To evaluate the association of p16/Ki-67 co-expression and persistence of high-risk human papillomavirus (HR-HPV) infection as well as cervical abnormalities.We performed a 3-year cohort study among which 2498 Chinese women aged 25 to 65 years were screened by different HPV tests in 2011. 690 women who were positive at any of the tests and a random sample of 164 women with all negative results received colposcopy, cervical specimens for cobas HPV test (Roche diagnostics) were collected before colposcopy; of this group, 737 cervical specimens were collected to perform cobas, Liquid-based cytology, HPV E6 test (Arbor Vita Corporation) and p16/Ki-67 dual staining (Roche diagnostics) in 2014. Colposcopy and biopsies was performed on women with any abnormal result.Compared to women without HR-HPV persistent infection, women in the HR-HPV persistence group had a higher risk of p16/Ki-67 positive, with an adjusted Odds Ratio(OR) and 95% confidence interval (CI) of 6.29 (4.07-9.72); moreover, adjusted odds ratio for women who had HPV16/18 persistent infection was nearly 4-folder higher than women with other 12 HR-HPV persistent infection . Additionally, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities, and resulted strongly associated with a CIN2+ diagnosis .p16/Ki-67 co-expressions associated strongly with HR-HPV persistence, especially with HPV16/18, and the presence of a CIN2+ lesion. Therefore, p16/Ki-67 could be considered as a suitable biomarker for cervical cancer screening, particularly in HPV-based screening programs. The rates of cervical cancer have been greatly reduced by organized cytology screening programs in developed countries. In China, where efficient screening strategies are lacked, the burden of cervical cancer remains high with an estimation of 98,900 incident cases and 30,500 deaths in 2015. At the INK4a (p16) protein is a negative regulator of proliferation in normal cells though downregulating the activity of cyclin-dependent kinase(CDK) 4 and CDK6 once the pRB has been inactivated. HPV E7 can promote cell cycle progression by inactivating pRB, resulting in the overexpression of p16 in affected cells. Ki-67 is a nuclear protein and a cellular proliferation marker. The expression of p16 and Ki-67 is mutually exclusive in normal cells. The simultaneous detection of both proteins within a cell would be indicative of deregulation of the cell cycle caused by HPV oncoproteins expressed mostly during viral persistence, which suggests a possible relationship between p16/Ki-67 co-expression and HPV persistent infection. However, no study has directly proven such an association to date.HR-HPV E6 and E7 oncoproteins are considered essential for the development of cervical cancer in persistent HPV lesions by interacting with p53 and pRB tumor suppressor proteins, which play an important role in the regulation of normal cell cycle [Here we used the CINtec PLUS Cytology test  targeted for detection of p16/Ki-67 co-expression to investigate whether the presence of p16/Ki-67 associates with HR-HPV persistence as well as histology in a clinical study cohort in China.As shown in Figure The baseline and follow-up HR-HPV positive rates for women included in this study are shown in Table Table All the participants were divided into different groups according to HPV infection status Table . ComparetrendP <0.001).As shown in Table Table Recently, for women aged 25 years and older, the use of cobas HPV Test was approved by FDA for primary cervical cancer screening. This is of great importance to China, where cytology based screening has been proven difficult to implement in rural areas due to the lack of skilled cytopathologists. Notably, such cytology based or VIA based screening program which targets for screening 70% of 270 million Chinese women by 2015, resulted into the addition of 546,000 new HPV tests as announced by Chinese Government in 2014. However, as 80%–90% of women with a positive HPV test are transient infections and will not have concurrent disease, the challenge of using HPV testing for primary screening is to find complementary biomarkers that can discriminate clinically relevant persistent infection in screen-positive women. The simultaneous detection of p16 and Ki-67, which is commercially available now prompted our choice to evaluate its usage for such purpose.We presented the first cohort study to examine the association between p16/Ki-67 co-expression and HR-HPV persistence. In a high risk screening population, we observed a significant higher positivity rate of p16/Ki-67 dual staining in HR-HPV persistence group. Compared to women without HR-HPV persistent infection, women in the persistent infection group had a higher risk of p16/Ki-67 positive, with an adjusted OR of 6.29 (95% CI: 4.07-9.72); moreover, adjusted odds ratio for women who had HPV16/18 persistent infection was 17.15 (95% CI: 7.11-41.33), which is nearly 4-folder higher than women with other 12 HR-HPV persistent infection . It may due to the higher carcinogenic potential of HPV16/18 oncoproteins than that of other carcinogenic HPV types . AlthougIn the present study, to avoid the verification bias, four screening tests were used and women who tested positive for any of the tests were selected for colposcopy and directed biopsy, four-quadrant cervical biopsies and ECC were performed to maximize ascertainment of disease if no visible lesions were found under colposcopy. The histopathology of all biopsies was reviewed by an expert in CICAMS and all CIN cases were p16-supported. Since the establishment of a correlation between the biomarker expression and severity of neoplasia is the initial step to assess the potential use of a biomarker for cervical cancer screening, we evalOur analysis has several limitations. One consists in the definition of HPV persistence used in our study; there is wide variation in definition of HPV persistence in the literature-30, but In summary, in a high-risk screening population, our study provides the first evaluation between presence of p16/Ki-67 and HR-HPV persistence. We observed p16/Ki-67 co-expression associated strongly with HR-HPV persistent infection, especially HPV 16/18. We found a strong association between dual staining and CIN2+ presence. Our results suggest that p16/Ki-67 is a suitable biomarker for cervical cancer screening, particularly in HPV-based screening programs. However, additional studies are needed to confirm these findings, particularly among populations with a large size.clinicaltrials.gov identifier: NCT01231945.The baseline study was approved by the institutional review boards (IRB) of the Cancer Institute/Hospital, Chinese Academy of Medical Sciences (CICAMS), of PATH , and of the US National Cancer Institute; the follow-up study was approved by the IRB of CICAMS.In 2011, a total of 2,498 women aged 25 to 65 years living in Xin-mi county were enrolled in a study named “Screening Technologies to Advance Rapid Testing for Cervical Cancer Prevention–Utility and Program Planning (START-UP)” Project based on the following criteria: 1) had a cervix; 2) had not been previously diagnosed with cervical cancer; 3) were not pregnant; 4) were physically able to undergo routine cervical cancer screening; 5) were able to provide informed consent. Women who were not married or never had sexual intercourse were excluded. Details on participants recruitment have been published elsewhere ,33. In 2TM Cervical Test  on a clinician-collected (cc) specimen, HC2 (QIAGEN) and careHPV testing (QIAGEN), both on a second cc and self-collected (sc) specimens, and visual inspection with acetic acid (VIA). One month later, women who tested positive for any of the 6 screening tests and an approximately 9.8% of randomly selected women who tested negative for all 6 tests (screen-negative women) were referred to colposcopy using a biopsy protocol as previously described[TM Cervical Test, and p16 /Ki-67 dual staining (to assess status of p16 /Ki-67 co-expression). Women who tested positive for any of the four tests underwent colposcopy evaluation and biopsy using a protocol as mentioned above.Under START-UP, women were screened by 6 different screening tests in 2011: OncoE6described. MeanwhiThe cobas HPV test was performed according to the recommendations of the manufacturer. This assay, based on the amplification of target HPV DNA by PCR followed by nucleic acid hybridization, can detect 14 HR-HPV genotypes: HPV-16 and HPV-18 individually and the other 12 types pooled .CINtec PLUS detects expression of p16 and Ki-67 as brown/cytoplasmic and red/nuclear reaction products, respectively. In accordance with the manufacturer's Instructions, sample is positive is classified on the basis of simultaneous brownish cytoplasmic staining (p16) and red nuclear staining (Ki-67) in one or more cells, independent of cellular morphology. Slides without any double-stained cells were called negative for p16/Ki-67 dual-stain cytology. All the slides were reviewed by a trained cytologist in CICAMS.Thin-layer cytology slides were prepared with ThinPrep Pap Test  and results were reported according to the Bethesda 2001 classification system. LBC results were considered negative when the result was negative for intraepithelial lesion or malignancy (NILM); all other results were considered positive and resulted in referral to colposcopy. All the cytological diagnoses were made by cytopathologists of CICAMS.INK4a by immunohistochemistry  as an additional adjudicator.The histopathological diagnosis was provided by a senior CICAMS pathologist and the worst of the biopsies or surgical specimen was used for the final diagnosis in these analyses. Additional sections of all initial biopsy diagnoses with CIN were cut and tested for p16Continuous and categorical variables were compared using t-tests and chi-square tests, respectively. The status of HR-HPV infection in the analysis was defined by the result of cobas HPV test. HR-HPV persistence was defined as HR-HPV positive both in 2011 and in 2014 via cobas HPV test. Associations between HR-HPV persistent infection and p16 /Ki-67 co-expression were examined using unconditional logistic regression models. Variables  associated p16 /Ki-67 co-expression were included as adjustment variables in logistic regression models. Chi square of trend for proportion was calculated to test linear associations between p16/Ki-67 dual staining and increasing severity of cytological and histological diagnoses. Odds ratios (OR) and 95% confidence interval (CI) by 2×2 tables were used to assess the association between p16/Ki-67 staining and histological outcomes. All P values less than 0.05 (two-sided) were considered to be statistically significant. SPSS 17.0  was used for the analyses.


Although the correlation of HPV genotype with cervical precursor lesions and invasive cancer has been confirmed, the role of HPV genotype in cervical cancer prognosis is less conclusive. This study aims to systematically investigate the independent prognostic role of HPV genotype in cervical cancer.A total of 306 eligible patients provided cervical cell specimens for HPV genotyping before therapy and had a median follow-up time of 54 months after diagnosis. Survival times were measured from the date of diagnosis to the date of cervical cancer-related death  and from the date of diagnosis to the date of recurrence or metastasis . Log-rank tests and Cox proportional hazard models were performed to evaluate the association between HPV genotype and survival times.P = 0.037) and HPV16-related species alpha-9 predicted better OS and DFS (both P < 0.01). After adjusting for age, FIGO stage, and therapy, HPV16 showed a hazard ratio (HR) of 0.36  for OS, and alpha-9 resulted in a HR of 0.17  for OS and 0.32  for DFS.A total of 12 types of high-risk HPV were detected and the leading ten types belong to two species: alpha-9 and alpha-7. HPV16 and 18 were the two most common types, with the prevalence of 60.8% and 8.8%, respectively. In the univariate analysis, HPV16-positive cases were associated with better OS  contains supplementary material, which is available to authorized users. Cervical cancer is the fourth most common malignancy in females worldwide, with an estimated 527,600 new cases and 265,700 deaths per year . The etiAlthough there has been much evidence on the role of HPV genotype in cervical precursor lesions and invasive cancer, it remains unclear whether they affect prognosis of cervical cancer. Furthermore, existing results on the relationship of HPV genotype with survival are heterogeneous. For example, early evidence showed that HPV16 positivity predicted poor prognosis and was associated with histological features of prognostic significance such as squamous cell carcinomas, pelvic node metastases, and lymphatic space invasion . But somTo better understand the role of HPV genotype in prognosis of patients with cervical cancer, we assessed the association of HPV genotype with overall survival  and disease free survival  among 306 cases of cervical cancer from China.Cervical cancer patients were consecutively recruited from Cancer Hospital, Chinese Academy of Medical Sciences from 2010 to 2012. We included patients who had a first diagnosis of histologically confirmed invasive cervical cancer, and the sampling of cervical exfoliated cells for HPV genotyping were taken by a gynecologist before therapy. Patients were excluded for the following criteria: a history of hysterectomy or conization, recurrent cervical cancer, other preexisting malignancies, and those with less than two months of survival after completing therapy. Patient’s age, FIGO stage, tumor histology, and pathologic variables were retrieved from medical records. This study was approved by the ethics committees of National Cancer Centre/Cancer Hospital, Chinese Academy of Medical Sciences, and all patients provided informed written consent before study enrollment.Genomic DNA was extracted from cervical cell specimens manually by using QIAamp DNA Mini Kit, according to the manufacture’s protocol . The quality of extracted DNA was assessed by PCR with a set of primers for the housekeeping gene β-actin . All β-actin positive specimens were tested for HPV DNA by following the manufacturer’s protocol of a HPV GenoArray Test Kit , which is a Chinese FDA-approved assay for HPV genotyping. A total of 21 HPV types could be detected simultaneously, including 13 high-risk (HR) types , two intermediate-risk types (HPV53 and 66), and six low-risk HPV types .Treatment information was retrieved from medical records and was summarized and grouped as follows: surgery alone ; surgery plus adjunctive chemotherapy (CT), radiotherapy (RT) or chemoradiotherapy (CRT); concurrent chemoradiotherapy (CCRT); CT or RT only. Each patient was followed up every 3 months in the first year and every 6 months in the next years by personal or family contacts, until June 2016. Hospital medical records were obtained in order to confirm the reported events. Only validated events were included in analysis. Overall survival (OS) was defined as the time between the date of diagnosis and the date of cervical cancer-related death or the last follow-up. Disease-free survival (DFS) was measured from the date of diagnosis to the date of recurrence, distant metastasis, or the last follow-up.P values presented were two-sided and were assumed significant as P < 0.05.The data were analyzed by using the Stata version 11.0 . To assess the potential of HPV type as a prognostic biomarker for cervical cancer patients with no matter single or multiple infections, all cases were included for HPV16 and 18 survival analysis. Multiple infections with only alpha-9 types were included for alpha-9 survival analysis. Survival curves were generated using the Kaplan-Meier method, and comparisons were performed using the log–rank test. Multivariate analyses of the factors associated with OS and DFS were done using Cox proportional hazard regression model. In the stratified analysis, the chi-square test-based Q-statistic was applied to test the heterogeneity between subgroups defined by age, FIGO stage, and treatment. All The demographic and clinical characteristics of cervical cancer patients are summarized in Table A total of 12 types of high-risk HPV were detected in this study  months. A total of 58 patients (19.0%) had experienced treatment failure, including 27 recurrences and 38 distant metastases (7 patients had both). In addition, 34 deaths (11.1%) were attributed to cervical cancer. The 5-year OS rate for the entire cohort was 87.1% (95% CI: 82.1–90.8%), and the corresponding DFS rate was 78.3% (95% CI: 72.5–83.1%).P < 0.001) and DFS (P < 0.001), while primary surgical treatment was associated with a better OS (P = 0.004) and DFS (P = 0.019). Of note, patients infected with HPV16 had a better OS than those with any other types (P = 0.037)  and DFS (P = 0.005), compared to alpha-7  Fig. . HPV16-ra-7 Fig.  and c. NP = 0.005) (Table P < 0.001) and DFS . Further analysis suggested that HPV52/33/31/58 group had more favorable OS  and DFS    was a predictor of better survival compared with alpha-7 species group including the other five HR types .Substantial differences in risk for high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer have been revealed between HR HPV types, in which HPV16 and HPV18 confer the highest risk , 12 HoweThe underlying mechanisms that result in the tumors caused by HPV16 and the alpha-9 species being less aggressive are still undetermined. Interestingly, HPV status has been recognized as a strong and independent factor for favorable survival of patients with oropharyngeal cancer (OPC) , 20. AccOur results demonstrated the independent prognostic value of HPV genotype in cervical cancer. HPV genotyping could potentially help to stratify cervical cancer patients for more effective therapeutic regimens. Patients with alpha-9-caused cervical cancer may receive less aggressive therapy to reduce side effects, while those with alpha-7 positivity may require more aggressive treatment and closer monitoring. Identifying the mechanisms by which the alpha-7 species leads to a poorer prognosis could help to improve the outcome of cervical cancer patients.


Lactobacillus spp. is involved in HPV acquisition and persistence and the development of cervical precancer and cancer. In this review, we summarise the current literature and discuss potential mechanisms for the involvement of vaginal microbiota in the evolution of CIN and cervical cancer. The concept of manipulation of vaginal bacterial communities using pre- and probiotics is also discussed as an exciting prospect for the field of cervical pathology.The vaginal microbiota plays a significant role in health and disease of the female reproductive tract. Next-generation sequencing techniques based upon the analysis of bacterial 16S rRNA genes permit in-depth study of vaginal microbial community structure to a level of detail not possible with standard culture-based microbiological techniques. The human papillomavirus (HPV) causes both cervical intraepithelial neoplasia (CIN) and cervical cancer. Although the virus is highly prevalent, only a small number of women have a persistent HPV infection and subsequently develop clinically significant disease. There is emerging evidence which leads us to conclude that increased diversity of vaginal microbiota combined with reduced relative abundance of  Chlamydia trachomatis infection. Emerging evidence indicates that cervicovaginal microbiota plays a substantial role in the persistence or regression of the virus and subsequent disease. This review will summarise this evidence, discuss possible mechanisms linking vaginal bacteria with cervical pathology and finally consider the potential for future therapeutic strategies.Cervical cancer, the commonest infection-associated neoplasm, and its premalignant precursor cervical intraepithelial neoplasia (CIN), are caused by strains of the human papillomavirus (HPV). Over 100 subtypes of HPV exist with 13 being identified as high risk  and causal of cervical cancer in 100 % of cases . The peaBacteria account for 50 % of the cells of the human body, and together with archaea and lower eukaryotes are collectively termed ‘human microbiota’ , 7. Due Lactobacillus [Lactobacillus crispatus, L. gasseri, L. iners and L. jensenii respectively and tend to have low species diversity and evenness. In contrast, CST IV is typically devoid of Lactobacillus spp. and instead enriched with strict anaerobic species often associated with bacterial vaginosis (BV) including Gardnerella, Megasphera, Sneathia and Prevotella. As will be discussed below, the structure of the vaginal microbiota (VMB) is dynamic and transitioning between CSTs can occur. In the vagina, the most common transition observed is from CST III to CST IV [L. iners may be less able to inhibit colonisation of strict anaerobes and pathobionts compared to other Lactobacillus spp. [L. iners appears more capable of surviving and adapting to a wide range of pH and other metabolic stress-related conditions due to the constitutive and inducible expression of genes not seen in other lactobacilli [The link between health, disease and the human microbiota is a fast-moving and contentious area of research, and an appreciation of the variation in microbiota composition amongst individuals is expanding our understanding of the pathophysiology underlying a variety of diseases affecting many body systems, from colorectal cancer , 12 to abacillus –18. Ravebacillus . In theibacillus , 19, 20.o CST IV , which slus spp.  or becauobacilli , 23.Lactobacillus spp. with associated overgrowth of anaerobes, particularly Gardnerella, Prevotella and Peptostreptococcus species [Lactobacillus and Gardnerella or Mobiluncus morphotypes [BV is a polymicrobial disorder characterised by a microbial community structure consistent with CST IV; that is, by diminished levels of  species . The pre species  and up t species . The Hayphotypes . BV has photypes –31. Howephotypes –34, whicphotypes  and the Lactobacillus spp. dominant microbiota, compared to Hispanic and Black women [Lactobacillus spp. occurs supported by maternal oestrogen. Reduced oestrogen levels 3–4 weeks post birth correspond with a reduction in vaginal Lactobacillus and increased species diversity with enrichment of strict anaerobe and enteric species, which is maintained until puberty [Lactobacillus spp. [Lactobacillus spp. depletion and increased diversity [Lactobacillus spp. dominance appears to be strongly influenced by oestrogen-driven maturation of the vaginal epithelium, which leads to the accumulation of glycogen in vaginal epithelia [Lactobacillus spp. colonisation [The composition of vaginal microbiota is influenced by numerous factors. Ethnicity is a major intrinsic factor known to be significantly associated with variance in community composition, with Caucasian and Asian women displaying a significantly greater prevalence of ck women . These dck women , and vagck women , and is ck women . Female  puberty . Increaslus spp. . Throughlus spp. , 42 whiclus spp. . Followiiversity . Consistiversity . The mecpithelia . Host α-nisation .L. crispatus and increase species diversity. Vaginal douching, particularly after menstruation, has been shown to significantly increase the risk of BV [The widespread use of synthetic hormones for contraceptive purposes also has an impact on the composition of vaginal microbiota. Meta-analysis has shown that hormonal contraceptive use is associated with a 31 and 32 % reduction in recurrent and prevalent BV and 18 % reduced risk of incidence . The stusk of BV , and cessk of BV . A recensk of BV . Interessk of BV , which mLactobacillus spp. presence compared to their uninfected twin [Sneathia spp. to be a microbiological marker of HPV infection , 92 high-grade squamous intraepithelial lesion (HSIL) and five invasive cervical cancer (ICC)) and showed that increasing severity of CIN was associated with higher VMB diversity and decreased relative abundance of lus spp.   of 34.1 for CIN in the presence of hrHPV, compared to hrHPV-negative women. The existence of A. vaginae alone was associated with an OR of 29.9 in HPV-positive women, compared to negative women in the cohort. Interestingly, the OR of CIN in hrHPV positive women with L. iners was 10.9  vs LSIL (n = 90); all women were hrHPV positive) and used the Dirichlet multinomial mixture model to partition samples into four different metacommunities (partitions 1–4), rather than the previously defined CSTs. Unlike the three previously described studies, this group did not find a high vaginal microbial diversity (CST IV/BV-like VMB) to be associated with HSIL. Partition 3 dominated L. iners and unclassified Lactobacillus spp. had higher HSIL levels as compared to those with diverse taxa unclassified Lactobacillus, L. iners, Bifidobacteriaceae, Clostridiales and Allobaculum (partition 1)   producing bacteria [Lactobacillus spp., L. iners is unable to produce H2O2, which has also been shown to have antibacterial and antiviral properties [2O2, which is subsequently present at low levels in the human vagina, and these physiological levels are unable to inhibit growth of BV-associated species in vitro [L. iners often predominates in the presence of HPV infection [Lactobacillus spp.-dominant CSTs [L. iners has many properties that differ from most other Lactobacillus spp., which could explain why this species does not prevent strict anaerobic growth well, and suggests additional or alternative protective mechanisms against dysplasia on the part of other Lactobacillus spp.Previous studies have shown higher rates of bacterial vaginosis in women with lower vaginal levels of hydrogen peroxide  is negatively correlated with CIN [o CST IV . This leo CST IV , they aro CST IV . It is twith CIN .L. gasseri but has since been found to be produced by other strains of L. crispatus and L. reuteri. [G. vaginalis [L. crispatus have been shown to excrete Lactobacillus epithelium adhesin (LEA), a compound that mediates adhesion to the intestinal and genital mucosa, but additionally inhibits pilus-mediated adhesion of G. vaginalis [L. iners produces many of the protective peptides mentioned above. Lack of protective peptides may account for the comparatively high rates of transition observed between L. iners-dominant microbial communities and CST IV [Besides influencing pH, as discussed above, species that are protective may inhibit pathogen growth through expression of bacteriocidal and bacteriostatic proteins such as bacteriocins  which arreuteri. –66. Biosaginalis . Recentlaginalis . There id CST IV . Microbid CST IV , 90, butG. vaginalis in particular has been shown to induce HIV replication in vitro [Lactobacillus spp. or a combination of the two, creates an environment that promotes the viral life cycle, persistence of the infection and ultimately development of dysplasia. BV is commonly diagnosed based on the presence of a characteristic thin, watery foul-smelling vaginal discharge [Prevotella and Bacteroides spp. and are found at significantly higher levels in women with BV [Recently, Borgdorff and colleagues proposed modulation of the vaginal epithelial barrier as an important driver of infection. Using samples derived from a cohort of 50 Rwandan sex workers, they examined changes associated with dysbiosis, which causes increased acquisition of HIV infection, using NGS and a proteomics approaches . Irrespein vitro . It is tischarge , which iischarge  and mucu with BV . Dysbios with BV  and a su with BV , 70 whic with BV .Dysbiosis has also been shown to result in higher levels of oxidative stress , which cG. vaginalis is commonly found in CST IV [G. vaginalis may play a role during this period of greater susceptibility. Sneathia spp. have frequently been identified in association with HPV positivity [Sneathia spp. have also been associated with adverse obstetric outcomes including miscarriage and preterm labour [Leptotrichia amnionii (now re-named Sneathia amnii) is associated with cervical cancer but not HPV infection or CIN [Sneathia spp. belong to the Fusobacterium genus, which has further been implicated in colorectal carcinogenesis [Fusobacterium spp. produce FadA, a virulence factor, which is capable of activating the WNT signalling pathway, a key cell survival and proliferation pathway which is found to be dysregulated colorectal carcinogenesis [F. nucleatum DNA levels have been shown to be inversely proportional to CD3+-T cell counts in colonic mucosa [It is currently unclear if dysbiotic vaginal microbial communities act in synergy with HPV to manipulate its cellular targets such as p53, pRB, survivin and hTERT  or whethn CST IV  and often CST IV . The immn CST IV . It is psitivity  and withsitivity , 55. Furm labour , 103. Prn or CIN . The speogenesis , 106 throgenesis . Fusobacogenesis  and cervogenesis . Fusobacc mucosa , which ic mucosa  and relac mucosa   and were significantly more likely to clear their cervical lesion (60 vs 31 %), compared to an untreated cohort [As described above, fections . This spf action , 118. Fud cohort .L. crispatus, L. jensenii and L. vaginalis in vitro but not Candida albicans, Escherichia coli or G. vaginalis, with the investigators using high-performance liquid chromatography (HPLC) to show that GOS and FOS could not be used as energy sources by the three latter pathobionts [Lactobacilli spp. colonisation in women with C. albicans infection [E. coli to human cheek epithelial cells in vitro [Prebiotics are indigestible carbohydrates, which include the fructo-oligosaccharide (FOS) and gluco-oligosaccharide (GOS) families, which promote the growth of healthy bacteria already present in the body. They have been most well studied in the gastrointestinal tract, where they have been shown to modulate microbiota composition, as well as exert immunomodulatory effects independent of the microbiota (reviewed in ). Severahobionts . GOS, aphobionts . Konjac nfection . Beyond in vitro , suggestin vitro . These rin vitro –130. Whiin vitro .Lactobacillus spp. The impact of other sexual practices such as oral intercourse, use of lubricants and having multiple sexual partners is poorly understood, and the information gathered in the current observational studies is very heterogeneous as highlighted in Table The ability to derive a causal link between vaginal microbiota and HPV infection and CIN/cervical cancer is limited by the cross-sectional nature of most studies undertaken in this area. This difficulty is further compounded by the slow natural history of the disease, with time from acute HPV infection to high-grade CIN taking years to decades. In addition, numerous other confounders can impact results including smoking  and vagiFurthermore, many of the published studies describe the VM in relatively small cohorts with absent or limited representation of a sizeable group of normal, HPV-negative controls for the described comparisons. Oh and colleagues used samples collected from women with both low-grade and high-grade preinvasive disease and as controls grouped women with normal and ASCUS cytology irrespective of their HPV status , includiA number of recent studies have led the scientific community to re-think the traditional conceptual model of the natural history of HPV. Previously regarded as a viral infection which simply causes transient infections or persists as a chronic infection based on studies conducted with protracted sampling intervals, it has now been shown that HPV status can fluctuate quickly over a short time period, based on studies involving very frequent testing over a short space of time , 133. WhAlternative approaches for the assessment of vaginal microbiota and its interactions with the host immune system may also offer useful means to monitor HPV acquisition, persistence and subsequent cervical dysplasia and neoplastic transformation. For example, metabonomics, defined as ‘the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification’ , is emerL. crispatus that colonised women with, and without BV, demonstrated considerable genomic diversity within the species and identified several genes exclusive to the presence or absence of BV [By definition, members of a particular bacterial species have a total nucleotide identity of >70 % across their genomes . The remce of BV . These gAfro-Caribbean women have a fourfold higher prevalence of CST IV VMBs compared to Caucasian and Asian women . They haPre- and probiotics clearly present an enticing novel therapeutic approach to this disease, because they are cheap, easy to administer, with a low side effect profile, unlike the current gold standard treatment for high-grade CIN, which involves a surgical method that carries significant risk to future reproductive outcomes –130. FurThe vaginal microbiota appears to play a role in the acquisition and persistence of HPV in the human vagina and in the subsequent development and progression of CIN. There is a need for further longitudinal studies to prove that these disease outcomes are influenced by VMB composition. This information may present the opportunity for development of novel therapeutic agents in the form of probiotics, to prevent HPV infection, promote its clearance in infected women and negate the risk of cervical dysplasia and future adverse reproductive outcomes that are associated with the current treatment methods –128. MecAlongside the bacterial microbiota, the virome is now a new emerging area of interest. Although we have known for many years that HPV is the aetiological agent in precancerous and cancerous pathologies of the cervix and lower genital tract, other viral genera present in the normal vagina, alongside papillomaviridae, may be involved in disease progression . Further


HIPK2 mRNA levels were analyzed in 20 normal tonsils and 20 TSCC specimens using real-time reverse transcription polymerase chain reaction. Immunohistochemistry of HIPK2 was performed in 79 resected specimens. HIPK2 was expressed in 57% of the TSCCs, and HIPK2 protein expression and HIPK2 mRNA levels were higher in TSCCs than in normal tonsils. HIPK2 overexpression was associated with poorly differentiated carcinoma and low alcohol consumption and was an independent prognostic factor for overall survival and disease-free survival (DFS) in TSCC and a negative independent prognostic factor for DFS in patients receiving postoperative radiotherapy. HIPK2 overexpression had a significant association with poorer DFS in HPV-positive TSCCs, but not in HPV-negative tumors. HIPK2 overexpression may be a potential prognostic marker for predicting prognoses and a high risk of recurrence, particularly in patients with HPV-positive TSCC.Tonsillar squamous cell carcinomas (TSCCs) are the most common human papillomavirus- (HPV-) associated oropharyngeal cancers with poor prognosis. Homeodomain-interacting protein kinase 2 (HIPK2) is a central regulator of p53, which participates in apoptosis during the DNA damage response. HIPK2 is involved in HPV-associated uterine cervical and cutaneous carcinogenesis through its binding of HPV E6, thereby preventing apoptosis and contributing to tumor progression. However, its clinical and prognostic significance in TSCC remains unclear. Tonsillar squamous cell carcinoma (TSCC) is the most common type of oropharyngeal cancer, accounting for approximately 70–80% of all cancers of this anatomical region, representing a highly aggressive malignancy with early lymphatic dissemination , 2. The The inactivation of the oncosuppressor protein p53 by the HPV E6 protein and the retinoblastoma protein (pRb) by the HPV E7 protein has been recognized as a pathogenic mechanism of HPV-associated tumor formation , 16. HIPAs the efficacy of HIPK2 expression as a prognostic and predictive factor for HPV-positive head and neck cancers is yet to be demonstrated, the present study was performed to address this clinically relevant question in tonsil cancers, stratified by HPV status and postoperative radiation therapy.n = 20) as a control group were obtained from patients who underwent tonsillectomy owing to chronic follicular tonsillitis. Tonsil cancer cohorts in the present study had been enrolled in previously published studies  = 2.958, 95% confidence interval [95% CI]: 1.353–6.465; P = 0.004, HR = 3.004, 95% CI: 1.415–6.377, resp.; Kaplan-Meier survival analysis showed that HIPK2-positive patients had shorter OS and DFS than HIPK2-negative patients  in the radiation therapy group. On the other hand, HPV was an independent prognostic factor for the OS of the patients receiving radiation therapy . However, there was no statistically significant difference between OS or DFS and HIPK2 overexpression in the patients with HPV-negative tumors who received radiation therapy (P = 0.277 and P = 0.152, resp.; Multivariate analyses including HIPK2 overexpression, HPV infection, age, pT category, and contralateral nodal metastasis revealed that HIPK2 overexpression was an independent prognostic factor for DFS (In recent years, there has been a growing interest in increasing the risk-benefit ratio in the clinical outcome of HPV-positive locally advanced oropharyngeal cancers. In the present study, we first investigated HIPK2 overexpression in patients with TSCC as a predictor of clinical outcomes related to HPV. HIPK2 overexpression was associated with poorly differentiated tonsil cancer, which is an unfavorable histological factor in patients with TSCC. HIPK2 overexpression was identified as an independent negative prognostic factor and was associated with decreased OS and DFS in patients with tonsil cancer, specifically in those with HPV-positive TSCC. However, these prognostic correlations associated with HIPK2 overexpression were not identified in patients with HPV-negative tonsil cancer. We also found that HIPK2 overexpression was associated with low alcohol consumption in patients with TSCC. This result appears to be partially comparable with those of previous studies referring to smoking and alcohol consumption as risk factors for the development of HPV-negative TSCC . As the  HIPK2 mRNA levels were significantly higher in TSCCs than in normal tonsil tissues. These findings may suggest that HIPK2 overexpression is relatively common in TSCC and may be involved in the tumor development of tonsil cancers. Concordantly, D'Orazi et al. [ HIPK2 mRNA levels in colorectal cancers are higher than those in normal colon cells [HIPK2 was highly expressed in 57% of TSCCs, and HIPK2 protein expression andi et al.  reportedon cells . HIPK2 ion cells , 28, 29,on cells . HIPK2 pon cells , 18, impon cells , 18. In on cells  have shoon cells . E6 inteon cells . Thus, Hon cells . HIPK2 gene, leading to the expression of mutated protein [HIPK2 may be an attractive biomarker and a therapeutic target , particu protein . Here, wThe retrospective procurement of archival samples and the limited number of patients may be limitations of the current study. Nevertheless, certain trends emerged from the results. HIPK2 overexpression may predict a poor prognosis and a high risk of recurrence in patients with TSCC, especially those with HPV-positive tonsil cancers and those receiving postoperative radiation therapy, implying that HIPK2 could be a potential prognostic and predictive biomarker for tonsil cancer.


For both any and high-risk HPV DNA, good agreement was observed between paired samples (Cohen’s Kappa of 0.660 (95% CI: 0.486–0.833) and 0.688 (95% CI: 0.542–0.835), respectively). In addition, significant positive correlations in HPV copies (per microliter of DNA extract) between paired samples were observed for HPV16 -adjusted p = 0.006), HPV18 , HPV31 , HPV53 , and HPV68 . First-void urine sampling using a first-void urine collection device was preferred over a clinician-collected cervical sample. And mostly, first-void urine sampling at home was favored over collection at the clinic or the general practitioner’s office. First-void urine sampling is a highly preferred, non-invasive method that ensures good agreement in HPV DNA (copies) with reference cervical samples. It is particularly interesting as a screening technique to reach non-participants, and its clinical performance should be further evaluated.The performance and acceptability of first-void urine as specimen for the detection of HPV DNA in a Belgian referral population was evaluated using an optimized sample collection and processing protocol. One hundred ten first-void urine and cervical samples were collected from 25- to 64-year-old women who were referred for colposcopy (January–November 2016). Paired samples were analyzed by the Riatol qPCR HPV genotyping assay. Acceptability data were gathered through questionnaires (NCT02714127). A higher high-risk HPV DNA prevalence was observed in first-void urine (The online version of this article (10.1007/s10096-017-3179-1) contains supplementary material, which is available to authorized users. To date, cervical cancer (CxCa) remains a significant problem worldwide, representing the fourth most common cancer in women . The idefirst-void urine) captures mucus and debris from exfoliated cells from the female genital organs, including the cervix. This explains why the first collected part of a urine void contains significantly more HPV DNA than the subsequent part [The use of first-void urine (FVU) as a liquid biopsy for HPV DNA testing is promising. High correlations have been established between urinary HPV DNA and cervical infections –28. The ent part , 29. Theent part , 31. In ent part –37. Thisent part , 39, or ent part , and  concordance between paired FVU and CS in a Belgian colposcopy cohort was investigated. Furthermore, we assessed the HPV copy number agreement between paired samples. Finally, we report on the acceptability of FVU sampling using a FVU collection device.Women (aged 25–64) who were referred to the colposcopy clinic at the Antwerp University Hospital  were screened for eligibility during January and November 2016 (NCT02714127). The criteria included referral due to abnormal cytology or infection with one or multiple HPV genotypes. Patients who underwent cervical conisation in the previous year were excluded. Women who fit the criteria were informed about the study and received an information brochure beforehand. Paired FVU and cervical HPV DNA genotyping results were available for 110 out of 127 eligible patients  prior to their visit with the gynecologist for a CS and colposcopy. Women were requested beforehand to not extensively wash their genitals before the visit at the clinic and to not urinate at least 1 h prior to this visit. Upon collection, FVU samples were immediately placed on ice and transported to the UZA laboratory, where they were aliquoted and preserved on dry ice before storage at −80 °C. For HPV DNA genotyping, one volume of urine conservation medium (UCM) [Upon arriving at the clinic, informed consent was obtained, and it was explained to women how to collect the FVU sample. Standard, illustrated instructions provided by the device company were also available. Women were asked to collect a FVU sample with the Colli-Peeum (UCM)  was addeData from CS ) and colposcopy  from D0 (day of the study visit) ± 3 months were retrieved from the medical records of each study participant. CS were collected with a Cervex-Brush® , which were transferred in 20 ml PreservCyt® collection medium  and analyzed with the ThinPrep® Pap Test (Hologic Europe). During colposcopy, the cervix was visually inspected for abnormalities and graded as normal, low-, or high-grade cervical abnormality. When indicated, a biopsy for histological confirmation was taken. Women scheduled for an ambulant conisation were also included, as were their histology results from the conus. Both LBC and histology were performed at the UZA pathology laboratory and were graded according to the Bethesda and Cervical Intraepithelial Neoplasia (CIN) classifications, respectively. Women graded with different colposcopic and histological outcomes were classified according to the most severe stage. CS were taken, and colposcopies were performed by two colposcopists.UCM-buffered FVU aliquots were thawed (after storage for 2–12 months) and DNA extraction was performed with an in-house protocol . BrieflyHPV DNA genotyping of CS was performed with the Riatol qPCR HPV genotyping assay (Riatol assay), quantifying 12 high-risk (HR) (HPV16/18/31/33/35/39/45/51/52/56/58/59), one probable HR (HPV68), three possible HR (HPV53/66/67), and two low-risk (LR) (HPV6/11) HPV genotypes  and β-glHPV DNA genotyping using FVU DNA extracts was performed with the Riatol assay , 42 accop-values were corrected for multiple testing using the false discovery rate (FDR) method [The percent correct and Cohen’s Kappa (κ) were calculated to assess the HPV genotype agreement between paired samples. In addition, the McNemar’s odds ratio (OR) and test were computed to compare the association between FVU or CS for a given HPV genotype. In case a zero cell count occurred, a constant of 1 was added to each cell in the table . The Wil) method . Statist) method  and IBM n = 32/107), 52.34% with low-grade (n = 56/107), and 17.76% with high-grade cervical abnormality (n = 19/107). Histology results were available from 33 women and revealed that 33.33% of women had cervical intraepithelial neoplasia grade 1 (CIN1) (n = 11/33), 18.18% with CIN2 (n = 6/33), 27.27% with CIN3 (n = 9/33), and 21.21% with no proven CIN (n = 7/33).The median participant age in our study population was 36 (interquartile range (IQR): 29–44 years old), with a gradual decrease in the number of participants per increasing age group. LBC and HR-HPV results are detailed in Table ®) resulted in a median collection volume of 19 ml FVU (IQR: 18–19 ml), collected within a median time span of 1 h and 56 min (IQR: 1:23–2:50) after the previous urination. Samples were frozen in aliquots after the addition of a DNA preservative (UCM) within a median time span of 12 min (IQR: 0:11–0:16) after urination. No invalid hDNA test results (β-globin negative) were reported in the 110 included CS, nor in FVU after in-house ultrafiltration and DNA extraction.The use of a FVU collection device (Colli-Peen = 88/110) and 75.45% (n = 83/110) in FVU and CS, respectively, with a good κ-agreement of 0.660 (95% CI: 0.486–0.833). The same pattern was observed for HPV16/18 and HR-HPV DNA, with a prevalence of 25.45 and 69.09% in FVU, and 23.64 and 66.36% in CS, respectively. A very good and good agreement for HPV16 and/or 18  and HR-HPV DNA  between paired samples was observed, with a McNemar OR of 3.000   and 1.500  , respectively  . In contrast, the observed odds of having a positive result for FVU versus CS was 7-fold higher in the case of multiple infections    good agreement was observed at the genotype level, except for HPV33/39/67 and 6, where moderate and fair κ-agreements were observed, respectively  nor FVU .For all 110 paired samples, β-globin was amplified to determine the amount of hDNA per µl of DNA extract. The median hDNA concentration in FVU and CS was 14.68 (IQR: 6.57–31.08) and 67.79 (IQR: 32.53–117.63) ng per µl of DNA extract, respectively. On the log scale, we detected 0.67 log more of hDNA in CS (1.83 ± 0.04 SE (standard error)) compared to FVU (1.16 ± 0.05 SE) on average. No linear correlation (Pearson) was observed between log hDNA and age for CS  in HPV copies per µl of DNA extract between paired samples were observed for HPV16 , HPV18 , HPV31 , HPV53 , and HPV68  (Online Resource Significant positive Spearman rank correlations (rp = 0.040) and 1.017 log (FDR-adjusted p = 0.040) increase in the median copy number per µl of DNA extract was observed in CS versus FVU, respectively.No significant Spearman’s rank correlations were observed between HPV copies per µl of DNA extract , and the number of cells present in either CS or FVU. For comparison, the log transformed HPV copies per µl of DNA extract and per hDNA equivalent for each individual genotype are represented in Fig. ®-collected FVU sample. The majority of the participants favored FVU sampling  over a clinician-collected sample . Five out of 83 women preferred the Colli-Pee® method if it were as reliable as a smear collected by a doctor/gynecologist. One woman only preferred this method if it could be performed at home. Another woman brought up the issue that no questions could be asked to the doctor/gynecologist. Although ‘no preference’ was not an option in the questionnaire, five out of 113 women (4.42%) wrote down that they did not have a preference between the two methods or checked both boxes. One woman  preferred yearly FVU collection with the Colli-Pee® and bi-yearly smear collection by the doctor/gynecologist. In addition, from the data that was collected, FVU collection at home was preferred over collection at the clinic or the general practitioner’s office. This was demonstrated by 115 out of 122 (94.26%) and 81 out of 120 (67.50%) of women agreeing that FVU sampling was a good method to perform at home versus at the clinic or the general practitioner’s office, respectively  and 66% (n = 73/110) in FVU and CS, respectively. These results are in line with studies where optimized collection and pre-analytical processing protocols were applied [This study provided evidence of a good HR-HPV agreement between paired FVU and CS . Furthermore, very good agreement was observed for HPV16, 18, and 31, with no significant difference in the observed odds of FVU and CS positive for the abovementioned genotypes, nor for HR-HPV. Where others reported a lower HR-HPV prevalence in urine samples compared to cervical specimens , we obse applied , 31, 50. applied , who col applied , 23. How applied  and inva applied , both HP applied . Further applied . The disThe HPV viral load in CS is typically defined as the number of HPV copies per cell, herein referred to as the hDNA equivalent. As similarly reported by Payan and colleagues , no signn = 83/113 (73%) favored FVU sampling). The acceptability of FVU sampling was also described by Leeman and colleagues, comparing the preference of a clinician-collected CS, vaginal self-sample , and FVU sample (Colli-Pee®) in 91 women providing all three samples, with an overall rating of 7.6, 8.1, and 8.6 out of 10, respectively [Lastly, the results obtained from the questionnaires confirmed data from previous studies , 16 repoectively . Notablyectively , these dectively . The useectively , 57, 58.ectively , demonstLimitations of our study need to be acknowledged, most notably the relatively small sample size. As our study was not designed to investigate HPV copy number trend and lesion severity, or to compare the association of FVU and CS for individual HPV-genotypes, one should be cautious with interpreting the results. The acceptability results should be interpreted with caution, as several women wrote down that they had no preference between the two methods, while the option ‘no preference’ was not given in the questionnaire. As this could bias our results, we choose to descriptively report the acceptability data obtained in this study. In addition, despite the heterogeneous character of our study population , participating women were well-characterized. The absence of a home-based setting, which is the purpose of using this liquid biopsy method , will beIn conclusion, in this study, we observed that, when appropriately sampled, stored, and processed, testing FVU for (HR-) HPV DNA ensured a good agreement with CS in a Belgian referral population. At the genotype level, significant positive correlations were observed between paired samples for the three most carcinogenic HPV types in our country, HPV16, 18, and 31, as well as for HPV53 and 68. The median copy number observed in paired samples did not differ except for HPV16 and 68. Furthermore, FVU sampling is highly preferred, non-invasive, and can be performed at home. This method is particularly interesting as a screening tool to overcome the hurdle of screening among non-responders. However, further research is imperative to standardize the procedures involved in HPV DNA detection in FVU and to evaluate its clinical performance in large population-based studies.ESM 1(DOCX 140 kb)


Combined exposure to NP and HSHFD induced macrovesicular steatosis with dilation and congestion of the central vein, liver inflammatory cell infiltration, and expression of genes regulating lipid metabolism, SREBP-1C, FAS, and Ucp2. These results demonstrate that NP aggravates NAFLD in HSHFD-treated rats by up-regulating lipogenic genes, and that HSHFD increases the toxic effects of NP. Thus subchronic NP exposure may lead to NAFLD, especially when combined with a high-sucrose/high-fat diet.Exposure to environmental endocrine disruptors (EEDs) contributes to the pathogenesis of many metabolic disorders. Here, we have analyzed the effect of the EED-nonylphenol (NP) on the promotion of non-alcoholic fatty liver disease (NAFLD) in rats fed high sucrose-high fat diet (HSHFD). Fifty Sprague-Dawley rats were divided into five groups: controls fed a normal diet (C-ND); HSHFD-fed controls (C-HSHFD); and rats fed a HSHFD combined with NP at doses of 0.02 μg/kg/day (NP-L-HSHFD), 0.2 μg/kg/day (NP-M-HSHFD), and 2 μg/kg/day (NP-H-HSHFD). Subchronic exposure to NP coupled with HSHFD increased daily water and food intake ( NAFLD is frequently associated with insulin resistance, dyslipidemia, obesity, and the metabolic syndrome2. Following the epidemics of obesity, NAFLD is emerging as an important public health issue in the world. The highest prevalence of NAFLD with figures ranging between 30 to 50% was reported in South American and Mexican populations3. In China, NAFLD is one of the leading etiologies of chronic liver diseases, with a prevalence ranging from 15.9%4 to 43.3%5 in the general population.Non-alcoholic fatty liver diseases (NAFLD) encompass a wide range of liver pathologies from simple steatosis to steatohepatitis and cirrhosis6. The potential effect of environmental endocrine disruptors (EEDs) on the increased incidence of NAFLD has raised a concern in recent years7. Nonylphenol (NP) is a well-known environmental endocrine disrupting chemical with weak estrogenic activity8, which is used in detergents, emulsifiers, and wetting agents in industry, but is also found in paints, pesticides, and household toiletries9. NP has been detected in human urine samples10 and adipose tissues11. Studies have suggested that NP has deleterious effects on endocrine12, reproductive13, immune14, and nervous16 systems in animals and humans.The pathogenesis of NAFLD is multifactorial with strong genetic and environmental contributions, such as unbalanced diets, over-nutrition, as well as sedentary lifestyles18, such as steatosis19, obesity21, insulin resistance22, and type 2 diabetes23. Wei et al. have demonstrated that bisphenol A exposure during the perinatal period exacerbated non-alcoholic steatohepatitis-like phenotype in high-fat diet (HFD)–treated male rat offspring24. In another study, Boucher et al. have found that polychlorinated biphenyls (PCB) interfere with the normal functioning of vital metabolic pathways in the liver and are associated with the development of NAFLD25. In addition, in our previous study, histopathological study in the liver revealed steatosis with hepatocellular ballooning degeneration in rats exposed to HSHFD plus NP26. Nonetheless, the effect of NP on the promotion of NAFLD development has not been demonstrated yet. Therefore, the present study was designed to test the hypothesis that subchronic NP exposure causes a predisposition to NAFLD. Specifically, we tested whether a combination of NP and high-sucrose/high-fat diet interacts in an additive or an antagonistic way.Recent studies have indicated that exposure to EEDs is associated with an increased risk of metabolic disordersAnimal experimental procedures were approved by Zunyi Medical University Ethics Committee. All methods were performed in accordance with guidelines and regulations of the Zunyi Medical University.Fifty Sprague-Dawley (SD) rats were obtained from the Animal Center of the Third Military Medical University , and received chow diet for 4 weeks. The rats  were randomly divided into 5 groups, each group had 10 rats. The rats were treated for 90 days. The first group fed with a normal diet (SCXK 2012–0012) (C-ND). The rats gavaged with groundnut oil alone (2 ml/kg/day). The second group fed with a high-sucrose/high-fat diet (SCXK2012–0012) (C-HSHFD). The third, fourth, and fifth groups were fed high-sucrose/high-fat diets with increasing concentrations of NP , which was dissolved in groundnut oil, at dose levels of 0.02 μg/kg/day (NP-L-HSHFD), 0.2 μg/kg/day (NP-M-HSHFD), or 2 μg/kg/day (NP-H-HSHFD). The ingredient of HSHFD was lard (15%), sugar (25%) and a normal diet (65%). The rats were raised under controlled temperature (20 ± 1 °C) and humidity (60 ± 5%), on a 12 – hr light (09:00–21:00 hr), 12 – hr dark (21:00–09:00 hr) cycle. Food and water were unlimitedly supplied to the animals.The body weight of rats was measured on day 90. The HSI was calculated as the ratio of the liver weight to the total body weight .27.On day 80, Doppler color ultrasound  was used to detect the liver oblique diameter of rat and identify the rats with fatty liverPentobarbital sodium at a dose of 20 mg/kg was intraperitoneally injected to rats to induce anesthesia. Blood serum was gathered from the abdominal aorta for biochemical studies on day 90. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), and low density lipoprotein cholesterol (LDL-C) activities were measured using a biochemical kit with an automatic biochemical analyzer .28.The rats were euthanized by cardiac arrests, and liver tissues were gathered on day 90. Liver specimens were fixed in 10% formalin and stained with hematoxylin-eosin (HE) to detect hepatic steatosis and inflammation29. The sequences of the primers used are listed in Table Total RNA of lung tissue was extracted using TRIzol . Reverse transcriptase was from Invitrogen , thermal cycler was from Biometra , and electrophoresis apparatus and gel imager were from Bio-Rad . The levels of FAS, SREBP1, and UCP2 were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2, 0.1% sodium dodecyl sulfate (SDS), 2 mM phenylmethylsulfonyl fluoride, 80 µg/mL leupeptin, 3 mM NaF and 1 mM dithiothreitol (DTT). Cell lysates (50 µg protein) were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane, blocked with 5% skim milk, and incubated with primary antibodies. PPARγ and C/EBPα antibodies were from Abcam  and the monoclonal β-actin antibody was from Chemicon . Horseradish peroxidase (HRP)-labelled mouse anti-rabbit IgG was from Jackson ImmunoResearch . After incubation with HRP-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent ECL Assay Kit  according to the manufacturer’s instructions30.Western blotting was performed according to standard procedures. Briefly, cells were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.4% Nonidet P - 40, 120 mM NaCl, 1.5 mM MgClSPSS18.0 packed programs for Windows  was used to perform all calculations. Data are expressed as mean ± standard deviation. The One-way analysis of variance (ANOVA) test and Student–Newman–Keuls (SNK) test were used for the evaluation of differences among the treatment groups. A P-value < 0.05 among groups was considered as the level of statistical significance.p = 4.39E-6). The weight gain in the NP-H-HSHFD group was higher than that in the C-HSHFD group  for 90 days had significantly increased body weight in comparison with control C-ND group  between the individual groups (p = 6.27E-20). Exposure to NP-L-HSHFD, NP-M-HSHFD and NP-H-HSHFD significantly increased the daily volume of water consumed in comparison with the C-HSHFD group . However, NP-M-HSHFD and NP-H-HSHFD exposure increased the daily food intake in comparison with the C-HSHFD group , which identified the rats with fatty liver. There was increased hepatic echogenicity in the NP-M-HSHFD and NP-H-HSHFD groups in comparison with the C-HSHFD group, indicating that NP-M-HSHFD and NP-H-HSHFD exposure results in worse fatty liver Fig. .Figure 5p < 0.05). Liver oblique diameter in the C-ND group (18.62 ± 0.45 mm) was smaller than that in the NP-L-HSHFD, NP-M-HSHFD and NP-H-HSHFD groups  and NP-H-HSHFD (22.12 ± 2.13 mm) groups rather than NP-L-HSHFD (21.15 ± 1.69 mm) was significantly larger than that in the C-HSHFD (20.35 ± 0.47 mm) group (p < 0.05). Exposure to NP-H-HSHFD increased plasma ALT and AST levels in comparison with the C-HSHFD group . There were the accumulation of lipid droplets in the cytoplasm, dilation and congestion of central vein, and inflammatory cell infiltration in the NP-H-HSHFD group Fig. .Figure 9p < 0.05). Similarly, expression of UCP2 mRNA was increased in the groups fed with NP compared to the C-HSHFD group (p < 0.05).Compared to the control C-ND and C-HSHFD groups, expression of liver SREBP1 mRNA was increased in the groups fed with NP, even though it reached statistical significance only in the NP-M-HSHFD group Fig. . Expressp < 0.05). PPARγ protein levels were increased only in the NP-M-HSHFD and NP-H-HSHFD groups compared to the C-HSHFD group  is a common environmental contaminant that is known to disrupt the endocrine system. NP can enter the body by ingestion or adsorption, and may mimic estrogenic action. Consequently, estrogen-like actions of NP have accounted for its adverse effects, such as reproductive malformationsOur results show that the combined exposure to NP and high sucrose-high fat diet results in metabolic disorders, including increased body weight, daily food intake and volume of water consumed, and hepatic echogenicity and liver oblique diameter, demonstrating that NP induces NAFLD in rats. In addition, our data demonstrate that HSHFD promoted the adverse effects of NP. Our data show hepatic steatosis with inflammatory cell infiltration in the NP-H-HSHFD group, indicating that a high-sucrose/high-fat diet combined with NP exposure could cause non-alcoholic steatohepatitis (NASH). Additionally, since the percentage of steatosis increased with the NP concentrations, these data indicate that the degree of NP exposure correlates with the degree of NAFLD steatosis in rats fed with HSHFD.33. The LDL is the major transport vehicle for TG in the blood. Our results showed that the administration of NP coupled with HSHFD increased the plasma levels of ALT, AST, TG, TC, and LDL-C, indicating that the exposure to NP + HSHFD causes a severe disturbance of lipid and protein metabolism. Our observations are in line with previous studies demonstrating the enhanced effect of other EEDs on lipid metabolism disorder in liver tissue35. Our study indicates that NP might regulate the lipid metabolism in adipocytes.Increased serum ALT and AST levels serve as markers of the liver damage caused by EEDs37. SREBP-1c-activated lipogenesis has been suggested to contribute to NAFLD38. FAS catalyzes lipid synthesis in the cytosol39, while UCP2, an anion carrier, inhibits mitochondrial membrane potential and ATP synthesis40. Our results demonstrate that NP exposure increases expression of fat synthesis-related genes, suggesting that NP may increase the lipid levels via the upregulation of SREBP1, FAS and UCP2 gene expression in the liver. These data are consistent with a previous study, which demonstrated that gene expression of SREBP-1C and FAS was increased in liver of EEDs-exposed rats41.Our results also showed that exposure to NP + HSHFD increased expression of SREBP1, FAS, and UCP2 mRNA. SREBP1 is an important liver transcription factor that regulates lipid synthesis and enzymes  catalyzing various steps in the FA and TG synthesis pathways42. C/EBPα is a key transcriptional regulator that plays an important role during adipocyte differentiation and function. A recent study has demonstrated that in 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of PPARγ and C/EBPα was increased43. The current study found that NP-HSHFD combined exposure was associated with the increased expressions of PPARγ and C/EBPα, suggesting that NP induces pre-adipocyte differentiation through altering the expression of these transcription factors. These observations are in accordance with previous publications demonstrating increased expression of PPARγ proteins in the fat tissues of EEDs )-treated rats44. Importantly, our findings provide the first evidence of altered gene expression in the liver after combined exposure to NP and high sucrose-high fat diet.PPARγ is predominantly expressed in adipose tissues, and is considered as the key regulator of adipocyte differentiation and adipogenesis45, which was far above actual environmental exposure level. For example, NP exposure doses changed from 50 mg/kg/day to 200 mg/kg/day in our toxicological studies47. Specifically, our previous study shown that the concentration of NP measured in Xiangjiang River in Zunyi of China ranges from 0.174 to 3.411 μg/L48. In light of the findings, the NP gavage doses of 0.02 μg/kg - 2 μg/kg in rats were designed in this study. In addition, exposure time for NP were short-term acute or subacute period in previous studies49, which was far shorter than the actual exposure time. In order to make up the above shortcomings, the current study designed low environmental exposure concentration and subchronic exposure time, respectively.The unit of NP exposure dose in prior studies was milligram (mg.)27 was to explore qualitatively whether NP exposure might induce NAFLD, therefore, only single NP exposure dose (180 mg/kg/day) was designed, and the unit of NP exposure dose was milligram. The current study is the extension and expansion of the prior study, which has two main differences from the former one: 1. the design of exposure dose. In the current study, NP exposure dose was close to human exposure level, which is microgramme level, and three levels of NP treatment dose were designed to observe a dose-effect relationship. 2. The exploration of mechanism. The detection indicators of lipid metabolism-related genes and proteins in liver were added on the basis of the former study.The objective of our prior studyIn conclusion, the present study demonstrates that prolonged exposure to low NP dose coupled with a HSHFD aggravates NAFLD in rats, as evidenced by increased body weight, increased daily volume of water consumed and daily food intake, increased hepatic echogenicity and oblique diameter of the liver, and increased plasma levels of ALT, AST, TC, TG and LDL-C. Moreover, exposure to NP induces inflammatory cell infiltration in the liver, and increases expression of SREBP1, FAS, and UCP2 in hepatocytes. Importantly, HSHFD promoted the adverse effects of NP. Together, our study indicates that subchronic NP exposure may lead to NAFLD, especially when combined with high-sucrose/high-fat diet.


INK4a staining served as a surrogate marker for HPV oncogene activity. Cell proliferation was determined by Ki-67 staining. Human immunodeficiency virus (HIV) status was evaluated by serology. Data on the consumption of alcohol and tobacco, and history of tuberculosis (TBC), oral thrush, and Herpes zoster, were obtained by questionnaire. Forty patients displayed ESCC, three displayed dysplastic epithelium, and 12 displayed normal epithelium. HPV16 was detected in six ESCC specimens and in one dysplastic lesion. Among HPV-positive patients, viral load varied from 0.001 to 2.5 copies per tumor cell. HPV DNA presence could not be confirmed by ISH. p16INK4a positivity correlated with the presence of HPV DNA (p = 0.03). Of particular note is that the Ki-67 proliferation index, in areas with diffuse nuclear or cytoplasmatic p16INK4a staining ≥50%, was significantly higher in HPV-positive tumors compared to the corresponding p16INK4a stained areas of HPV-negative tumors (p = 0.004). HPV infection in ESCC was not associated with the consumption of tobacco or alcohol, but there were significantly more patients drinking locally brewed alcohol among HPV-positive tumor patients compared to non-tumor patients (p = 0.02) and compared to HPV-negative tumor patients (p = 0.047). There was no association between HIV infection, history of TBC, Herpes zoster, oral thrush, or HPV infection, in ESCC patients. Our indirect evidence for viral oncogene activity is restricted to single tumor cell areas, indicative of the role of HPV16 in the development of ESCC. The inhomogeneous presence of the virus within the tumor is reminiscent of the “hit and run” mechanism discussed for β-HPV types, such as HPV38.This study was designed to explore the role of human papillomavirus (HPV) in esophageal squamous cell carcinoma (ESCC). Fifty-five patients receiving diagnostic upper gastrointestinal endoscopy at Zomba Central Hospital or Queen Elizabeth Hospital in Blantyre  in 2010, were included in our study. Formalin-fixed paraffin-embedded biopsies were collected for histopathological diagnosis. HPV DNA was detected using multiplex Quantitative PCR (qPCR) and in situ hybridization (ISH). p16 Esophageal cancer (EC) is the eighth most common cancer diagnosis, with an incidence of 456,000 cases worldwide in 2012. More prevalent than other tumors, it shows high geographical variation, with high occurrence in a variety of regions, including the Asian esophageal cancer belt in Southcentral Asia, Turkmenistan, and parts of Southern Africa. Malawi belongs to the high incidence area of EC in Africa ,4.In Malawi, ESCC is endemic, with an incidence rate of 24.2 per 100,000 residents, compared to 3.2 per 100,000 in the United States or 6.6 per 100,000 in the U.K. . HPV-relSince the discovery of HPV type 16 in 1984, the role of HPV in human malignancies has become well established . HPV is INK4a reflects the oncogene activity of the deregulated E7 oncoprotein, which inhibits retinoblastoma (Rb) [Tests for the detection of persistent HPV infections in tissue samples are manifold, and include in situ hybridization and target nucleic acid amplification. In particular, polymerase chain reaction (PCR) is used for HPV detection, genotyping, and viral load determination. In situ hybridization detects the viral genome within the histopathological context of a lesion . Determioma (Rb) ,19. INK4a to determine HPV load, HPV prevalence within the histopathological context, the proliferation index, and oncogene activity, respectively. Moreover, further risk factors such as smoking, alcohol consumption, and human immunodeficiency virus (HIV) infection, were also considered. To further explore the role of HPV in ESCC we applied different methods to investigate HPV infection in the tissue samples of patients with ESCC. Our study is the first to combine real-time qPCR, in situ hybridization, and the immunohistochemical staining of Ki-67 and p16A total of 55 patients attending upper gastrointestinal endoscopy in 2010 were included in this study. The tumor group consisted of 40 histologically proven ESCC patients. Three patients showed a dysplastic epithelium in their histopathology. For 12 patients, no histopathological signs of ESCC were found in their biopsies, therefore these patients were defined as non-tumor patients. T in a multiplex real-time PCR assay, in HPV-positive samples, varied from 0.001 to 1.01 HPV copies per cell. When normalizing for the proportion of tumor cells within the sections, viral load varied from 0.001 to 2.5 HPV copies per tumor cell. In four samples, viral load ranged from 1.3 to 2.5 HPV copies per tumor cell.All 55 samples were examined using a multiplex real-time PCR assay for the detection of HPV16, 18, 31, and 45, in two independent runs. Since we could only detect HPV16 DNA, three further independent duplex real-time PCRs for the detection of HPV16 and β-globin were performed for most of the samples. For five samples, HPV16-positivity was reproducible in all runs, and for two samples it was reproducible in 4 of 5 and 3 of 5 runs, respectively. Thus, HPV16 DNA was detected in seven (12.7%) samples at least three times. These seven samples were considered HPV16-positive . One of p = 0.3). Information about grading was available for 39 tumor patients. Grading of ESCC samples among HPV-positive tumor samples was significantly lower compared to HPV-negative tumor samples (p < 0.001). Six of the 7 HPV16-positive cases were tumor patients with histopathologically confirmed ESCCs. One HPV16-positive case showed dysplastic epithelium. The difference in HPV prevalence between tumor patients and non-tumor patients was not statistically significant , of which one sample was HPV-positive  was found in 25 samples, of which 22 were tumor samples.Three tumor samples showed a diffuse nuclear p16positive , Table 1INK4a staining ˂20%, as well as biopsies with only focally stained cells, were defined as p16INK4a-negative. All HPV-positive tumor samples showed either diffuse nuclear or diffuse cytoplasmatic p16INK4a staining ≥20%. Positive p16INK4a staining was significantly associated with HPV status (p = 0.03). This association was still significant when positive nuclear or cytoplasmatic p16INK4a staining ≥50% was taken as the threshold level for p16INK4a-positivity (p = 0.03). Biopsies showing diffuse nuclear or diffuse cytoplasmic p16p = 0.03). Additionally, we determined the Ki-67 proliferation index exclusively in tumor areas with diffuse nuclear or cytoplasmatic p16INK4a staining . This association was also significant when the Ki-67 proliferation index of HPV-positive samples  with nuclear or cytoplasmatic p16INK4a staining ≥20% was compared to HPV-negative tumor samples . All HPV positive samples, overall, showed a high Ki-67 proliferation index. HPV-positive samples  had a higher proliferation index compared to HPV-negative tumors, including dysplastic samples  and non-tumor patients HPV-positivity among ESCC patients was not significantly associated with positive HIV status, when HPV-positive tumor patients were compared to HPV-negative tumor patients  and non-tumor patients . HPV-positivity among ESCC patients was not significantly associated with smoking, when HPV-positive tumor patients were compared to HPV-negative tumor patients  and non-tumor patients . There were significantly more patients who drank locally brewed alcohol among HPV-positive tumor patients compared to HPV-negative tumor patients  and non-tumor patients . HPV-positivity in ESCC patients was not significantly associated with the consumption of alcohol, when HPV-positive tumor patients were compared to HPV-negative tumor patients .The prevalence rate of HPV in both ESCC and benign esophageal papillomas, varies greatly all over the world ,20 and eCt value for HPV16 in qPCR showed that HPV was not present in every cell and, if present at all, only a few viral genomes per cell persisted. Viral load exceeded 1.0 copy per cell in four of the tested HPV-positive samples. The maximum number of copies per cell was 2.5. This is consistent with results from another study examining viral load in ESCC with a multiplex real-time PCR assay. Si and colleagues found less than 10 copies per genome equivalent in 65% of the HPV16-positive esophageal cancer samples [To define the prevalence of HPV within the histopathological context of the tumor samples and to determine viral load, quantitative PCR and HPV-DNA in situ hybridization was carried out. In cervical cancer, the HPV genome is usually present and transcriptionally active in every tumor cell, and a viral load of several hundred copies per cell has been reported ,25. Addi samples . Reasons samples . The wea samples . INK4a-positive samples showed high-risk HPV by in situ hybridization [In situ hybridization was carried out on 13 samples. In situ hybridization staining patterns among HPV-positive tumor samples did not differ from HPV-negative tumor samples . This isdization . In anotdization . Cooper dization . A punctdization . INK4a immunohistochemistry. Transforming viral infections are characterized by an upregulation of p16INK4a, due to the inactivation of Rb as a result of deregulated E7 gene expression. Strong, homogeneous and diffuse staining of p16INK4a throughout the whole cancer tissue is a well-defined surrogate marker for HPV-induced cervical cancer [INK4a staining of the entire lesion is not characteristic for ESCC. Unfortunately, the definition of p16INK4a-positivity in ESCC varies greatly and, thus, complicates direct comparison between studies. In our study, we showed that diffuse p16INK4a staining of ≥20% of tumor cell areas was significantly associated with HPV positivity (p = 0.03). This correlation remained significant when raising the threshold level for p16INK4a-positivity to ≥50% of tumor cell areas (p = 0.03). Our results are consistent with another study investigating p16INK4a expression in ESCC. Cao and colleagues found a correlation between HPV and p16INK4a (86.2% p16INK4a-positive samples among HPV-positive tumors). Strong nuclear and/or cytoplasmatic staining over 70% was defined as positive in that study [INK4a staining of only parts of the tumor tissue is also described for tumors of other sites, such as vulvar neoplasias [INK4a in tumor cells can be lost in the cells due to molecular events, such as promotor methylation or loss of heterozygosity [To assess viral E7 oncogene activity in tumor samples, we performed p16l cancer . In the at study . p16INK4oplasias , carcinooplasias , anal caoplasias , and coloplasias . Moreovezygosity . INK4a-positivity and HPV status in ESCC was evident [INK4a-positivity ranged from ≥10% positive cells to 100%. Clearly, focal staining for p16INK4a, or even patchy staining, most likely reflects numerous physiological processes unrelated to HPV E7 expression [INK4a staining in at least 20% of tumor cell areas, or even in over 50% of tumor cell areas, was significantly higher in HPV-positive tumors than in the corresponding p16INK4a stained areas of HPV-negative tumors . It may be speculated that this increase in the Ki-67 proliferation index is due to the functional inactivation of Rb by the HPV E7 oncoprotein, which would provide support for a role of HPV in ESCC. No other published studies have compared the Ki-67 proliferation index in p16INK4a-positive tumor cell areas in relation to HPV status. Clearly, elevated levels of p16INK4a expression are not exclusive for HPV-induced cancer. As reviewed by Witkiewicz and colleagues, there are two complementary models for the occurrence of tumors with high expression of p16INK4a. First, oncogenic stresses induce p16INK4a, thereby limiting tumorigenic progression. This is followed by the inactivation of Rb, which facilitates disease progression. Second, loss of Rb yields oncogenic stress, which induces p16INK4a [INK4a stained areas differed significantly between HPV-positive and HPV-negative tumors, the first model most likely applies for HPV-negative ESCC. This would align with the observations of Bai and colleagues, who showed that expression of p16INK4a increases during esophageal squamous cell cancer progression [However, there are several studies in which no correlation between p16 evident ,32,42,43pression . Howeverp16INK4a . Since tgression . p = 0.02) and HPV-negative tumor patients (p = 0.047). Locally brewed alcohol may act synergistically with the HPV infection in the pathogenesis of ESCC. The carcinogenic effect of locally brewed alcohol products could be mediated by acetaldehyde, the main metabolite of ethanol [The risk of developing cervical intraepithelial neoplasias increases in patients with an impaired immune system . Howevers p = 0.0. Locally ethanol . Limitations of our study include the low number of patients, especially the low number of HPV-positive tumor patients in the statistical analysis of further risk factors, and the long storage of the biopsies in formalin before embedding in paraffin. A self-generated questionnaire was used for the assessment of further risk factors, as no validated questionnaire for these factors was available in 2010. Not all questions could be answered by all patients. Furthermore, in Africa, patients with ESCC usually present themselves at a late stage of disease . TherefoINK4a-overexpressed tumor areas in HPV-positive cancers, suggest that HPV infection might play a role in a subset of ESCC patients. However, since the HPV genome does not seem to be present in every tumor cell, the theory of “hit and run” [INK4a expression do not correlate [Several observations in our study, such as the presence of HPV16 DNA in cancers and dysplasias only, and the increased proliferation rate in p16and run” , in analand run” . β-HPVs and run” ,52,53,54and run” . In β-HPorrelate . In cervorrelate , which wIn conclusion, the role of human papillomaviruses in the carcinogenesis of esophageal squamous cell carcinomas, still remains elusive. To further investigate the role of HPV in ESCC, more studies, with a stronger focus on molecular events in the context of intratumoral heterogeneity and other risk factors, need to be conducted.INK4a and Ki-67. Fifty-five patients receiving diagnostic upper gastrointestinal endoscopy at Zomba Central Hospital or Queen Elizabeth Hospital in Blantyre in 2010, were included in our study. Biopsies of all 55 patients were taken and immediately fixed in buffered formalin. Sera were collected from 42 patients. Further investigation was done at Jena University Hospital, where the biopsies were embedded in paraffin. Formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut as consecutive sections. The first and the last sections (3 µm) were stained with hematoxylin and eosin for histopathological diagnosis. The sections in-between (15 µm) were used for DNA extraction and subsequent qPCR. Renewed sectioning was performed for in situ hybridization and immunostaining for p16All patients gave their written consent prior to endoscopy (signature or finger print). The study was approved by the College of Medicine Research and Ethics Committee (COMREC) . Consecutive serial tissue sections were prepared for DNA extraction. The first and last sections were hematoxylin-eosin-stained for histopathological diagnosis and to estimate the fraction of tumor cells in the tissue. Ten 15 µm tissue sections were deparaffinized with xylene, re-hydrated through a graded ethanol series, and dissolved in a 500 µL digestion buffer , and 0.5% SDS (Sodium dodecyl sulfate)). Proteinase K (100 µg/mL) digestion was done overnight at 56 °C. For DNA extraction, an equal volume of phenol/chloroform/isoamylalcohol was added and vortexed intermittently for 30 s. After centrifugation, the DNA in the aqueous phase was precipitated by adding 1/10th volume of 7.5 M ammonium acetate and 2 volumes of ice-cold ethanol, followed by another centrifugation step. The pellet was dissolved in 25 µL 10 mM Tris/HCl pH = 8.0. DNA concentration and quality were determined by spectrophotometry .β-globin in one of the multiplex reactions. PCR was performed in an end volume of 25 µL, containing 9.5 µL DNA , 12.5 µL Platinum Quantitative PCR SuperMix-UDG  to prevent amplification of carry-over products [A multiplex real-time PCR assay (TaqMan format ), designproducts , 10 pmolproducts .Reproducibility of the PCR results was assessed by repeating the assay on different days. All samples were tested for HPV16-positivity at least three times. All samples were tested with a primer mixture of reaction 1 twice, and once with a primer mixture containing primers for HPV16 and β-globin only (duplex PCR). For 30 samples, there was enough DNA left to repeat the duplex PCR assay twice . Ct values of the HPV16 genome and β-globin were used to calculate the viral load of HPV16, as copies per cell in 6 of 7 HPV-positive samples, according to the following formula: Ct HPV16: 21.8, Ct β-globin: 23.93, viral load: 2.2 copies per cell), HPKII , and MRI .To assess validity, viral load was determined the same way in HPV16-positive cell lines with a known viral load: SiHa . Hybridized probes were detected by streptavidin-HRP. The FFPE sections were deparaffinized with xylene, re-hydrated through a graded ethanol series, and washed in phosphate buffered saline (PBS). In situ hybridization was conducted using Dako GenPoint HPV Biotinylated DNA probe according to the manufacturer’s protocol . GenPoint HPV Probe contains HPV genomic clones in the form of double-stranded fragments of 500 base pairs or less and multiple biotinylated oligonucleotides from 25 to 40 bases in length. Briefly, the deparaffinized tissue sections were incubated with 0.8% pepsin at 37 °C for 5–10 min, rinsed in deionized water, immersed in 0.3% HINK4a (E6H4) was used according to the manufacturer’s protocol for qualitative detection of p16INK4a proteins in FFPE tissue sections. After adding the substrate-chromogen solution, slides were washed with distilled water and stained with Harris Hematoxylin, followed by washing with tap water. Finally, the sections were dehydrated and mounted with coverslips. Similar to cervical precancers (CIN2/3) and cancer, samples showing a diffuse nuclear p16INK4a staining pattern were considered to be positive. Furthermore, samples showing a diffuse cytoplasmic p16INK4a staining pattern were also defined as p16INK4a-positive. However, in contrast to cervical cancer, p16INK4a staining showed considerable intratumoral heterogeneity. To account for this, p16INK4a-positivity was scored in the context of all tumor cell areas, i.e., biopsies in which 20–50% and ˃50% of tumor cell areas showed diffuse staining were scored as positive and strongly positive, respectively. Biopsies with focal staining or with less than 20% diffuse staining were defined as p16INK4a-negative. The expression of p16 was analysed by immunohistochemistry. A CINtec Histology Kit  containing the monoclonal antibody anti-p16INK4a-positive areas. At least 200–300 cells were counted. Ki-67 staining indices were grouped into negative (<10%), weakly positive (10–20%), positive (20–50%), and strongly positive (>50%).To assess the proliferation activity of tumor cells, the Ki-67 index was determined. Ki-67-positive cells were counted in the area with the highest Ki-67 staining. In addition to this overall Ki-67 index, we determined the Ki-67 index in p16® HIV Integral II, Siemens, Erlangen, Germany) according to the manufacturer’s protocol. The tests were performed in a BEPIII system . Both HIV specific antibodies and the HIV p24 antigen, were detected, ensuring very high diagnostic sensitivity. The HIV status of each patient was determined using a commercially available ELISA system , and oral thrush, were collected using a self-generated questionnaire. Not all questions could be answered by all patients. INK4a, and Ki-67 immunohistochemistry were done by ALG and an experienced pathologist (AG) at Jena University Hospital. Histopathological diagnosis, grading and analysis of p16p < 0.05. To test the reproducibility of the PCR results, Fleiss’s Kappa was assessed. Statistical analysis was performed with IBM SPSS Statistics 22. To assess the relationship between the epidemiological data and clinical data, an exact Fisher test was used for nominal data and a Mann–Whitney U test was used for metric data. Therefore, median and interquartile ranges were determined to describe the variance of the metric variables. Results were considered statistically significant when


Cervical cancer is the second leading cause of death among female patients with cancer in the world. High risk human papillomavirus has causal roles in cervical cancer initiation and progression by deregulating several cellular processes. However, HPV infection is not sufficient for cervical carcinoma development. Therefore, other genetic and epigenetic factors may be involved in this complex disease, and the identification of which may lead to better diagnosis and treatment. Our aim was to analyze the expression of microRNAs in cervical cancer cases positive or negative for HPV E6/E7 mRNA, and to assess their diagnostic usefulness and relevance.The expression of three different microRNAs  in 52 formalin-fixed paraffin-embedded (FFPE) primary cervical cancer tissue samples and 50 FFPE normal cervical tissue samples were evaluated.P < 0.001). MiR-21 and miR-155 expression combined with the HPV E6/E7 mRNA assay in HPV E6/E7 negative cervical cancer showed increased AUC of 0.7267 and 0.7000, respectively , demonstrating their potential as diagnostic tools. Moreover, miR-21 and miR-155 were predictors showing a 7 fold and 10.3 fold higher risk for HPV E6/E7 negative patients with cervical cancer  while miR-155 was a predictor showing a 27.9 fold higher risk for HPV E6/E7 positive patients with cervical cancer (P < 0.0001).MiR-9, miR-21, and miR-155 were significantly overexpressed in cervical cancer tissues compared to normal tissues (There is a strong demand for additional, alternative molecular biomarkers for diagnosis and management of precancer patients. MiR-21 and miR-155 may be helpful in the prediction of both HPV positive and HPV negative cases of cervical cancer. Cervical cancer is the third most common malignancy in women worldwide . High riRecently, several studies reported the development of cervical cancers that are HPV negative despite increased sensitivity of HR-HPV detection methods. Through meta-analyses of HPV detection methods, both Tjalma, et al. and Giorgi, et al. found that 4.2 to 8.2% of cases were HPV negative in 574 invasive cervical cancers and 3162 invasive cervical cancers . SimilarEpigenetic instability is affected by microRNAs (miRNA or miR-). MiRNAs are 19 to 25 nucleotides (nt) in length, and have a role in transcriptional and epigenetic regulation through binding the 3′-UTR of the target-mRNA , 12. It Many miRNAs studies have tried to confirm the utility of each miRNAs in cervical cancers with different methods. Lui et al. used miRNA direct sequencing analysis with six human cervical carcinoma cell lines and frozen cervical tumor tissues . Lee et Among these several miRNAs, three miRNAs  having their revealed targets that might be related to cancer were selected. Ma et al. showed miR-9 increased cell motility and invasiveness by targeting Cadherin 1(CDH1) and lead to cancer metastasis . AsanganAlthough the roles of these three miRNAs  have been studied in cervical cancer, their potential diagnostic or prognostic value in a clinical setting has not been examined. In addition, it is not known whether there is an association between these three miRNAs and HR-HPV infection status in clinical tissue specimens. Therefore, the purpose of this study was to investigate miR-9, miR-21, and miR-155 expression levels in cervical cancer and normal tissue samples, and determine their possible relation to HR-HPV E6/E7 oncogene expression.A total of 52 FFPE cervical cancer tissue samples and 50 FFPE normal cervical tissue samples were used from the Department of Pathology, Yonsei University Wonju Severance Christian Hospital, Wonju, Republic of Korea, between January 2010 and December 2014  was added and vortexed, followed by incubation for 3 min at 56 °C. RNA extraction was performed using the Qiagen RNeasy FFPE kit  according to the manufacturer’s protocol. Total RNA purity and concentration were determined by measuring the ratio of the absorbance at 260 and 280 nm using an Infinite 200 spectrophotometer . All preparation and handling procedures were conducted under RNase-free conditions. Isolated total RNA was stored at −70 °C until used.Complementary DNA (cDNA) was synthesized using a TaqMan microRNA Reverse Transcriptase kit  according to manufacturer’s instructions. Briefly, 5 to 10 ng of total RNA was used for cDNA synthesis. The reverse transcriptase (RT) reaction mixture contained 0.15 μL of 100 mM dNTP mix , 1 μL of 50 U/μL reverse transcriptase, 1.5 μL of 10× reverse transcriptase buffer, 0.19 μL of 20 U/μL RNase inhibitor, and adjusted the total reaction volume to 15 μL with nuclease free water. The cDNA synthesis reaction was performed as follows: 16 °C for 30 min followed by 42 °C for 30 min, and 85 °C for 5 min.T) which is the number of PCR cycles required for the fluorescence to exceed a value significantly higher than the background fluorescence, using the TaqMan small RNA assay (Applied Biosystems by Life Technologies) with miRNA specific primers according to manufacturer’s instructions. Briefly, 1.4 μL of cDNA was added to 10 μL of probe qPCR mix and 7.6 μL of nuclease free water. The following TaqMan small RNA assay (Applied Biosystems) primers were used: hsa-miR-9-5p, hsa-miR-21-5p, hsa-miR-155-5p, and RNU6B. All analyzed miRNAs are of human (Homo sapiens) origin and therefore, the prefix “hsa” is omitted throughout the text. RT-qPCR reactions were performed using a CFX96 Real-Time PCR System Detector . Samples were run in duplicate for each experiment. Data were analyzed using the comparative Ct (2-ΔΔCT) method using the small nuclear RNA, RNU6B, as an endogenous control. To monitor reagent contamination, negative controls were included for each primer pair. PCR cycling conditions were as follows: 95 °C for 3 min 40 cycles of 95 °C for 15 s and 60 °C for 60 s.MiRNA expression was quantified by determining the cycle threshold . PCR primers and the corresponding TaqMan probes were designed for three different sets of HPV regions, with each set of probes targeting their conserved sequence .RT-qPCR reactions consisted of 10 μL of 2 × Thunderbird probe qPCR mix , 5 μL of primers and TaqMan probe mixture, 2 μL of template cDNA, and distilled water for a final reaction volume of 20 μL. The multiplex RT-qPCR assay detected the HPV E6 and E7 genes simultaneously in a single tube by incorporating two targets (E6 and E7) using specific TaqMan probes, which were labeled with different fluorophores . Positive and negative controls were included throughout the procedure. PCR cycling conditions were as follows: 95 °C for 3 min 45 cycles of 95 °C for 20 s and 60 °C for 40 s. To avoid false negatives because of mRNA degradation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.U test were used to determine statistical significance between cervical cancer and normal cervical tissue samples as well as investigate miRNA expression in patients according to HPV infection status. Receiver operating characteristic (ROC) curves were generated to assess diagnostic accuracy of each miRNA, and the area under the ROC curve (AUC) was calculated to measure discriminatory capacity. The best sensitivity/specificity pair was selected based on the maximum likelihood ratio. Univariate and multivariate logistic regression by odds ratio (OR) and 95% confidential interval (95% CI) were performed to assess predictors for cervical cancer diagnosis using the XLSTAT software . All statistical tests were two-sided, and a P value ≤0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism software version 5.02  and MedCalc 9.0 software . Student’s t-test and Mann Whitney Prior to investigating miRNA expression levels, we first examined HPV E6/E7 mRNA expression in 52 FFPE cervical cancer tissue samples and 50 FFPE normal control samples. Fifteen (28.8%) of the 52 FFPE cervical cancer tissue samples were negative for HR-HPV E6/E7 expression (termed HR-HPV E6/E7-negative), while 37 71.2%) samples were positive (termed HR-HPV E6/E7-positive). We found that all 50 FFPE normal cervical control samples were negative for HPV E6/E7 mRNA expression (Table .2% samplP < 0.0001) , 82.7% (95% CI = 69.7–91.8), and 65.4% (95% CI = 50.9–78.0) respectively, while the specificity was 80.0% (95% CI = 66.3–90.0) for miR-9, 72.0% (95% CI = 57.5–83.8) for miR-21, and 96.0% (95% CI = 86.3–99.5) for miR-155. Positive predictive values (PPVs) of miR-9, miR-21, and miR-155 were 77.8%, 75.4%, and 94.3% respectively, while the respective negative predictive values (NPVs) were 70.2%, 80.0%, and 71.6% (Table P < 0.0001), while miR-21 and miR-155 were up regulated in HPV E6/E7-negative cancer tissue samples compared to normal controls . We found no significant difference in miR-9 expression levels between HR-HPV E6/E7-negative cancer samples and normal cervical samples , 0.8135 (95% CI 0.7257–0.9013), 0.8215 (95% CI 0.7349–0.9082), 0.8935 (95% CI 0.8243–0.9626) in HPV E6/E7, HPV E6/E7 + miR-9, HPV E6/E7 + miR-21, and HPV E6/E7 + miR-155, respectively. Especially, in HPV negative cervical cancer, miR-21 and miR-155 in conjunction with the HPV E6/E7 mRNA was shown increased AUC value of 0.7267 (95% CI 0.5776–0.8757) and 0.7000 (95% CI 0.5152–0.8646) respectively, compared to HPV E6/E7 assay  followed by expression of miR-155 , miR-21 , and miR-9 . Ages below 60 years were not a significant risk factor but being over the age of 60 years did confer a higher risk to cervical cancer  and 10.3 (95% CI = 1.5–70.7) fold risk to cervical cancer, respectively   showed significantly higher expression in cervical cancer tissues compared to that found in normal cervical tissues  and interleukin 6 (IL-6) and decreased expression of PDCD4 in cell proliferation and initiates inflammation-associated carcinogenesis via nuclear factor kappa-light chain-enhancer of activated B cells (NF-kB) and interleukin-6 (IL-6) signaling pathways in colon and cervical cancer cells and Asangani et al. found miR-21 down-regulates PDCD4 in colon cancer and functions as stimulating invasion, intravasation, and metastasis , 29. ForIn terms of diagnostic value, miR-21, and miR-155 expression in combination with the HPV E6/E7 mRNA assay may be useful in the diagnosis of cervical cancer independent of HPV infection because these miR-21 and miR-155 may have the discriminatory power to detect HPV negative cervical cancer cases Table . In partSome studies have investigated miRNAs for association with HPV infection as potential diagnostic and prognostic indicators. Xiaohong Wang et al. revealed that miR-92a and miR-378 expression was associated with cancer progression in HPV positive tissue samples . SimilarOur findings showed that miRNA RT-qPCR assays for specific miRs may be useful tools in the diagnosis of cervical cancer and especially, HPV negative cases of cervical cancer. In addition, these findings are important towards determining the possible role of miRNA expression in cervical cancer development, and the relationship between miRNA and HPV infection. Further study is needed in pre-cancer lesions to understand the role of miRNAs in tumor carcinogenesis, and more tests using normal and cervical cancer samples will be necessary to clearly demonstrate the potential utility of these assays for cervical cancer screening and diagnosis.