previous randomized efficacy trial of an HPV16/18 AS04-adjuvanted virus-like particle vaccine (Belgium) . Only maBelgium) ,39. BrieBelgium) ,39.Women who had entered the vaccine trial were aged 15–25 years and were cytologically negative, seronegative for HPV16 and 18 antibodies by ELISA, and HPV-DNA-negative by PCR for 14 oncogenic HPV types  no more than 90 days before study entry. Health-care providers obtained cervical swab specimens for cytology and HPV DNA testing at screening and months 6, 12, and 18. At months 0 and 6, and subsequently every 3 months, women self-obtained cervicovaginal samples with two sequential swabs for HPV DNA testing, for up to 27 months . CervicaAt the start of the current study for HPV16 variants, a subset of 5,144 cervical swab samples from 548 women assigned to the placebo group  had beenhttp://clinicaltrials.gov under study numbers NCT00689741 and NCT00120848. The protocol, consent forms and amendments were approved by the central review board, as well as 32 institutional review boards at study sites in Canada, USA and Brazil, as already described by Harper et al ,39. WomeDNA was isolated from 200 μl of cervical swabs in PreservCyt suspension by the MagNA Pure LC instrument , using the MagNA Pure LC Total Nucleic Acid isolation kit (Roche Diagnostics) according to the manufacturer’s instructions. DNA was eluted in 100 μl of elution buffer and 10 µl was used for each PCR reaction. Cervical tissue materials were processed according to a standardized sandwich method . BrieflyThe stained slides were used for histopathological assessment by an expert pathologist. The three intermediate sections were digested overnight by 100 µl of proteinase K lysis buffer at 56°C. Proteinase K was heat-inactivated for 20 minutes at 95 °C. Ten µl of extracted DNA was used for each PCR reaction. If necessary, extracted DNA was diluted ten times prior to PCR to circumvent inhibition of amplification.Cervical tissue from one woman was processed by LCM technology for localized HPV analysis. The H&E- and p16-stained whole tissue sections (WTS) were scanned using digital microscopy . Separate, small regions of (borderline) CIN1 and CIN2 lesions as well as normal cervical epithelium were assigned by a pathologist, generally in the first WTS stained by H&E (Section 1). At least one region was selected per lesion grade, obtaining at least 5% of the complete lesion area. The selected areas were excised using the Zeiss P.A.L.M. microbeam ultraviolet (UV) laser microdissection and catapulting system and transferred to an AdhesiveCap500 opaque tube (Zeiss) . LCM was10 PCR primer set to amplify a broad spectrum of HPV genotypes, as described earlier [10 amplimers were used to identify the HPV genotype by reverse hybridization to the LiPA25 genotyping strip . This line probe assay contains probes for 25 different HPV genotypes, i.e. HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70 and 74. Cervical swab and (micro-)dissected tissue samples were tested by the SPF earlier ,42. Brie2 concentration was increased from 2 mM to 2.5 mM, as well as the amount of each primer from 15 to 20 pmol. Furthermore, the annealing temperature was raised from 52 to 55 °C. The amplimers were cleaned-up by ExoSAP-IT reagent and analysed by reverse hybridization on the HPV16 variant strip according to the manufacturer’s instructions.Identification of the HPV variant in HPV16-positive swabs was performed using the E6-based HPV16 variant reverse hybridization assay , as described previously . The HPV The baseline prevalence and distribution of HPV16 variants and presence of mixed variant infections was determined in the first HPV16-positive event of each woman, regardless if the sample was self-obtained or collected by the health-care provider. Three definitions for persistence of HPV16 variant infection detected at consecutive time-points were applied. The HPV16 variant infection was considered persistent if the same variant was detected in cervical samples over a minimum of 10 months (12-month definition), 5 months (6-month definition) or 2.5 months (3-month definition).Only materials from women in the placebo group were selected to rule out influence of the vaccine on the natural history of HPV16 variant infections. At the start of the current study, 5,144 cervical samples from women in the placebo group (n=548)  had been10 PCR/DEIA, and were genotyped by LiPA25. The most prevalent genotypes among HPV-positive swabs were HPV52 (15.4%), 16 (13.5%), 51 (11.5%), 53 (9.5%) and 66 (7.8%). Among the LiPA25-positive samples, 60.9% contained a single genotype, and the remaining 39.1% contained 2 to 7 HPV genotypes.In this subset of 5,144 swabs from the total study population, 1,595 (31.0%) samples were found HPV-positive by SPFSubsequently, we selected 61 women from the placebo group who had at least one HPV16-positive cervical swab during the study period. A total of 207 HPV16-positive swab specimens were available for variant analysis. Among the 207 HPV16-positive swabs from 61 women, 136 (65.7%) were positive and 71 (34.3%) were negative by the HPV16 variant RHA. In total, 47 women tested positive for a HPV16 variant in one or more swabs. In fourteen women, the variant could not be identified in any of their HPV16-positive swabs. All these women had been HPV16-positive by LiPA25 at only a single time point during follow-up. This suggests that these women experience transient HPV16 infections with low viral loads, below the detection limit of the HPV16 variant RHA.The distribution of HPV16 variants found at baseline among 47 women is shown in Among the women with a single HPV16 variant (91.5%), European variants were the most prevalent (76.6%). African (8.5%) and North American/Asian-American variants (6.4%) were less common in this population.Among 35 out of the 47 women that were positive for a HPV16 variant (74.5%), the variant was identified at multiple time points during follow-up. In this group, the dynamics of the HPV16 variant infection was investigated over time. The women in this group had an average total of 10.4 swabs (range 4-13) per individual, obtained during an average follow-up period of 23.1 months (range 9-27) at 3-6 month intervals.In 32 of the 35 women (91.4%), no HPV16 variant changes were observed throughout the follow-up period. Among these 32 women, variant ‘persistence’ was observed for at least 12 months in 10 women (31.3%), for 6 months in 14 women (43.8%) and for 3 months in 7 women (21.9%). In one woman (3.1%), the interval between two HPV16 Eu 350T-positive swabs was over 12 months, but without any HPV16-positive swabs in between. Thirty women (93.8%) were infected with a single HPV16 variant and two women (6.2%) had multiple variants. In 3 women, a change in HPV16 variants was observed during follow-up. In 2 of 35 women (5.7%), a mixture of HPV16 variants was observed in the first positive sample, and the composition of this mixture changed over time. A mixed infection with two European variants (Eu 350T and Eu 350G) progressed towards a single infection (Eu 350T). In the second woman, a mixed infection of Eu 350T and Af2 resolved to a single infection (Af2). In addition, 1 of 35 women (2.9%) was initially positive for Eu 350G and cleared this infection. After a HPV16-negative period of six months she was positive for a different European variant, Eu 131G. 10 LiPA25 and were on average obtained 22 months after the baseline swab (range 8-39 months). For eight women, tissue specimens were collected between two HPV16 variant-positive swabs. From the remaining four women the tissue sample was taken after the last available HPV16 variant-positive swab. From 12 women in the placebo group who had a HPV16 variant identified in a cervical swab, cervical tissue specimens were also collected. All specimens tested positive for HPV16 by SPFIn 10 of 12 women (83.3%), the HPV16 variant was identified in the processed whole tissue section (WTS), i.e. Eu 131G (n=4), Eu 350T (n=4), and Eu 350G (n=2), while two women (16.7%) remained negative by the HPV16 variant RHA. In all cases, the variant identified in the WTS matched the variant found in the swabs taken before, and -if available- after the biopsy was obtained.The HPV16-positive WTS of the 12 women had been histopathologically examined for the worst lesion according to the CIN  classification system. Five individuals had normal cervical tissue and the specific HPV16 variant, i.e., Eu 131G, could be determined in three of them. Seven women were diagnosed with lesions graded as either CIN1 (n=1), CIN2 (n=4) and CIN3 (n=2). For 4 of the 7 women with a CIN lesion, the WTS was single positive for HPV16, i.e., Eu 350T (n=3) and Eu 350G. No other HPV genotypes were detected by the LiPA25 algorithm. This strongly suggests that HPV16 most likely caused the lesion in these women. The other three women had at least one other HPV type in addition to HPV16  in the WTS . An example of persistence of the same HPV16 infection is shown in The tissue sections of one of these three women were available for localized HPV analysis by LCM technology, in an attempt to assign a specific HPV type to the lesion. Cervical tissue (CIN3) of this woman had been embedded in four blocks. Each block was processed by the sandwich method and macro-dissected into a total of 11 areas for HPV analysis. HPV16 (Eu 131G), 18, and 39 were detected in these areas, often as multiple infections. Three macro-dissected tissue sections could be examined by LCM. In total, 11 regions were assigned by a pathologist, excised by LCM, and analysed for HPV by the LiPA25 algorithm  in HPV16-positive women, who were assigned to the placebo group of an HPV16/18 vaccine trial. The previously evaluated HPV16 variant RHA  proved tRemarkably, the frequency of HPV16 positive swabs containing multiple HPV16 variants was very low, and this is in contrast to the frequency of samples containing multiple genotypes. The reason for this is unknown, but one could speculate that infection with a certain genotype is protective against super- or co-infection with another variant of that same genotype. Also, other studies investigating HPV variants reported surprisingly few mixed variant infections -46. ThisEuropean HPV16 variants were highly prevalent in cervical swabs of our study population (76.6%), while African (8.5%) and North American/Asian-American variants (6.4%) were less common. In women with biopsy-confirmed CIN, only European variants were observed. The sample size of women with CIN is too small to investigate associations between specific variants and risk for CIN. The high prevalence of European HPV16 variants could be explained by the large proportion of women of European descent in this population, as suggested by the recruitment site  and ethnicity of recruited women . Our findings are consistent with previous work by Zuna et al, who found high prevalence of European HPV16 variants (86%) among HPV16-positive women in the USA . In most women, consecutive HPV16-positive swabs consistently contained the same variant. Variant changes were rarely observed in successive HPV16-positive swabs. In two women, a ‘persistent’ mixed HPV16 variant infection resolved to a single variant infection. Perhaps the immune response was able to clear one particular variant infection, but not the other. Another woman cleared a HPV16 variant but was infected with a different variant after six months. The primary infection did not induce a protective effect against the secondary infection, or perhaps the immune system was suppressed.Data from our study show that most HPV16-positive swabs contain the same HPV16 variant over time, which provides further evidence for a persistent infection. From a group of women with multiple HPV16-positive swabs, also HPV16-positive cervical biopsy specimens were available. In 83.3% of these women, the same HPV16 variant was identified in swab samples as well as tissue specimens, which often contained CIN. The HPV16 variant was not identified in tissue samples of the other women (16.7%). This could indicate a slightly lower sensitivity of the HPV16 variant RHA compared to LiPA25 in FFPE tissue specimens. Although the number of samples analysed was small, these findings suggest that a HPV16 genotype consistently detected in follow-up cervical swabs and tissue samples usually involves a persistent infection with the same variant. A persistent infection with the same oncogenic HPV type, most notably HPV16, increases the risk for the development of (pre)cancerous lesions in the cervical epithelium, whereas re-infection with HPV16 after previous clearing probably is not associated with an increased risk.Cervical tissues were further investigated if the persistent HPV16 variant infection was causative for development of CIN lesions. HPV16 was often found as the single genotype in whole-tissue sections of specimens with a diagnosed CIN lesion. This indicates that HPV16 was driving the lesion. In tissue of one woman, LCM technology was a suitable technique to assign causality of a CIN lesion to HPV16, when also other genotypes were present in the complete tissue sample. This study had limitations. Only a subset of clinical materials available from the previously described vaccine study group  was analIn summary, the HPV16 variant-specific reverse hybridisation assay can be used to investigate prevalence of single and multiple HPV16 variants, to survey the dynamic behaviour of mixed variant infections in consecutive cervical swabs, to monitor persistence of a particular variant, and its association with cervical precancerous lesions. The HPV16 genotype consistently detected in follow-up cervical swabs and tissue samples usually involves a persistent infection with the same variant.


C. trachomatis infection.Vaginal pH is related to genital tract inflammation and changes in the bacterial flora, both suggested cofactors for persistence of human papillomavirus (HPV) infection. To evaluate the relationship between vaginal pH and HPV, we analyzed data from our large population-based study in Guanacaste, Costa Rica. We examined vaginal pH and the risk of HPV infection, cytological abnormalities, and C. trachomatis infection was assessed by logistic regression. Results were stratified by age at visit.Our study included 9,165 women aged 18-97 at enrollment with a total of 28,915 visits (mean length of follow-up = 3.4 years). Generalized estimating equations were used to evaluate the relationship between vaginal pH and HPV infection  and low-grade squamous intraepithelial lesions (LSIL), the cytomorphic manifestation of HPV infection. The relationship between enrollment vaginal pH and C. trachomatis DNA was associated with increased vaginal pH in women < 25 years (OR 2.2 95% CI 1.0-5.0).Detection of HPV was positively associated with vaginal pH, mainly in women < 35 years . Elevated vaginal pH was associated with 30% greater risk of infection with multiple HPV types and with LSIL, predominantly in women younger than 35 and 65+ years of age. Detection of Our findings suggest a possible association of the cervical microenvironment as a modifier of HPV natural history in the development of cervical precancer and cancer. Future research should include studies of vaginal pH in a more complex assessment of hormonal changes and the cervicovaginal microbiome as they relate to the natural history of cervical neoplasia. Lactobacilli are key components of the vaginal defense system [C. trachomatis, trichomoniasis [The development of cervical cancer is linked to persistent infection with at least one of about a dozen carcinogenic genotypes of human papillomavirus (HPV). Most HPe system . Changese system ,10 whilemoniasis , urinarymoniasis , and mycmoniasis . TherefoLactobacillus species that dominate the vaginal microflora and produce lactic acid from anaerobic glycolysis [Lactobacilli in the vaginal mucosa [Vaginal pH reflects a combination of factors affecting the vaginal microenvironment. In healthy, reproductive aged women, vaginal pH is primarily determined by the lactic acid produced by the metabolically active epithelium and from ycolysis . Both prycolysis . A pH raycolysis . After ml mucosa . It has l mucosa .C. trachomatis DNA measured from cervical cells obtained in a random subset of cohort members at enrollment.Our study examined the relationship between vaginal pH and risk of HPV infection and HPV-related cytological abnormalities, with particular attention to possible differences between women in varying age groups in a large screening study of HPV infection and cervical neoplasia in Guanacaste Province, Costa Rica. In addition, we evaluated whether there is an association between increased vaginal pH and Vaginal pH was measured during enrollment and follow-up screening visits of a population-based cohort study of cervical neoplasia in Guanacaste, Costa Rica. Detailed methods of recruitment, screening, and follow-up have been reported elsewhere ,16. BrieExfoliated cervical cells for split-sample conventional Pap smear and ThinPrep cytology  were collected using a Cervex broom-like collection device  during the pelvic exam. Additional cervical cells were collected using a Dacron swab and stored in Specimen Transport medium  for HPV testing. Cervical abnormalities were identified by visual inspection, cytology, or cervicography. Cervical cytologic abnormalities were classified as cancer and/or high-grade squamous intraepithelial lesions (HSIL+), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells of undetermined significance (ASCUS), reactive changes, or normal. As the current analysis focused on HPV-related cytomorphologic changes, a woman's cytology was interpreted as LSIL if her conventional smear and/or ThinPrep results met the criteria for LSIL and the diagnosis of the alternative method was either ASCUS or LSIL.Specimens from the enrollment visit were tested initially in the United States by the Hybrid Capture Tube test . However, because this method had limited sensitivity (10 pg/mL) and only detected 11 of the carcinogenic HPV types, all specimens were retested using a polymerase chain reaction (PCR) test. As previously described ,18, DNA Vaginal pH was measured with a pHydrion strip . After insertion of a sterile speculum the pHydrion strip was placed on the left lateral vaginal wall between the speculum blades until moistened and color change was immediately compared with the colorimetric scale and the measurement was recorded across a range of 3.0-5.5 in increments of 0.5 pH units.C. trachomatis was measured with an assay for C. trachomatis DNA as previously described [C. trachomatis DNA was detected in enrollment samples using a C. trachomatis PCR-DEIA assay . After the PCR, C. trachomatis DNA was detected by C. trachomatis--specific probe hybridization in a DNA enzyme immunoassay that used 10 μL of the PCR product. The mixture of probes present in the C. trachomatis DNA enzyme immunoassay can recognize all C. trachomatis serovars and genovariants that have been deposited in GenBank.Detection of escribed  in an agThe unit of analysis was the study visit. Based on their age at each visit, women were stratified into the following categories: < 25 years, 25-34 years, 35-44 years, 45-54 years, 55-64 years and 65+ years. To standardize frequency of visits by time, each clinic visit was assigned to a bin defined by years after enrollment. Bins for each woman began with bin 0 (enrollment date), continued for every year until they were censored  or they were lost to follow-up. If a woman was tested more than once in a bin and her HPV results were discordant on positivity, the result from the positive visit was taken to acknowledge the possibility of HPV measurement error. Similarly, if pH was tested more than once in a bin, the visit with the highest vaginal pH recorded was chosen. Regarding cytology, if more than one result was available, the more severe reading was chosen.Our main analyses focused on risk of HPV positivity with vaginal pH as the primary independent variable. We calculated the odds of testing HPV positive vs. testing negative and the odds of testing positive for multiple HPV types vs. a single HPV type. For all of the above-mentioned analyses, we used the method of generalized estimating equations (GEE) with an independent correlation structure . This apC. trachomatis at enrollment. Analyses were performed using Stata 11.0 analytic software .When appropriate to increase statistical power and stability of estimates, we included vaginal pH as a dichotomous variable (pH < 5.0 and pH ≥ 5.0). We used logistic regression to examine the association between vaginal pH and Nine thousand one hundred sixty-five women were included in this study, with a total of 28,915 visit data points. Overall, the mean age at the time of visit was 41.8 ± 15 years (range 18-100 years).Ordinal logistic regression analysis revealed a significant relationship between vaginal pH and age at visit (OR 1.6 95% CI 1.6-1.6). Figure The prevalence of HPV was highest in the 25-34 year age group and decreased somewhat steadily among the older age groups, with a slight increase in women aged 65+ years in all visit data (data not shown).Detection of HPV was positively associated with vaginal pH in women in all age groups under age 55 with about a 10-20% increased risk of HPV detection for a vaginal pH ≥ 5  and detection of more than one HPV type among all women (HPV positive and negative)  when adjusted for age at visit Table . Age-strWhen adjusted for age at visit, a vaginal pH of ≥ 5.0 was positively associated with a diagnosis of LSIL vs. normal cytology including reactive changes  .C. trachomatis DNA detected in cervical exfoliated cells and vaginal pH at enrollment in the subgroup of women previously described. Among women < 25 years, we found a positive association between vaginal pH and presence of C. trachomatis DNA (OR 2.2 95% CI 1.0-5.0), with women with a pH of > 5.0 (n = 37) more likely to have a C. trachomatis infection (32.4%) versus women with a pH of < 5.0 (n = 135), of whom only 17.8% were positive (data not shown). This positive relationship was present among women younger than 45 years, however in women aged 45+, an inverse trend was observed, though these results were not significant.To assess whether vaginal pH could reflect (or predict) the presence of other sexually transmitted infections, we analyzed the relationship between the presence of We report a positive association between vaginal pH and HPV positivity in a very large cohort of randomly selected women. The relationship between HPV and vaginal pH was particularly pronounced in pre-menopausal women (< 25 and 25-34 years). The presence of multiple HPV type infections was also significantly related to elevated vaginal pH overall and particularly in women under 35 years and in women aged 65+ years. In line with these results, we also found a significant positive association between increased vaginal pH and LSIL, a cytomorphic indicator of HPV infection. This relationship was particularly significant in the youngest (< 25 and 25-34 years) and the oldest (65+ years) age groups.Our study confirmed that vaginal pH rises with age, starting in the peri-menopausal age range. These results are in line with a previous cross-sectional study evaluating the epidemiologic determinants of vaginal pH in the same-population based sample from Guanacaste (enrollment data only) in which vaginal pH was strongly related to age and menopausal status . HoweverGardernella vaginalis, predominate in the vaginal cavity [C. trachomatis DNA and vaginal pH among women < 25 years. Previous in vitro studies have demonstrated a significant reduction of C. trachomatis replication when exposed to acidic pH [C. trachomatis infection was associated with higher vaginal pH even when controlling for BV. Taken altogether, these findings suggest a common link between increased vaginal pH and susceptibility to acquiring sexually transmitted infections, and warrant further exploration.HPV is a sexually transmitted infection and the association of HPV prevalence with elevated vaginal pH could be due to some unmeasured aspect of sexual behavior. However, changes in the vaginal microenvironment associated with pH in younger women affect the immunological balance within the cervical tissue and provide a biologically plausible mechanism for increased HPV prevalence ,25. A hel cavity . The abscidic pH ,27 and acidic pH  found thAlthough the relationship between elevated vaginal pH and overall HPV positivity was not significant among women aged 65+ years, we did find a positive association between vaginal pH of ≥ 5.0 and detection of carcinogenic HPV, multiple HPV type infections, and diagnosis of LSIL in these women. Risk of HPV infection may be exacerbated by an age-related attenuation of the immune response and reductions of the natural immune defenses of the skin, typical of menopause . The vagThere were several limitations in our study. We realize that we did not have adequate information regarding vaginal conditions such as BV which could have been useful in characterizing underlying causes of elevated vaginal pH. Furthermore, this study did not include data on female and/or male sexual behavior, an especially important determinant of HPV prevalence in women in Guanacaste. Therefore, we were unable to address whether the relationship between vaginal pH and HPV was confounded by sexual activity. Finally, we did not measure pH and HPV frequently enough to assess the temporal direction of any possibly causal relationship. Thus, we are not claiming that the associations we found suggest that elevated pH increases HPV prevalence. This is meant to be the report of a novel epidemiologic association.C. trachomatis DNA in women aged < 25 years. Future studies should obtain closely spaced measurements, permitting a focus specifically on the temporal sequence of HPV acquisition and increased vaginal pH, and should include data on pH modulators such as the vaginal microflora, chronic inflammation, and/or pre-and-peri-menopausal hormone levels. If increased vaginal pH is found to precede an HPV infection, this might elevate the clinical significance of measuring vaginal pH. However, utility of this or any biomarker must be firmly established before introduction into clinical practice. In addition to studying vaginal pH, we anticipate that by using high-throughput sequencing of a shared region of bacterial ribosomes  [Our data suggest that elevated vaginal pH is related to detection of HPV, particularly multiple type infections, and LSIL in certain age groups and with detection of mal RNA) , we can The authors declare that they have no competing interests.MC: Analyzed data, drafted manuscript. ACR: Made substantial contributions to conception and design of study and acquisition of data and provided critical review of the manuscript. JG: Contributed substantially to analyses and interpretation of data and provided critical revisions of manuscript. RH: Made substantial contributions to conception and design of study and acquisition of data and provided critical review of the manuscript. AH: Made substantial contributions to conception and design of study and acquisition of data and provided critical review of the manuscript. SW: Biostatistician, involved in the design of the study and provided critical revisions of manuscript. RB: Laboratory collaborator, performed HPV typing, provided critical revisions of manuscript. MS: Principal Investigator, involved in the conception and design of the study and provided critical revisions of manuscript. All authors read and approved the final manuscript.National Institutes of Health contracts  and grant (CA78527 to R.D.B.). The Guanacaste cohort  was funded by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. HPV testing was supported by Dr. Burk's grant support.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/33/prepub


We present a case report of secondary vaginal cancer after complete hysterectomy due to myoma that was complicated by low-grade cervical intraepithelial neoplasia (CIN I) and human papilloma virus (HPV) infection. After complete hysterectomy, the HPV DNA level in the vaginal tissue was obviously increased, and vaginal cancer was diagnosed 6 months later. We conclude that HPV infection can cause vaginal cancer after complete hysterectomy in cases complicated by CIN. Therefore, HPV should be regularly assessed during the postoperative follow-up period. HPV viral subtypes can also cause other cancers, including vulval, vaginal, anal, penile and oropharyngeal carcinomas.2 Women over 60 years of age who have CIN lesions are at an elevated risk, and should be followed closely. Follow-up should continue even after a hysterectomy.3 Here, we present a case report of secondary vaginal cancer after complete hysterectomy due to myoma, which was complicated by low-grade cervical intraepithelial neoplasia (CIN I) and human papilloma virus (HPV) infection. A review of the relevant literature is presented to increase awareness of the importance of monitoring HPV infection.The incidence of vaginal intraepithelial neoplasia (VAIN) and vaginal cancer is very low, at 0.2-0.3 and 0.42 per 100,000, respectively, in the USA.A 62-year-old para 2, gravida 2 married patient was admitted to our unit due to intermittent vaginal bleeding over the preceding year. Four months prior to admission, she had undergone a thin-prep cytological test (TCT) which revealed low-grade squamous intraepithelial lesions (LSILs) of the cervix . Her hybThe patient was found to have uterine fibroids, and these were treated surgically. After anesthesia was instituted, a vaginal conventional hysterectomy was performed. Histopathological examination of the resected uterus revealed uterine fibroids, endometrial atrophy, chronic cervical inflammation, squamous cell hyperplasia, localized low-grade CIN I, and interstitial gland tumor-like hyperplasia . The shoThe patient was reviewed in clinic 6 months post-operatively. A vaginal cytology test by the TCT method showed dyskaryosis, with grade III high-grade squamous vaginal intraepithelial neoplasia . The res2 Brinton et al found that low education, low income, previous abnormal Pap smears, genital warts and a history of hysterectomy were important risk factors for VAIN and vaginal cancer; other risk factors included a current or past history of CIN, immunosuppression, previous radiotherapy and exposure to diethylstilbestrol.4VAIN and vaginal cancers are rare diseases with unclear epidemiologies. Conclusive evidence has demonstrated that a large number of cervical cancers occur due to infection with HPV.5 The outcomes of conservative treatment with cervical cone excision and total hysterectomy are similar. Therefore, to reduce the increased risk of injury associated with more extensive surgery, a hysterectomy for CIN II or CIN III is not usually indicated.5 We report the case of an elderly women with persistent HPV infection and CIN I who presented with vaginal bleeding secondary to uterine fibroids. She accepted the surgical intervention of hysterectomy with the aim of preventing future cervical cancer. Due to the patient’s age, and the high HPV viral load, we investigated her postoperatively with testing of vaginal exfoliated cells for HC2-HPV DNA. Vaginal cancer was identified after six months; with timely treatment, and her condition was well-controlled. Therefore, we advocate that patients with CIN, including those who have undergone hysterectomy, should undergo cervical conization and routine HC2-HPV DNA testing.A history of hysterectomy for CIN is a known risk factor for secondary VAIN; the incidence of VAIN ranges between 0.9%–6.8% after hysterectomy for CIN.5 recommended that cervical and vaginal colposcopy should be performed prior to hysterectomy due to CIN. The histology of exfoliated vaginal wall cells should be followed-up postoperatively for at least 18 months with samples taken every six months. If the histology at 18 months is negative for carcinoma, then follow-up can be discontinued.6 After treatment, colposcopy should be re-examined each year for up to 4 years after surgery. Patients with CIN I should be followed with annual colposcopy inspections for 2 years while a 10 year follow-up is recommended for patients with CIN II-III. This policy of post-treatment cytological surveillance ensures early detection of treatment failure and repeat conservative treatment as necessary for those who have a preserved uterus with cervical conization. However, some scholars believe that vaginal cancer does not develop after the hysterectomy, but the primary cancer in the vagina was already present prior to surgery. Therefore, a full examination should be conducted prior to the hysterectomy to prevent a missed diagnosis. Schockaert et al2 HC2-HPV-DNA testing is crucial for the successful overall outcome of conservative management of cervical CIN or in those who undergo total hysterectomy. Therefore, for resections due to CIN by cervical cone or hysterectomy patients, follow-up should include the HC2-HPV-DNA test and TCT, with HC2-HPV-DNA testing given higher priority. If the HC2-HPV-DNA test is positive, she should undergo cytology and, if abnormal cytology is present, colposcopy can then assist with the early detection of disease recurrence. If colposcopy is negative for HC2-HPV-DNA on the initial examination, repeated HC2-HPV-DNA testing and cytology inspection at 12 months will be adequate to discern disease recurrence. We believe that persistent HPV infection is the most salient risk factor for the recurrence of cervical CIN, from which VAIN and vaginal cancer can develop. The decision to commence antiviral therapy and the choice of therapy are therefore important questions that require accurate diagnostic tools and follow-up.In conclusion, HPV infection is the cause of cervical cancer, vaginal cancer, vulvar cancer, VAIN and other diseases. HPV testing has a powerful negative predictive value; if patients are followed-up for 6 months, a negative HC2-HPV-DNA test indicates that the risk of CIN recurrence is very small.


Human Papillomavirus (HPV) is the most common cause of cervical cancer. Cervical cancer being the second most common cancer after lung cancer, affecting women of different age groups; has a prevalence of about 20% in young sexually active women. Among different types of HPV, HPV16 the major strain causing this cancer and is sexually transmitted had been unnoticed for decades. Keeping in mind the multiple risk factors related with cervical cancer such as early age sexual activities, teenage pregnancies, smoking, use of oral contraceptives, having multiple sex partners, hormone replacement therapies and various other unknown factors lead to the onset of the disease. Awareness for various diagnostic procedures such as Pap smears screening prove to be an effective way in eradicating the oncogenic potential of HPV. Human papilloma viruses (HPV) are small non enveloped virus containing double stranded DNA as their genetic material and are about 55 nm in size. Their genome has three functional coding regions; E: a gene coding early viral function, L: a gene coding late viral function and LCR: a long control region which lies between E and L. They appear to show similarity with plyoma viruses and consist of about 72 capsomeres. The papilloma viruses and its viral nature were first seen in human warts in 1907, and the first papilloma virus was isolated from a rabbit that was identified by Richard Shoppe in the year 1983. Even it was an early start for the detection of human papilloma viruses; this topic remained closed till 1970's. Studies related with papilloma viruses were allowed to move forth only when cervical cancer's proximity with HPV was proved and with its increasing significance in the field of molecular virology [Human papilloma virus is known to be the main reason for cervical carcinomas. Back in 1976 HPV was seen with its involvement in the successful development of cervical tumours and since then various epidemiological and biological statistics has justified this observation . HPV virCervical cancer as the most prevalent cancer in women in the coming years is the main cause of death, especially in young women. Its ratio varies all over the world, but a significantly high ratio has been seen in the western countries. By statistical and geographical analysis it proves to be higher in Native American women than in Caucasian women. It was also found common in Hispanic and African American women . AccordiAbout 100 different subtypes of HPV with distinguished variations in its genetic and oncogenic potential are known. The subtypes which specifically affect the anogenital tract are HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66 and 69 [HPV16 is one of the most prominent strains of HPV which is typically classified under high risk type infection as mentioned earlier. It is associated with additional infections namely lichen sclerosis and not only it leads to cervical cancer but is also related with vaginal, vulval, anal and penile cancers or benign growths specifically in these areas, but the most prominent and hazardous damage caused by HPV 16 strain is cervical cancer . The HPV1 and E2 proteins are supposed to be the recognition factors and regulators for early viral transcription. Icosahedral capsid for virion generation are formed by L1 and L2 [Replication of HPV 16 is initiated with the replication of viral DNA in contrast with the cellular chromosomes. In this process of division, one daughter cell moves apart from the basal lamina to undergo differentiation. Infection in the basal lamina can persist for years because, of the restriction of virion production to only differentiated cells. The E5, E6, E7 respectively. These proteins are associated with cervical cancer studied both in vitro and in vivo [6 and E7 are important for the inhibition of tumour suppressor genes namely p53 and pRb [5 is concerned it is known to have weak oncogenic properties which result in increased activity of epidermal growth factor receptor (EGFR) and there by inhibiting the major histocompatibility complex expression [2 and E1 are the two proteins which play a leading role in control of transcription and replication and in the absence or loss of activity of these proteins deregulation of E6 and E7 onco-proteins results [HPV 16 affects approximately 20% of the adult population particularly in western countries . It some in vivo . The pro and pRb . As far pression . For a c results .6 protein comprises of about 151 amino acids, these proteins are confined to the nuclear matrix and non-nuclear membrane fraction. Their major function is in zinc binding. This E6 protein is related with high risk HPV associated with wild type p53 tumour suppressor protein. To get associated with p53, the E6 protein needs a cellular protein of about 100-kDa and is known as E6-AP [6 protein favours abnormal cell growth by the process of binding and rapidly targeting the p53 protein [6-E6 AP complex specifically target ubiquitin targeted substrates. It also shows p53 disruption and p53 mediated apoptosis [7 proteins are small proteins consisting of 98 amino acids and zinc binding phosphoproteins are confined in the nucleus. This high risk HPV E7 protein has a higher binding affinity for pRb protein [HPV I6 Eas E6-AP . The E6  protein . This p5poptosis . HPV E7  protein .2 F-1and cMyc proteins. These cMyc and E2 F-1 proteins activate some of the essential genes which are required for DNA synthesis. The HPV infected cell typically shows that pRb is inactive throughout in association with binding of the E7 proteins. This decreased or inappropriate release of transcription factors favours the deregulated expression of genes which are related with normal control and cell division [The mutational analysis reveals that an E7 protein plays a major role in transformation and has proved its ability with interference with pRb function. This interference of E7 protein with the pRb function contributes in the development of malignancy. pRb is also a tumour suppressor gene and is the main mutant allele of Rb present in the germline of an individual which is linked to a predisposition to retinal tumour . The majdivision .7 possesses and has the capability of overcoming DNA damage induced p53-dependant G1 arrest. In the precancerous lesions the loss of control point favours and allows the DNA damaged cells to enter the normal cell division initiating to cancerous or tumourigenic mutations. This evidence of mutations is well supported by recent reports that deregulate expression of E2 F in rat fibroblasts which leads to S phase entry in the cell cycle and apoptosis in p53 [6 protein by targeting the p53 protein for degradation inhibits the transcriptional activation of the kinase inhibitor and secondly expression of the E7 proteins can result in the constitutive inactivation of pRb and hence the deregulation of E2 F1 takes place [More striking evidence reported by several studies is that Es in p53 . Moreovees place .6 and E7 proteins and it is very essential to bring into light the detailed analysis of these studies on the specific regions of HPV16 E7 proteins [7 are also related with in vivo functions which include immortalization and transformation alone or in cooperation with ras.Many reviews and studies had focused on the mechanisms and significant properties of Eras, fos and other related oncogenes has also been detected in cervical cancer cell lines but their in vivo role and proliferation is still unpredicted [As far as the causes of HPV16/HPV18 are concerned sexual behaviour is the strongest factor for this increasing malignancy. Despite sexual behaviour other factors might also be involved in its infection. It is important to note that all HPV infections does not lead to a malignancy however, the two strains of HPV that are HPV16 and HPV18 mainly contributes in the development of cervical cancer. Other factors include smoking, extensive use of oral contraceptives and history of having sexually transmitted diseases . Mutatioredicted .Other factors that enhance cervical cancer include impaired immune response, persistence of virus, smoking and administration of steroid hormones . Loss ofHaptoglobulin which was an acute phase glycoprotein present in body fluids is reported to be in relation with tumour and infection which modulate the Th1/Th2 response -29. The HPV affects not only women, but this virus is even contracted by men. Men do have the ability to transmit it but unlike women in men there is no such confirmed lab test available such as women do have Pap smear tests .HPV16 which ultimately leads to cervical cancer stays and incubates in the body for a prolonged period of time before the onset of malignant cervical cancer . Sometim6 and E7 proteins and their expression has been seen in the cell lines [Cervical cancer can also be defined as the cancer originating at the opening of the womb which progressively migrates to the whole of cervix . HPV16 ill lines . IgG andBack in 1977 statistical data suggested that HPV viruses are present in cervical epithelium . In diff1 and E2 gene expression which control the genes and deregulation of E6 and E7 open reading frames. The normal function of tumour suppressor genes p53 and Rb is blocked by the products of E6 and E7 coding regions [1 and G2 points. By this data it was confirmed that HPV16 provided the solid evidence for its development in cervical invasive cancer. HPV DNA was found and present in about 90% of all cervical cancers [The most striking and promising evidence that human papillomaviruses play a pivotal role in oncogenesis is based on different patterns of HPV DNA in cervical cells and its integration. The result of integration is disruption of E regions . As a re cancers .Keeping in view the association between HPV and Cervical Cancer, early age is thought to be a primary factor in HPV infection. Its probability increases with the number of sexual partners at any time of that person's life. Epidemiological data shows that the exogenous and endogenous sex hormones also make a women more prone in developing cervical cancer in combination with HPV infection . By analRisk factor for Cervical Cancer also increases with the increase use of Oral Contraceptives (medications taken by mouth for the purpose of birth control). Women who use oral contraceptives more than 5 years of duration had a 3 fold increased in the development of cervical cancer than those who do not use such type of medications for birth control. To cap it all, according to epidemiological studies, the incidence of cervical cancer and increased risk in multifarious women and with women using hormonal contraceptives for a long time were consistent and confirm the hypothesis that female hormones favours the risk of cervical cancer. It was more common in women who avoided Pap smears and also in women who had orthodox religious beliefs such as church goers ,45.Human semen is also considered as the major biological basis in sexual behaviour that enhances HPV infection and cervical cancer. Chronic inflammation of cervix is due to local cellular and molecular changes that are reported to be triggered by multiple exposures to semen ,47. HumaHPV infection can be clinically diagnosed by the presence of warts in low risk types. Genital warts which are sexually transmitted can be present in the form of cauliflower like smooth popular, small elevated warts. Progressive cervical disease initiates as a benign non invasive squamous intraepithelial lesion or CIM. Differential diagnosis can be done with three procedures of molecular assays namely: signal amplification, target amplification, non amplified hybridization assays . Pap smeIt is now well established fact that Cervical Cancer is due to HPV infection which is a sexually transmitted disease. Cervical cancer is among the second most common cancer in women worldwide. It is a multi-factorial disease. Various factors are involved in its progression of cervical cancers such as smoking, sexual behaviour, age and number of sexual partners -64. Infe1 proteins at present. As VLPs, has no viral DNA therefore these are not associated with the development of infection even though their structure is similar to actual virus morphologically. By intra-muscular (I/M) injection, high levels of systemic anti HPV L1, Immunoglobulin G antibodies are formed due to VLPs [Risk of infection with HPV is directly proportional to age. Prophylactic HPV Vaccines is found to be effective in treating HPV infection and it is shown by number of randomized clinical trials -69. Gard to VLPs .1 viral protein which shows increased effectiveness and mostly adopted. Sexual naïve adolescents should be routinely vaccinated as recommended by World Health Organization (WHO). These vaccines can prove to be effective for future infection instead of previously exposed individuals. As Papillomavirus is non-lytic virus and can divide only in epithelial keratinocytes so, prophylactic vaccines seem to be ineffective in HPV neoplasia. Type of HPV is correlated with the clinical outcome of infection. For some infections, host's immune system is strong enough to eradicate and they get cleared instantly.Prophylactic HPV Vaccines targeting LSeveral vaccination trials have shown that both bivalent against HPV 16, 18 and Quadrivalent against HPV 16, 18, 6 and 11 shows remarkable immunological response which is related to nearly 100% efficacy for a maximum 5 years in Naïve women -75. HerdIncrease in risk of HPV infection has a tremendous role in women related sexual problems and the hazardous effect is in the form of cervical cancer which is the major cause of death among all the cancers in developing countries. Avoidance of cervical screening and socio cultural taboos with special reference in the South East Asian belt promotes HPV and its strong relation with cervical cancer. Women are not aware, literate enough to go for regular pap screening and negative myths or facts for sexual issues in this region. Women don't go for the treatment of genital warts and the basic purpose of this review article is to highlight the dire need of awareness of this deadly cancer.HPV: Human Papiloma virus; FTP: Full Term Pregnancy ASCUS: atypical squamous cells are of undetermined significance; CRN: cervical intraepithelial neoplasia.The authors declare that they have no competing interests.RF and AZ reviewed the literature, and wrote the manuscript. KK helped RF & AZ in literature review and statistical analysis. MI edited the manuscript. All the authors read and approved the final manuscript.


A defective DNA damage response can result in genomic instability (GIN) and lead to transformation to cancer. As p53-binding protein 1 (53BP1) localizes at the sites of DNA double strand breaks (DSBs) and rapidly forms nuclear foci (NF), the presence of 53BP1 NF can be considered to be an indicator of endogenous DSBs reflecting GIN. Our aim was to analyse the presence of DSBs by immunofluorescence for 53BP1 expression in a series of cervical lesions, to evaluate the significance of GIN during carcinogenesis.INK4a overexpression. The number of 53BP1 NF in cervical cells appeared to increase with progression during carcinogenesis. The distribution of 53BP1 NF was similar to that of the punctate HPV signals as determined by in-situ hybridization and also to p16INK4a overexpression in CIN, suggesting an association with viral infection and replication stress.A total of 80 archival cervical tissue samples, including 11 normal, 16 cervical intraepithelial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14 squamous cell carcinoma samples, were analysed for 53BP1 NF, human papillomavirus (HPV) infection, and p16Immunofluorescence analysis of 53BP1 expression can be a useful tool with which to estimate the level of GIN. During cervical carcinogenesis, GIN may allow further accumulation of genomic alterations, causing progression to invasive cancer. Any genotoxic agents can induce DNA damage and consequently result in oncogenic alterations. The importance of the DNA damage response (DDR) pathway in tumour suppression is well recognized. A defective DDR can result in genomic instability (GIN), which is generally considered to be central to any carcinogenic process.in vitro with immunofluorescence that 53BP1 exhibits diffuse nuclear staining in untreated primary cells, whereas, after exposure to radiation, 53BP1 localizes at the sites of DSBs and forms discrete nuclear foci (NF).The p53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins with C-terminal BRCA1 C-terminus domains.E6 and E7, have been shown to be the main contributors to the development of human papillomavirus (HPV)-induced cervical cancer, and increased expression resulting from integration of the viral DNA into the host genome has been detected in invasive cancers and a subset of high-grade lesions.E6 and E7 together cause polyploidy soon after they are introduced into cells, so GIN is thought to play an essential role in the cellular transformation of cervical epithelium during carcinogenesis. The most manifest function of the E6 protein is to promote the degradation of p53 through its interaction with a cellular protein, E6-associated protein, an E3 ubiquitin ligase.1 cyclin-dependent kinases releases E2F, leading to cell cycle progression into the S phase. E7 is able to bind unphosphorylated pRb, and this may induce cells to prematurely enter the S phase by disrupting pRb–E2F complexes. One cyclin-dependent kinase inhibitor, p16INK4a, which prevents the phosphorylation of pRb family members, is overexpressed when pRb is inactivated by E7.INK4a results in cell cycle arrest, but with E7 expression, this is overcome. Thus, overexpression of p16INK4a has been suggested as a useful biomarker for evaluating HPV pathogenic activity in cervical lesions.Uterine cervical cancers are believed to develop through a multistep process. Furthermore, it is well established that persistent infections with high-risk human papillomavirus (HR-HPV) represent a necessary cause of high-grade premalignant lesions and subsequent invasive cancer of the uterine cervix. The HR-HPV viral oncogenes, INK4a overexpression during cervical carcinogenesis. Like other tumours, GIN was shown to be induced in cervical epithelium at a precancerous stage, and increased significantly with progression to cancer.The present study analysed the presence of endogenous DSBs by immunofluorescence for 53BP1 expression in a series of cervical tissues from patients to evaluate the significance of GIN and its association with HPV infection and p16in-situ hybridization (ISH) studies.Eighty archival uterine cervical tissue samples were selected for this study from the archives of the Department of Obstetrics and Gynaecology, University Hospital. Accuracy of diagnosis was confirmed by a gynaecological pathologist (H.N.) and a general pathologist (M.N.). Histologically, the 80 primary cervical tissue samples were as follows: 11 normal cervical tissue samples from uteri that were surgically resected because of leiomyoma; and 16 cervical intraepithelial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14 squamous cell carcinoma (SCC) samples. All samples were formalin-fixed and paraffin-embedded tissues, from which sections were prepared for immunofluorescence and 2O2 in methanol for 20 min, and denatured and hybridized in a humidified chamber at 37°C for 60 min. Detection of hybridized probe was performed with the GenPoint Tyramide Signal Amplification System for Biotinylated Probes , with application of primary peroxidase-conjugated streptavidin, biotinyl-tyramide secondary peroxidase-conjugated streptavidin, and the chromogenic substrate diaminobenzidine (DAB). The slides were counterstained with haematoxylin. Evaluation of the HPV ISH signals was performed according to the criteria described previously.ISH was performed with the GenPoint Catalyzed Signal Amplification System  for HR-HPV , according to the manufacturer's protocols. After deparaffinization and rehydration, the sections were incubated in 0.8% pepsin for 10 min at 37°C and treated with proteinase for 5 min at room temperature. Sections were immersed in 0.3% HAfter antigen retrieval with microwave treatment in citrate buffer, deparaffinized sections were preincubated with 10% normal goat serum. Tissue sections were then reacted with anti-53BP1 rabbit polyclonal antibody  at a 1:200 dilution. The slides were subsequently incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody . Specimens were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride , analysed, and photographed with a High Standard All-in-One Fluorescence Microscope . Signals were analysed at a ×1000 magnification.2/95% air environment. After fixation with methanol and preincubation with 10% normal goat serum, cultured cells were treated according to the same immunofluorescence method as mentioned above. The pattern of 53BP1 immunoreactivity was classified into four types  staTo assess the association between localization of 53BP1 NF and HR-HPV integration into the host genome in neoplastic cells, dual immunofluorescence for 53BP1 expression and ISH to detect HR-HPV were performed on the same tissue sections. Each section was first labelled by immunofluorescence, subjected to ISH as mentioned above, and photographed with a High Standard All-in-One Fluorescence Microscope . The images obtained were merged and analysed by use of the accompanying image analysis software with Biorevo BZ-9000.2O2/methanol, sections were preincubated with 10% normal goat serum. After antigen retrieval, tissues were incubated overnight at 4°C purified mouse anti-human p16  at a 1:300 dilution. The slides were subsequently incubated with biotinylated goat anti-rabbit antibody for 1 h at room temperature, and then avidin–peroxidase, and then visualized with DAB.After immersion in 0.3% HWe also carried out double-label immunofluorescence staining for 53BP1 and Ki67 expression to clarify the association between type of 53BP1 expression and cyclingecells. For double staining, tissues were incubated with a mixture of rabbit anti-53BP1 and monoclonal mouse anti-Ki67  antibodies at 1:50 dilutions, and subsequently incubated with a mixture of Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 546-conjugated goat anti-mouse antibodies. Specimens were counterstained with DAPI-I (Vysis), and visualized and photographed with a High Standard All-in-One Fluorescence Microscope . Signals were analysed at a ×1000 magnification.P-value of <0.05 was accepted as statistically significant.The Jonckheere–Terpstra test was used to assess associations between the type of HPV ISH signal  or the type of 53BP1 expression  and histological grade of cervical neoplasia. Associations between the types of 53BP1 expression and HPV ISH signal were also assessed with the Jonckheere–Terpstra test. The PHREG procedure in the SAS 8.2 software  was utilized for calculation. All tests were two-tailed, and a P < 0.0001). The incidence of mixed and punctate types significantly increased in the order of normal, CIN1, CIN2, CIN3, and SCC.The ISH results for each type of cervical lesions are summarized in P < 0.0001).The results of the immunofluorescence staining patterns for 53BP1 in cervical lesions are presented in P < 0.0001). The incidence of the DDR and large NF types significantly increased in the order of negative, diffuse, mixed and punctate types of HR-HPV ISH signal.Associations between types of 53BP1 expression and HR-HPV ISH signal in the cervical epthelium are summarized in The results of the dual immunofluorescence staining for 53BP1 expression and ISH to detect HR-HPV are shown in INK4a expression in cervical lesions is shown in INK4a overexpression in each cervical lesion.The comparison of 53BP1 expression and p16CIN1 expressed the stable or low DDR type of 53BP1 immunoreactivity, and little Ki67 nuclear staining, mainly at the basal layer . In contet al.Ionizing radiation (IR) effectively induces DSBs in normal cells and activates DDR pathways to maintain genomic integrity. Although non-irradiated cells typically show diffuse 53BP1 nuclear staining, many 53BP1 NF are immediately induced after exposure to a low dose of IR.INK4a as a surrogate marker of HR-HPV and CIN has been well established in recent years, with studies showing both increased immunoexpression of p16INK4a in dysplastic cervical epithelium and a positive correlation with HR-HPV infection and the degree of CIN.E6 and E7. Several risk factors for cervical neoplastic progression are likely to contribute to viral oncogene deregulation, particularly integration of HR-HPV into the host genome.INK4a overexpression; this represents a byproduct of viral infection, and suggests an association between the presence of GIN and replication stress that allows enhancement of cell proliferation and avoidance of cell death. As co-localization of 53BP1 NF and the punctate type of HR-HPV ISH signal was rare, most viral integrated sites might have been bypassed by the host's DDR machinery, and integrants may have a growth advantage over others in a mixed population of cervical cells.The value of p16This study also demonstrated little co-localization of the DDR type of 53BP1 expression and Ki67 nuclear staining as a marker for cycling cells in precancerous lesions. An intact DDR pathway is activated, leading to cell cycle arrest by p53 activity, so precancerous/dysplastic cells may still preserve DDR function, at least partially, and this may induce sporadic regression of dysplasia. In contrast, cancer cells showing 53BP1 NF were frequently co-localized with Ki67 staining, suggesting a disruption of the DDR pathway that subsequently leads to an irreversible malignant transformation. Taken together, these findings indicate that GIN may have already occurred at the precancerous stage during cervical carcinogenesis, and that increasing GIN based on a disrupted DDR may allow further accumulation of other genomic alterations, causing progression to invasive cancer through acceleration of cell growth/replication stress. Further research is needed on the molecular mechanism of the uncoupling of cell cycle progression in the face of increasing severity of DNA damage at the advanced phase of carcinogenesis.In summary, this study has demonstrated a number of 53BP1 NF in cervical lesions resected from patients that were similar to those found in irradiated cells with a DDR pathway activated to eliminate DSBs, suggesting the occurrence of endogenous DSBs. GIN seems to be induced at the precancerous stage through integration of the HR-HPV genome into the host cell genome. Furthermore, invasive cancers exhibited 53BP1 NF in cycling cells, which suggested that the disrupted DDR subsequently led to further amplification of the genomic injury. A recent study on immunohistochemical detection of DDR-associated molecules, including 53BP1, suggests that GIN is an early event that occurs in pulmonary hyperplasia prior to changes in the p53 tumour suppressor gene during lung carcinogenesis in patients, suggesting that GIN may serve as a causative link between precancer and cancer.