Country-level HPV genotyping data may be sought by decision-makers to gauge the genotype-specific burden of HPV-related diseases in their jurisdiction and assess the potential impact of HPV vaccines. We investigated, by country, the availability of published literature on HPV genotypes in cervical, vaginal and vulvar cancers and intraepithelial neoplasms  and on prevalence and incidence of genital HPV infections among women without clinically manifest disease.Primary sources of publications were the PubMed/Medline and EMBASE databases. Original studies or meta-analyses published from 2000, covering genotypes 16 and 18 and at least one of genotypes 31/33/45/52/58, were included. Key exclusion criteria were language not English, cervical lesions not histologically confirmed (cytology only), special populations  and, for cervical studies, small population (<50). A total of 727 studies reporting HPV genotype-specific data were identified: 366 for cervical cancers and CINs, 43 for vulvar or vaginal cancers and VINs/VaINs, and 395 and 21 for infection prevalence and incidence, respectively, in general female population samples. A large proportion of studies originated from a small set of countries. Cervical cancer/CIN typing data was scarce for several regions with the highest cervical cancer burden, including Eastern, Middle and Western Africa, Central America, South-East Asia, South Asia, and Eastern Europe. Data for vulvar/vaginal disease was limited outside of Europe and North America.Although a large body of published HPV genotype-specific data is currently available, data gaps exist for genotype-specific infection incidence and several world regions with the highest cervical cancer burden.The online version of this article (doi:10.1186/s13027-015-0008-y) contains supplementary material, which is available to authorized users. Human papillomaviruses (HPV) belong to the most prevalent sexually transmitted infections worldwide , with geAt the country level, decision-makers are likely to seek data on the local genotype-specific burden of HPV-related diseases for baseline information against which the impact of HPV vaccination may be assessed. Focusing on the 7 high-risk (HR) genotypes covered by the 9-valent vaccine , we investigated, by country, the availability of published literature on HPV genotypes in cervical, vaginal and vulvar cancers and intraepithelial neoplasms  and on prevalence and incidence of genital HPV infections among women without clinically manifest disease.The primary sources of data were the National Library of Medicine’s PubMed/Medline and EMBASE databases. Bibliographies were screened to identify additional sources of information. The literature searches covered literature published from 2000 to May 2014; key search terms included epidemiology, human papillomavirus, genotyping, cervix, vagina, vulva, and intraepithelial lesions, cancer, in various combinations.Publications meeting these criteria were included: original study or meta-analysis reporting (1) HPV genotype distribution in histologically confirmed CINs, VINs or VaINs or cervical, vulvar or vaginal cancers, or (2) genotype-specific genital HPV prevalence or incidence in women without clinically manifest disease . Studies were included if they reported on genotypes 16 and 18, and at least one of the additional HR genotypes covered by the 9-valent vaccine . Key exclusion criteria were: publication not in English, small study (n < 50) for prevalence and cervical lesions/cancer studies, special populations , duplicate publication. For each study, data was systematically extracted for country, world region, type and date of study, type of population, age, lesion type, sample type and collection method, HPV assay, number of subjects with valid HPV test results, and HPV genotyping results.Study selection and data extraction were performed by three investigators. One of these investigators performed quality control for accuracy and conformity with selection criteria.http://www.statsilk.com/software/statplanet).Availability of HPV genotyping data was compiled by world region and country . For HPVThe literature searches yielded 11,474 records, of which 1,765 were screened in full-text and 727 included  of all publications originated from just six countries .A large body of data  of these studies originated from Europe, with very few available from Africa, Asia/Pacific and Latin America and the Caribbean Table . Among fA total of 395 publications reported HPV genotype-specific genital prevalence among women without clinically manifest disease.  For ten countries, including Brazil and Turkey, ten or more such publications were available Figure . HoweverA total of 238 larger prevalence studies, reporting genotype-specific prevalence in at least 500 subjects without clinically manifest disease, were identified for 68 countries, which, however, excluded most countries in Africa . Sub-regions that have a notable scarcity of cervical cancer/CIN genotyping data relative to their cervical cancer burden, as estimated by GLOBOCAN 2012 , includeAn additional finding is that, although there may be ample cervical lesion typing and prevalence data in many jurisdictions, genotype-specific incidence data is limited to few available prospective studies. Data, such as age-specific rates of HPV acquisition, are needed for informing dynamic models to obtain more accurate predictions of the population health impact of vaccination.This study has both strengths and limitations. We did not include anal disease in our study, as anal cancer affects both genders and we intended to focus on females. However, as the burden of anal cancer is reported to be rising in Western countries , genotypIn conclusion, although a large body of HPV genotype-specific data on distribution in CINs and cervical cancers and female genital prevalence is available, significant data gaps exist for multiple world regions as well as for type-specific incidence.Additional file 1:Supplementary Material. Full list of 727 references by world region.


Persistent high-risk human papillomavirus (HR-HPV) infection has been implicated in the development of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. Invariant natural killer T (iNKT) cells produce large amounts of cytokines to regulate immune responses. However, the role of iNKT cells in human persistent HPV-infected cervical tissues is unknown.In our study, 201 patients with diagnoses ranging from normal ectocervical tissue to CINIII from June 2010 to May 2012 were enrolled. HPV DNA and HPV types were detected using the hybrid capture-2 HPV DNA test. Flow cytometry was used to investigate iNKT and CD3+ T cell infiltration into cervical tissues. Real-time quantitative reverse transcription-polymerase chain reaction was used to study IFN-γ expression and immunohistochemistry was used to determine CD3+ T cell distribution.p < 0.05), especially in CINII-III (p < 0.01). IFN-γ expression was also increased in HPV-positive cervical tissues (p < 0.05). CD3+ T cells were detected among both epithelium and stromal layers in cervical tissues, and the percentage of CD3+ T cells in HPV-positive cervical tissues was similar to that in HPV-negative cervical tissues (p > 0.05).A significant increase in iNKT cells was observed in HPV-positive cervical tissues (The iNKT cell aggregation in cervical tissues during the progression from HPV infection to CIN indicates that iNKT cells might play an important role in suppressing immunity. IFN-γ expression could also be related to the HPV infection status. Preventing the accumulation or functioning of iNKT cells in cervical tissues may be a viable method to prevent the development of CIN.http://www.diagnosticpathology.diagnomx.eu/vs/2521874671514142The virtual slides for this article can be found here:  Cervical cancer is the third most common cancer worldwide in females, with approximately 529,800 new cases each year, nearly 85% of which occur in developing countries . InfectiInvariant natural killer T (iNKT) cells are a sub-population of T cells characterized by the expression of a restricted αβ T-cell receptor (TCR) repertoire: Vα24-JαQ paired with Vβ11 in humans. iNKT cells also co-express natural killer (NK) cell receptors such as NK1.1 . DistincSeveral recent studies have demonstrated the pivotal anti-tumor effects of iNKT cells and have reported reduced numbers of iNKT cells in patients with malignant diseases, including colon cancer, head and neck cancer, breast cancer, renal cell cancer and melanoma . In a moThus far, nothing is known about the specific role of iNKT cells in regulating local cervical immunity in humans. In this study, we asked whether iNKT cells play a role in persistent human HPV-infected cervical tissues. We demonstrated that in the progression from HPV infection to CIN, iNKT cell accumulation might play an important role in suppressing immunity. Further, IFN-γ expression could also be related to HPV infection status. Preventing the accumulation or functioning of iNKT cells in cervical tissues may be a viable way to prevent the development of CIN.All clinical specimens were obtained from the outpatient department of gynecology in West China Second University Hospital, Sichuan University. Written informed consent was obtained from all patients. Patients with abnormal Pap tests who needed to be diagnosed by biopsy using a colposcope were eligible for this study. All patients were older than 25 years old; patients included in this study had not received any prior cervical treatment within 12 months from study enrollment and did not suffer from other infections (such as herpes simplex virus [HSV] or chlamydia); malignancies and gravidas were excluded. This study was approved by the Medical Ethics Committee of West China Second University Hospital, Sichuan University .HPV DNA specimens were collected with cervical brushes  prior to the application of acetic acid and iodine using a colposcope. HPV DNA was analyzed using the Digene Hybrid Capture® 2 High-Risk HPV DNA Test kit  according to the manufacturer’s instructions. Thirteen HR-HPV types were detected, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.Five hundred microliters of denaturation reagent was added to the specimen transport medium, and the sample was placed in a 65°C water bath for 45 minutes. Then, 75 μl of the mixture and 25 μl of the high-risk HPV probe were transferred to a hybridization microplate, vortexed for 3 minutes, and incubated in a 65°C water bath for 30 minutes. The entire contents were pipetted into a capture microwell and vortexed for 60 minutes at room temperature (RT). After discarding the liquid from the wells, 75 μl of detection reagent 1 was added to the wells for 30 minutes at RT and discarded again. After washing the capture wells with wash buffer 6 times, 75 μl of detection reagent 2 was added to the wells for 15 minutes in the dark. The negative high-risk calibrator and the quality control reagent were tested simultaneously. Finally, the results were analyzed using the Digene assay analysis software DML 2000. Signal strengths with relative light units (RLU)/cutoff (CO) ratios ≥1.0 were considered positive.The biopsy samples were obtained under a colposcope according to a standard protocol that included conventional visual assessment, the application of 5% acetic acid and iodine, the recognition of the transformation zone and the identification of the abnormal area. In each patient, one biopsy was taken from a suspected CIN lesion of the cervix, and another biopsy was taken from a normal area. We harvested the suspected CIN lesion from the most typical and severe parts of the lesions under a colposcope to avoid the possibility of different grades presented in the same specimen to the greatest extent. Each biopsy sample was divided into 2 parts for histopathological diagnosis in the pathology department and for laboratory research. The histopathological diagnosis was reviewed by two senior experts from pathology department in our hospital. The biopsy samples for flow cytometry were transported in media at 4°C and analyzed within two hours. The biopsy samples for real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) were immediately frozen in liquid nitrogen. The biopsy samples for immunohistochemistry (IHC) were immersed in 10% formalin for 24 hours and embedded in paraffin.Anti-human monoclonal antibodies (Ab) to CD3 (Clone UCHT1), TCR Vα24 (Clone Cl5), TCR Vβ11 (Clone C21), IgG1 (Clone 679.1Mc7) and IgG2a (Clone 7 T4-1 F5) were purchased from Beckman Coulter . The FcR Blocking Reagent was purchased from Miltenyi Biotec . The Live/Dead Near-IR Fixable Dead Cell staining kit was purchased from Invitrogen .5 - 1106 cells and were stained with anti-CD3, anti-TCR Vα24 and anti-TCR Vβ11 antibodies or anti-CD3, IgG1 and IgG2a as isotype controls for 30 minutes at 4°C in the dark. Prior to the antibody staining of cells, FcR block and viability stains were added to each tube for 30 minutes on ice in the dark. Negative controls and single-color compensation controls were prepared simultaneously. Stained cells were analyzed on a FACSCalibur flow cytometer .The cervical tissue was cut into the smallest pieces possible in ice-cold media at 4°C. The tissue suspension was transferred to a 70-μm cell strainer. After centrifuging the cell suspensions at 30 g for 3 minutes to remove cell aggregates and debris, the supernatant was then centrifuged at 400 g for 5 minutes at 4°C. The cervical tissue cells were used for flow cytometry by discarding the supernatant. The resuspended cells were divided into 2 tubes with an optimal volume of 100 μl for 510The cervical tissue was ground as finely as possible in liquid nitrogen using a pestle. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). To avoid contamination with genomic DNA, cDNA was synthesized from total RNA using the PrimeScript® RT reagent Kit with gDNA Eraser  and was stored at -80°C.Real-time quantitative PCR reactions were performed in a total volume of 50 μl in the presence of 5 μl of cDNA, 300 nM of forward and reverse PCR primers and 25 μl of iQ SYBR Green Supermix (BIO-RAD). Each cDNA sample was analyzed in duplicate using an ABI Prism 7300 Sequence Detector (Applied Biosystems) under the following conditions: an initial step of 3 minutes at 95°C; 40 cycles of 15 seconds at 95°C, 15 seconds at 55°C and 30 seconds at 60°C; and a final melting-curve step of 15 seconds at 95°C, 30 seconds at 60°C and 15 seconds at 95°C to prevent nonspecific fluorescence derived from unintentional products such as primer-dimers. SYBR Green real-time PCR primers for IFN-γ, CD3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: IFN-γ , forward2O2 solution in methanol at RT for 10 minutes to block endogenous peroxidase activity. The sections were incubated with 10 mM citrate buffer (pH 6.0) at 95°C for 10 minutes to unmask the antigen epitopes and with normal goat serum (NGS) in phosphate-buffered saline (PBS) at RT for 1 hour to prevent non-specific antibody binding. The primary antibody, mouse monoclonal anti-human CD3 , was applied at a 1:50 dilution to sections and incubated in a humidified chamber at 4°C overnight. For primary antibody detection, specific goat polyclonal antibodies were used at a dilution of 1:200 for 30 minutes at RT. After washing in PBS, streptavidin-horseradish peroxidase (Sav-HRP) conjugates were applied to the sections in the dark at RT for 30 minutes, followed by a diaminobenzidine (DAB) substrate solution to reveal the color of the antibody staining. For negative controls, sections were stained with PBS instead of the primary Ab. Normal human normal lymph nodes were used as a positive control. All slides were counterstained with hematoxylin. Positive cell numbers were analyzed by microscopic counting (400×) from 10 randomly selected areas.The formalin-fixed, paraffin-embedded cervical tissue blocks were sectioned at 4-μm thickness and mounted onto SuperFrost/Plus glass slides. The slides were deparaffinized and rehydrated and then incubated in a 3% Ht tests and analysis of variance with Levene’s test. If the distribution was extremely skewed, a nonparametric two-tailed Mann-Whitney U test was used. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD) post hoc test. When the P value was <0.05, differences were considered significant.The statistical analysis was performed using the SPSS software, version 13.0. Results groups are shown as the means ± standard deviations (SDs). Comparisons between groups were performed using two-tailed Student’s From June 2010 to May 2012, a total of 201 patients were enrolled in the study. According to the pathological examinations, the biopsies of all of the cervical tissues were diagnosed as normal ectocervical tissue (NCT), chronic cervicitis, CINI, CINII or CINIII. In the study, 134 patients were classified as HPV-positive (66.7%) by HC-2, 67 of whom were diagnosed with high-grade CIN by a pathologist. In the HPV-negative group, none of the subjects were diagnosed with high-grade CIN. The patient characteristics are summarized in Table n = 48) and an HPV-negative group (n = 24). The proportion of CD3+ T cells in the HPV-positive group was similar to that in the HPV-negative group    in cervical tissues by flow cytometry from an HPV-positive group  Figure A, B. We ) Figure C, D, whie et al. .Figure 1vs. 0.868%, p = 0.528)   and HPV-negative cervical tissue n = 23) for CD3. Immunoreactivity with an anti-CD3 Ab was noted in both epithelium and stromal layers from formalin-fixed, paraffin-embedded cervical tissue sections. There were no significant differences in CD3 expression between HPV-positive and HPV-negative tissues     compared to the HPV-negative group (n = 24)  compared to the HPV-negative group (n = 20)   but was similar to expression in the NCT to low-grade CIN subgroup (n = 27) . IFN-γ expression in the NCT to low-grade CIN subgroup was similar to expression in the HPV-negative group  , are considered a suppressive factor in tumor immunosurveillance ,38. We dFurthermore, we sought to investigate how iNKT cells function in cervical tissues. INKT cells produce IFN-γ in several diseases ,20. In tIn addition, IFN-γ expression in < CINII cervical tissues of the HPV-positive group was higher than that in controls but only slightly lower than that in CINII/III cervical tissues, with no statistical significance. We hypothesize that in the early stages of HR-HPV infection, other factors that produce IFN-γ may have little function in CIN progression during persistent HR-HPV infection. However, our study did not confirm a direct relationship between iNKT cell accumulation and IFN-γ expression. To clarify this point, future experiments are needed.NK cells, as large granular lymphocytes, play an important role in the early stages of immune responses. In addition to cytotoxicity, NK cells function by producing cytokines, especially of IFN-γ . One preIt is possible that different grades of CIN may be present in the different parts of biopsy specimens. In our study, each biopsy sample was divided into two parts for histopathological diagnosis in the pathology department and for laboratory research. As a result, the possibility of grouping bias is inevitable, as the flow cytometry and real-time quantitative RT-PCR destroyed the morphology of the specimens. However, we found that the immunohistochemistry results were consistent with the histopathological grading from the pathology department. Therefore we believe that the grouping bias was at an acceptable level.In conclusion, the results of this investigation indicate that iNKT cells are capable of suppressing the local immune environment in persistent HR-HPV-infected cervical tissues by producing IFN-γ to induce high-grade CIN. Therefore, blocking iNKT cell accumulation may be useful for either treating persistent HPV infections or preventing the progression from CIN to cervical cancer. Further studies are required to confirm our findings.iNKT cell accumulation in cervical tissues during the progression from HPV infection to CIN indicates that iNKT cells may play an important role in suppressing immunity. IFN-γ expression may also be related to the HPV infection status. Preventing the accumulation or functioning of iNKT cells in cervical tissues may be a viable method to prevent the development of CIN.


This retrospective study aimed to investigate the prognostic significance of pretreatment lymphocyte-to-monocyte ratio (LMR) in locally advanced cervical cancer and its effect on overall survival.The usual blood routine test was quantitatively performed to detect LMR. Signal strengths of human papilloma virus (HPV) type DNA in detected cervical cancer samples using hybrid capture 2 were analyzed in relative light units (RLU) compared with 1 pg/mL of HPV type 16 DNA-positive control (RLU/PC) samples. A total of 1.0 RLU/PC (~1 pg/mL) was used as the threshold for a positive result. The HPV-positive specimens were typed using reverse-hybridization line probe assay.The LMR and HPV DNA were found to be independent prognostic markers for 5-year overall survival (OS) and progression-free survival, respectively. Their joint detection may further enhance the predictive value for OS. In the positive HR (high risk)-HPV DNA status subgroup, LMR had a positive effect on improved OS but not in the non-HR HPV DNA status subgroup.The LMR and HR-HPV DNA status can be identified as independent prognostic factors. The different influences of LMR in combined chemoradiotherapy on survival may be related to HR-HPV DNA status. The combined detection of LMR and HR-HPV DNA status may contribute to screening prognosis. Cervical cancer is the third most commonly diagnosed type of cancer and the fourth leading cause of cancer death in women, accounting for 8 % 275, 100) of total cancer deaths among female individuals in 2008 [00 of totInflammation has long been associated with the development, progressive process, and long-time treatment outcomes of cancers. Pretreatment peripheral differential leukocytes  are associated with prognosis of various malignancies, such as breast cancer , gastricTo date, only one study has examined the relationship between differential leukocyte levels and prognosis of patients with cervical cancer, but this study only investigated the prognostic value of the neutrophil-to-lymphocyte ratio (NLR) in cervical cancer . The proTherefore, we performed a retrospective study on the prognostic value of pretreatment LMR in locally advanced cervical carcinoma patients and then investigated its correlation with HPV DNA status and the influence of HPV DNA status on the prognosis of LMR level in the disease. To the best of our knowledge, this is the first study on the association between LMR and prognosis of cervical carcinoma patients.Patients with locally advanced cervical carcinoma, who were initially treated at the Sun Yat-sen University Cancer Center from January 2003 to December 2004, were enrolled in this study. Patient eligibility was based on the following inclusion criteria before treatment: (1) biopsy-confirmed cervical cancer of clinical stage IIb-IVa according to the International Federation of Gynecology and Obstetrics (FIGO) 2009 Staging System; (2) histological types that were squamous cell carcinoma (SCC) or adenocarcinoma (ADC) or their mixture in cervical carcinoma; (3) definitive radiotherapy combined with chemotherapy in any sequence; (4) with complete clinical data; (5) performance status ranging from 0 to 2 based on the Eastern Cooperative Oncology Group system; (6) normal renal, cardiac and liver functions; and (7) with sufficient pretreatment tumor biopsy specimens for measurement of HPV DNA levels before treatment.The exclusion criteria were as follows: (1) distant metastasis under clinical examination or imaging at the time of diagnosis; (2) histological types that were not squamous cell carcinoma (SCC) or adenocarcinoma (ADC) or their mixture in cervical carcinoma; (3) previous chemotherapy; (4) previous radiotherapy to the pelvis region; (5) previous surgery on the primary tumor site before being enrolled in this study; (6) history of other malignant tumors or simultaneous multiple tumors; and (7) a positive pregnancy test result for women of childbearing age.A total of 424 patients were enrolled in this study. Patient consent and approval from the Institute Research Ethics Committee were obtained prior to the use of these clinical materials for research purposes. The clinical characteristics of the locally advanced cervical carcinoma patients are described in detail in Table All enrolled patients received any of the three treatment modalities: inductive chemotherapy plus radiation therapy (IC + RT), concurrent chemoradiotherapy (CCRT), and inductive chemotherapy (IC) plus CCRT.Images taken from computed tomography (CT) with immobilization tools were used to plan external beam radiation (EBRT) and brachytherapy. Each patient received EBRT using 6 MV photons with a 7-field intensity modulated radiotherapy (IMRT) technique to spare some of the small bowels anterior to the iliac nodes. EBRT was administered to the whole pelvis for a total dose of 50 Gy. The daily fraction size was 2.0 Gy, which was administered five times per week for 5 weeks. High-dose rate intracavitary brachytherapy was started 3–4 weeks after EBRT initiation. The median dose of 24 Gy was delivered to point A with 4 Gy per fraction twice a week for 3 weeks. The total dose delivered to point A was equal to or greater than 85 Gy.2) and paclitaxol (175 mg/m2). All concurrent chemotherapy regimens were cisplatin-based. Cisplatin (50 mg/m2) alone or a combination of cisplatin (30 mg/m2) and paclitaxol (135 mg/m2) was administered weekly for 5 weeks during EBRT, beginning on the first day of radiation.IC was administered 3 weeks per cycle for two or three cycles, with a combination of cisplatin . All patients had no self-reported acute infections or hematologic disorders, indicating that the cell counts can represent the normal baseline value. The peripheral LMR was calculated from the differential count by dividing ALC by AMC.Paraffin wax-embedded blocks from 424 patients with locally advanced cervical cancer were collected. Deparaffinization was performed with Xylene to prepare samples for DNA isolation, and DNA was retrieved using the Qiagen DNeasy Blood & Tissue Kit . DNA was extracted using proteinase K digestion and then purified by a modified protocol using the Qiagen DNeasy Kit, as described by Steinau . The extThe HPV-positive specimens were typed using reverse-hybridization line probe assay with 23 type-specific hybridization probes , 14. We Follow-up examination was performed approximately every 3 months for the first 2 years, every 6 months for the next 3 years, and every year thereafter. During the routine follow-up, imaging studies, including pelvic magnetic resonance imaging, thorax and abdomen CT, and bone scan using single photon emission CT, were performed at the predetermined time. When tumor recurrence and/or distant metastasis was suspected based on clinical findings or imaging studies, biopsy of that lesion was performed on a case-by-case basis.The observations ended on December 31, 2012. The assessed endpoints of this study were overall survival (OS) and progression-free survival (PFS). OS was characterized by the time of the initial treatment to death from cervical carcinoma or the last follow-up visit. PFS was characterized by the last date of follow-up or the end date of observation from the initial treatment to distant metastasis or the relapse noted on images in patients who completely responded to treatment, and the locoregional or distant progression of disease in patients who partially responded.p value <0.05 was considered significant.These endpoints were analyzed and compared using the Kaplan–Meier method and log-rank test. Chi square test was used to compare qualitative variables. Univariate analysis was performed to determine the significance of variables using the Cox regression model for PFS and OS. Multivariate analyses with the Cox proportional hazards regression model was used to determine the independent prognostic factors. Spearman’s rank correlation test was used to evaluate the relationship between LMR level and HPV DNA status in terms of some clinical characteristics. The statistical test was a two-sided test performed using SPSS 16.0 programs . A two-tailed According to the detected HPV DNA status from the pretreatment cancer tissue of patients with advanced cervical carcinoma, the patients with positive HR HPV DNA were classified as the HR-positive HPV DNA group, whereas those with negative or low risk-HPV DNA were classified as the non-HR HPV group.Receiver operating characteristic curve analysis was used to define the most appropriate cutoff value for absolute lymphocyte and monocyte counts and LMR to stratify patients at HR of malignancy-related death or progression, thereby avoiding the predetermined cut point. The score selected as the best cut-off value was the one closest to the point with maximum sensitivity and specificity.In this study involving 424 patients with unresectable advanced cervical carcinoma treated with chemoradiotherapy, none had distant metastasis at the time of diagnosis. The median follow-up period was 73 months for all patients and 88 months for patients who did not die of their disease. A total of 129 patients  had distant metastasis throughout the follow-up period, of which 52, 40, 25, and 12 developed bone metastases, lung metastases, liver metastases and distant lymph node metastases in the abdominal or thoracic cavity, respectively. Moreover, 80 patients developed recurrent disease at either local or regional lesion site; the local–regional recurrence rate was 18.87 % (80/424). The 3- and 5-year OS rates were 70.3 and 54.76 %, respectively. Of 148 patients, 52 died from metastatic disease, 54 from recurrent and metastatic disease, and 42 from recurrent disease.9/L  with sensitivity of 77.73 % and specificity of 72.4 %, 0.38 × 109/L , and 5.28  with sensitivity of 85.37 % and specificity of 75.40 %, respectively .A total of 424 patients were eligible for this study. The optimal cut-off values of ALC, AMC, and LMR were 2.35 × 10p > 0.05) compared with the WHO pathologic type. However, the LMR level was significantly different between different lymph node status classifications and FIGO classification (p < 0.05), as summarized in Table p < 0.01).Of the 424 patients with advanced cervical carcinoma, 390 had HR-positive HPV DNA in the cancer tissue samples tested using HC2. As shown in Tables A , IIB , IIIA  and IIIB + IVA , but also on OS  and PFS .In this study, the LMR levels were closely associated with HPV DNA status. Most of the patients with high LMR levels had HR-positive HPV DNA. The significant association between high LMR level and HR-positive HPV DNA rate was observed not only in terms of FIGO classifications II9) and low ALC (<2.35 × 109) groups were analyzed and found to be 82.2 and 64.2 %  and low AMC (<0.38 × 109) groups on 5-year PFS, with 79.5 and 71.8 %, respectively  and low AMC (<0.38 × 109) groups, with 75.5 and 71.5 %, respectively  and low LMR group (<5.28) were analyzed using the he Kaplan–Meier survival analysis and were found to be 75.0 and 61.5 %, respectively. The difference between the two groups was significant in terms of the log-rank test (p = 0.032) and 83.9 versus 50.9 % (p = 0.027) for the high LMR group versus the low LMR group before treatment   and 82.5 versus 52.0 % (p = 0.032) for the HR-positive HPV DNA group versus the non-HR HPV DNA group before treatment . This group was followed by patients with high LMR/non-HR HPV DNA or low LMR/HR-positive HPV DNA, and finally by those with low LMR/non-HR HPV DNA.Each combined expression of LMR level and concurrent HPV DNA status in a single patient before treatment formed three different combined groups, namely, high LMR/HR-positive HPV DNA, high LMR/non-HR HPV DNA or low LMR/HR-positive HPV DNA, and low LMR/non-HR HPV DNA group. As shown in Fig. p = 0.039), although they were not significantly related to PFS  were related to OS, and PFS, respectively, as summarized in Table p = 0.035) of OS, for all the enrolled patients  and superior PFS  and PFS , respectively.In the multivariate analysis, among the three combined modality as mentioned above, the low LMR and concurrent non-HR HPV DNA status were the negative prognostic factors for OS  have been observed to be associated with prognosis of various cancers. Survival outcomes in cervical carcinoma patients are influenced by immune cells , 18 in t9/L) was closely correlated with better OS in unresectable advanced cervical carcinoma under univariate and multivariate analysis. This result is consistent with the study of Choi et al. who reported that high lymphocyte count (≥2.145 × 109/L) remains an independent factor for favorable survival in patients with unresectable advanced cervical carcinoma [The prognostic influence of lymphocyte and monocyte, as the major components of inflammatory response, have been extensively investigated on cancer patients. In the current study, the increased circulating lymphocyte count , for all the enrolled patients. Moreover, high LMR level was confirmed to be an independent prognostic factor for superior survival  did exist. For example, firstly, elevated level of pretreatment ALC and LMR were provided to be significantly correlated with superior OS and PFS under an univariate analysis Table ; SecondlPrevious studies have demonstrated that the hyperactivation of the inflammatory pathways subsequent to viral infection is a driving force that accelerates cancer development, malignant traits, and progression of cervical cancer , 44. MorWe classified the enrolled patients according to the detected HPV DNA status from pretreatment cancer tissue, in order to elucidate the influence of HPV DNA status on the prognosis of LMR level in cervical cancer. We observed that higher LMR level was an independent prognostic factor for superior survival in the HR-positive HPV DNA group. In the non-HR HPV DNA group, the findings were contrary to those in the HR-positive HPV DNA group. No significant difference on PFS and OS was found between the two groups, thereby supporting the concept that the prognostic effect of LMR in advanced cervical carcinoma may be correlated with the HPV DNA status.p < 0.01, both). Moreover, high LMR and concurrent HR-positive HPV DNA were found to be independent predictive markers of OS.We further investigated the clinical value of joint detection of pretreatment LMR level and HPV DNA status in a single patient. Patients with high LMR and concurrent HR-positive HPV DNA showed the best prognostic factor because of their longer survival compared with other groups on OS and PFS (This study has several limitations given its retrospective nature. First, selection bias cannot be completely eliminated in the study cohort. Therefore, caution should be taken while interpreting the results of this study. Second, during stratified analysis, the sample size was too small to provide sufficiently persuasive evidence to support any given conclusion. Based on these limitations, a prospective study should be conducted to provide more evidence to support the conclusions of this study.This study is the first to demonstrate that pretreatment elevated peripheral blood LMR can predict favorable prognosis and is an independent prognostic factor for OS and PFS in locally advanced cervical cancer, along with HR-HPV DNA status. The different effects of LMR in combined chemoradiotherapy on survival may be related to the HR-HPV DNA status of pretreatment cancer tissue in patients with locally advanced cervical carcinoma. The combined detection of LMR and HR-HPV DNA status can contribute in the correct stratification of patients with unresectable advanced cervical carcinoma at HR of unfavorable prognosis. We acknowledge that this finding is limited to a retrospective study in a single center. Thus, further studies are necessary to be performed in a multicenter or prospective manner to validate these important findings.


A formidable threat to the health of women, cervical carcinoma can be prevented in many cases with adequate screening. The current guidelines for cervical carcinoma screening were created as joint recommendations of the American Cancer Society (ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP) and the American Society for Clinical Pathology (ASCP) in 2012, and later accepted and promoted by the American Congress of Obstetricians and Gynecologists (ACOG). The 2012 recommendations underscore the utility of molecular testing as an adjunct to cytology screening for certain women and provide guidance to clinicians based on different risk-benefit considerations for different ages. This manuscript will review screening techniques and current recommendations for cervical cancer screening and human papilloma virus (HPV) testing, as well as possible future screening strategies. Routine screening of cervical cytology with the Papanicolaou (Pap) smear has been deeply ingrained in clinical practice for the past century. Originating in the 1940s and recognized as the standard of gynecologic care by the 1960s, annual cervical cytology screening has been an integral part of the well-woman exam for decades . Mortaliet al., high-risk HPV is a necessary but not sufficient condition for the development of almost all cases of cervical cancer [The development of cervical cancer is strongly associated with previous infection with the human papilloma virus (HPV) and cervical cancer represents 53.4% of the total number of HPV-associated cancers in women [Data suggest that more than half of cervical cancer cases could be prevented through adequate screening. Fifty percent of women diagnosed with cervical cancer have never undergone cervical cytology testing and another 10% have not received screening in the five years preceding their diagnosis . FurtherThe current guidelines for cervical carcinoma screening were created as joint recommendations of the American Cancer Society (ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP) and the American Society for Clinical Pathology (ASCP) in 2012, which were accepted and promoted by the American Congress of Obstetricians and Gynecologists (ACOG) later that same year . These gProtocols for cervical cancer screening have varied since their inception, yet these changes have not demonstrated an impact on the life expectancy for patients with cervical cancer . The lacDespite its benefits, cervical cancer screening is not infallible, nor is it universally available. Women without access to medical care, without health insurance, or below the poverty line have an increased risk of developing cervical carcinoma . AdditioAs the prevalence of cervical carcinoma decreases, clinicians as well as the expert panels who advise them must thoughtfully consider the risks involved with cancer screening. These unfavorable consequences include unnecessary colposcopies or biopsies for lesions which might regress, healthcare costs, the psychological impact of a diagnosis of HPV, and an unknown impact on reproductive outcomes . AuthorsThe association between LEEP and adverse pregnancy outcomes has been a subject of debate for many years. A meta-analysis found no increase in perinatal mortality associated with a history of LEEP . In 2010This manuscript will review screening techniques and current recommendations for cervical cancer screening and HPV testing, as well as possible future screening strategies.Newer liquid-based or conventional smear methods of cervical cytology collection are acceptable for screening purposes ,6. With Cytologic screening, however, is far from perfect. The ATHENA trial of 47,000 women demonstrated that 10% of women who tested positive for HPV 16 and/or 18 had high-grade cervical disease that was undetected by cytology . In addiSince healthcare providers may view the Pap smear as the motivation for annual patient visits, clinicians may be reticent to adopt the practice of less frequent testing. However, ACOG still recommends annual appointments for a well-woman exam  . It is ii.e., inconclusive evidence of cervical cancer or its precursors). However, the joint recommendations released in 2012 recommend HPV testing in conjunction with routine cytology as well as in reflex testing starting in women ages 30 to 65 [Historically, molecular HPV “reflex testing” has been performed to evaluate the need for colposcopy in women with typical squamous cells of undetermined significance (ASC-US) on cytology  aligns with the ASCCP 2012 updated recommendations for the management of abnormal tests [The cobasntervals . The appal tests . In respal tests . The intThere is a growing body of evidence in support of these expert panel interim recommendations. The Joint European Cohort study demonstrated that the six-year risk of CIN3 or higher following a single negative HPV test was significantly lower than the risk following a negative Pap in a group of 24,295 women . An obseAs additional tests are approved by the FDA for primary screening of cervical cancer, the choice of HPV assay may present challenges. A 2014 study of 5064 SurePath samples of women participating in routine cervical screening found considerable disagreement between four human papillomavirus assays, Hybrid Capture 2, cobas, CLART, and APTIMA . The extNevertheless, multiple studies provide mounting evidence for the utility of HPV testing as primary screening and may continue to lead to further changes in recommendations, particularly as the actual impact of new screening guidelines on cancer prevention is investigated . FurtherAccording to unanimous agreement of ACOG, ACS, ASCCP, ASCP, and USPSTF, cervical carcinoma screening should begin at 21 years of age. Young women are commonly infected with HPV within just a few months to a few years after coitarche, but the infection is nearly always cleared by a young, immunocompetent person within one to two years . Women uWomen between the ages of 21 and 29 should receive cervical cytology screening alone every three years  . Co-testversus every two years (four to six cases per 1000) [The evidence for the increase in the interval of screening stems from modeling studies that found no significant difference in the predicted lifetime risk of cervical cancer in women screened every three years (five to seven new cases per 1000) er 1000) . Furtherer 1000) .Women between 30 and 65 years of age should be co-tested with cytology and HPV testing every five years  ,6. Alteret al.’s model suggests that implementation of five-year co-testing will lead to one additional cervical cancer diagnosis out of 369 appropriately-screened women and one additional cervical cancer death among 1639 screened women, as compared to three-year co-testing [Justification for lengthened screening intervals is based on the principle of equal management for equal risk. Because multiple possible screening results are associated with similar risks of the presence of cervical cancer precursors or cancer, as well as the risk of colposopies and other procedures, patients should be managed consistently based on that risk . Data su-testing .According to data from Kaiser Permanente Northern California (KPNC) based on screening results from 1,008,855 women, 2734 additional cervical cancer diagnoses and 615 deaths from cervical cancer would be prevented if the three-year co-testing interval is maintained in lieu of the five-year interval. If instead a two-year co-testing interval was adopted, each cancer prevented would require 92 additional colposcopies and each death prevented would require 408 additional colposcopies [According to the latest consensus guidelines, all screening should be discontinued for women after the age of 65 who have had adequate negative screening ,6. AdequEven if a woman older than 65 has a new partner, the low risk of HPV persistence and progression to cancer with newly acquired HPV infection renders any screening in women over 65 with adequate negative prior screening results unnecessary. Post-menopausal epithelial atrophy, common in this age group, can lead to false-positive results and thus abnormal cervical cytology has a very low positive predictive value . FurtherThe ACS, ASCCP, and ASCP did not find sufficient evidence to suggest that the frequency of screening in any age group should be altered based on prior negative cytology . A matchFor women with a history of cervical cancer, previously treated CIN2, CIN3, existing HPV or HIV infection, immunodeficiency, or a history of diethylstilbestrol exposure in utero, recommendations for screening are more intensive. According to the CDC, HIV-infected women should be screened by cervical cytology twice in the first year following diagnosis and yearly thereafter, regardless of whether or not the diagnosis of HIV is made prior to the age of 21 years . The conAmong women who have had a total hysterectomy, that is, inclusive of removal of the cervix, and who have never had CIN2 or higher, routine cytology screening and HPV testing should be completely discontinued . ContinuConsistent with the 2013 ASCCP updated guidelines for the management of abnormal screening results , the ACSet al., suggests that many HPV infections will be cleared by the immune system within one year [In contrast, if the HPV test is positive in the context of negative cytology, clinicians may either repeat co-testing in one year or perform an HPV genotyping test. Immediate colposcopy is indicated in cases of either subsequent detection of high-risk HPV genotypes (16 or 16/18) or a cytology result of LSIL or higher with a repeat positive HPV test upon one-year follow-up . For casone year .For women previously treated for CIN2 or higher, there is a 2.8-fold increased risk of persistent or recurrent disease for 20 years following treatment that necessitates continuation of routine age-based screening . This reRegardless of HPV vaccination status, the current recommendation is to screen women according to standard age-based guidelines ,6. WhileWhile additional studies are still necessary, it is reasonable to suggest that among individuals vaccinated before the initiation of sexual activity, future guidelines may permit screening in women beginning at age 25 . VaccinaScreening guidelines will continue to evolve with increasing understanding of the relationship between HPV oncoproteins and cervical dysplasia. While highly sensitive, current HPV testing methods lack specificity and positive predictive value . Women wversus Pap followed by HPV testing (with the latter group comprised of women with ASC-US on cytology) [Because HPV testing is more sensitive than cytology, although less specific, a reasonable future screening strategy may be testing for HPV first followed by a conventional Pap test in women with a positive HPV test result. The multi-stage HPV FOCAL trial of 6154 women compared HPV followed by Pap testing ytology) . The firAs molecular screening and vaccination slowly decrease the need for traditional cytological testing, clinical practice guidelines will continue to change. Significant research advances have been made in the prevention, screening, and treatment of cervical carcinoma and will likely continue. HPV testing received a strong endorsement in the 2012 consensus guidelines, yet has not been fully accepted by healthcare providers. A recent survey of 266 obstetricians and gynecologists demonstrated that compliance with guidelines is suboptimal, with solo practitioners especially unlikely to follow both vaccination and screening guidelines relative to those in group practices .In the survey, many physicians also cited patient and personal discomfort with extended screening intervals. Therefore, provider and patient education about the benefits of lengthened screening intervals  in women who are at low-risk for cervical carcinoma is needed . To miniDespite the improvements in screening technology, women without access to adequate health care will not benefit from these advances. Targeted HPV vaccination efforts in lower socioeconomic populations may be the key to reducing high rates of cervical cancer incidence and mortality . The inc


Objective: Uptake of human papillomavirus (HPV) vaccinations by 17- to 18-year-old girls in England is below (<35%) target (80%). This trial assesses (a) the impact of financial incentives on uptake and completion of an HPV vaccination program, and (b) whether impacts are moderated by participants’ deprivation level. It also assesses the impact of incentives on decision quality to get vaccinated, as measured by attitudes toward the vaccination and knowledge of its consequences. Method: One thousand 16- to 18-year-old girls were invited to participate in an HPV vaccination program: 500 previously uninvited, and 500 unresponsive to previous invitations. Girls randomly received either a standard invitation letter or a letter including the offer of vouchers worth £45  for undergoing 3 vaccinations. Girls attending their first vaccination appointment completed a questionnaire assessing decision quality to be vaccinated. Outcomes were uptake of the first and third vaccinations and decision quality. Results: The intervention increased uptake of the first  and third  vaccinations. Impacts were not moderated by deprivation level. Decision quality was unaffected by the intervention. Conclusions: Although the intervention increased completion of HPV vaccinations, uptake remained lower than the national target, which, in addition to cost effectiveness and acceptability issues, necessitates consideration of other ways of achieving it. Human papillomavirus (HPV) is a ubiquitous sexually transmitted virus that could lead to cervical cancer , the secSince September 2008, a national program has been implemented in England and Wales, United Kingdom, aiming to vaccinate girls aged 12 to 13 years against HPV. A 2-year “catch-up” campaign targeting 17- to 18-year-old girls has also been initiated. The objective of these vaccination programs is to provide three doses of the HPV vaccine to females before they become sexually active, when the risk of HPV infection and subsequent cervical cancer development increases. It is estimated that if this objective is met and vaccination coverage is sufficiently high (80% of the target population), up to 400 deaths per year in England could be prevented . AlthougOffering girls financial incentives to undergo the HPV vaccination could increase uptake rates. Financial incentives are increasingly being considered and used in health care policies in the United Kingdom and elsewhere in an attempt to improve health . They arThe exact mechanisms by which financial incentives operate to influence behavior, including vaccination uptake, are currently unknown. In theory, they are likely to work via learning-theory principles, by linking the target behavior to a positively evaluated stimulus , thus strengthening the value associated with it . From anEven if effective in increasing uptake of HPV vaccinations, the use of financial incentives in HPV-vaccination programs will need to be considered in the context of their possible negative consequences. Unlike most interventions designed to change behavior, the use of financial incentives raises particular concerns regarding their potentially adverse effects on the quality of people’s decisions to engage in incentivized behaviors. For example, it has been argued that the prospect of receiving a financial reward could result in the risks associated with the incentivized behavior being overlooked . This isAssessing the quality of health-related decisions is important, as it allows predictions to be made regarding the psychological and physiological adjustment of patients . One wayThe present study is the first trial that addresses the aforementioned uncertainties with regard to the use of financial incentives in HPV vaccination programs. The specific aims of this trial are (a) to assess the impact of financial incentives on initial uptake and completion of an HPV-vaccination program, and (b) to examine whether the impact of financial incentives on uptake and completion of an HPV-vaccination program is moderated by recipients’ level of social deprivation. The trial further aims to assess the impact of financial incentives on the quality of decisions to be vaccinated, as measured by participants’ attitudes toward the vaccination and their knowledge of its health consequences. It was hypothesized that girls who were offered financial incentives to get vaccinated against HPV would be more likely to receive the first and third HPV vaccinations. Participants’ level of social deprivation was hypothesized to moderate the effects of financial incentives on uptake of the first and third HPV vaccinations, with larger effects of the incentives being predicted for the most socially deprived. In line with existing concerns, it was further hypothesized that compared with nonincentivized girls, those offered financial incentives would have less positive attitudes toward the HPV vaccination and would be less knowledgeable about its health consequences.The present study is is a parallel-group randomized controlled trial. Further details of the study methods are available in the trial protocol .2Between 2008 and 2009, the uptake rates in England for the “catch-up” HPV-vaccination campaign, targeting females aged 17 to 18 years, was 62.2% for the first vaccination, 54.2% for the second vaccination, and 31.8% for the third vaccination . The equParticipants were one thousand 16- to 18-year-old girls: 500 had not yet received an invitation to attend the vaccination program (first-time invitees), and 500 had previously received an invitation to get vaccinated, but had failed to attend the first vaccination appointment (previous nonattenders). All girls lived in Birmingham, United Kingdom, and (a) were registered with general practitioners within the BEN and Heart of Birmingham Primary Care Trusts; (b) were eligible to be vaccinated through the participating community clinics ; and (c) had not been vaccinated against HPV before.Participants were selected randomly from a list of names of all girls aged 16 to 18 years registered with participating general practitioners see . After eAll participants received letters, addressed to them, inviting them to attend their first HPV vaccination session. The letters included the date, time, and venue of their allocated vaccination appointment. Participants were given the option to reschedule their appointment or attend a different immunization clinic by contacting the immunization team at a designated telephone number, included in the letter.Along with the invitation letters, all participants were sent a leaflet containing information about HPV and the HPV vaccine. This was the standard leaflet used and distributed by the NHS.Participants in the intervention groups received an invitation letter, which included the offer of Love2Shop vouchers worth £45  for receiving the three vaccinations.The vouchers could be exchanged at numerous stores in the United Kingdom, including general merchandise and department stores; fashion and footwear retailers; specialist retailers ; jewelry shops; sports, outdoor, and motoring stores; home improvement and soft furnishing stores; restaurants; and leisure facilities .The total amount was based on the only existing study of which we are aware, which assessed the impact of incentives on uptake of a vaccination requiring completion of three doses . In the Participants in the intervention groups received text messages reminding them of their second and third vaccination sessions. These were sent during the intervals between the first and second vaccinations, and between the second and third vaccinations, and 2 days prior to the next session. The wording of these messages was, “(Name), don’t forget your HPV jab on (day) at (time) at the (venue). Thank you.” Participants were not able to reply to these messages. Participants in the control groups did not receive these reminder text messages.Uptake of each vaccination was recorded at the community clinics where vaccinations took place and was measured as the proportion of those invited who received each vaccination.Area-level social deprivation was measured using participants’ postcodes to calculate English Index of Multiple Deprivation (IMD) scores, which range from 0.37 (least deprived) to 85.46 (most deprived) . The IMD is a measure of deprivation in England based on area of residence. It measures deprivation at the small-area level, that is, the Lower Layer Super Output Area. It is derived from seven indices of deprivation, including income, employment, health deprivation and disability, education skills and training, barriers to housing, and services and crime. These are combined into a single deprivation score for each small area in England.1not at all good) to 7 (extremely good) and (b) 1  to 7 (extremely harmful).” Scores obtained on the latter item were reverse coded.Two items rated on a 7-point scale, assessing attitudes toward the HPV vaccination: “For me, having the HPV vaccination is (a) 1  of three statements relating to the vaccination: “If I have the HPV vaccination . . .,” (a) “I am less likely to get cervical cancer”; (b) “I am less likely to get other sexually transmitted diseases”; (c) “I am less likely to get pregnant.”To assess the impact of financial incentives on the quality of decisions to undertake the HPV vaccinations, a short modified version of a validated measure of informed choice was used , consistParticipants were also requested to state their main reason for getting vaccinated.The original measure of informed choice was developed for use in the context of prenatal screening and consists of eight items assessing knowledge and four items assessing attitudes . The comThe financial incentive scheme was run by the BEN Primary Care Trust (in partnership with the Young Foundation). Participants were selected and recruited into the scheme by the Birmingham Primary Care Shared Services Agency, who holds and controls all patient data in Birmingham, United Kingdom. Recruitment took place between February and March of 2010.The vaccination sessions were conducted at three community clinics  between March and September 2010, by nurses working with Heart of Birmingham Primary Care Trust. When attending their first session, participants signed a consent form, completed the measure of informed choice, and selected a date for their next vaccination. Delivery of each vaccination was contingent on completion of all previous doses. After getting vaccinated, participants in the intervention groups were handed the appropriate vouchers.The intervention was run by Healthy Incentives, a social enterprise arising as a result of a partnership between the Young Foundation and the NHS BEN Primary Care Trust. The use of vaccination invitation letters, information leaflets, and reminder text messages were part of the latter’s standard health services. Also part of the Primary Care Trust’s standard practice is the use of records reflecting attendance in vaccination programs of all eligible patients registered within the Trust. These records are routinely used to create reports. The offer of financial incentives was introduced as part of the Primary Care Trust’s innovation in health service development and delivery. Permission to implement the incentive scheme was granted by the Primary Care Trust, which was responsible for all related insurance and indemnity arrangements. Participants’ anonymity and privacy were protected by the Data Protection Act . The desTo assess the impact of the intervention on initial uptake  and completion of the HPV-vaccination program , logistic regressions were performed separately for first-time invitees and previous nonattenders. To test the moderating effect of social deprivation on the impact of the intervention, the interaction between IMD scores and intervention was added to the logistic regression models. To test whether there was a difference in the effect size of the intervention in the two samples, data sets were combined and another logistic regression was conducted, in which sample  was added as a predictor of the model, along with the intervention. The chi-square test was used to test for differences in attrition rates from the first to the third vaccination between the intervention and control groups. Differences in knowledge of the HPV vaccination between intervention and control groups were tested using the chi-square test, and differences in attitudes toward the HPV vaccination were examined using one-way analysis of variance. All tests were assessed at the 5% level of significance.All groups were comparable in age and social deprivation see . Data meOR = 1.63, 95% CI ; see OR = 1.63, 95% CI ; see OR = 0.611, 95% CI , p > .05).Financial incentives significantly increased initial uptake of the HPV-vaccination program by approximately 10% see  in both OR = 2.152, 95% CI ; see OR = 4.283, 95% CI ; see OR = 0.494, 95% CI ; p > .05).The combination of financial incentives and text messages significantly increased completion of the HPV-vaccination program by about 10% see  in both 2 = 0.50, p > .05. For previous nonattenders, the attrition rate was 11.2% in the intervention groups versus 7.4% in the control group, χ2 = 2.39, p > .05  or previous nonattenders .The effect of the intervention on uptake of the first and third vaccinations was not moderated by social deprivation in either first-time invitees  and knowledge similarly high , χ2 =  = 9.017, p > .05.The quality of decisions to undergo the HPV vaccination was similar in the two trial arms: attitudes were similarly positive .Consistent with the hypotheses, the offer of financial incentives increased the proportion of girls undergoing an initial HPV vaccination, and the combination of financial incentives and reminder text messages increased the proportion of girls completing the course of three HPV vaccinations. Contrary to predictions, these effects did not vary with level of social deprivation. Also contrary to predictions, there was no evidence to suggest that girls’ attitudes toward the HPV vaccination and their knowledge of its health consequences were affected by the offer of financial incentives.Findings from the current study are consistent with previous research demonstrating the effectiveness of financial incentives in promoting immunizations . IncentiThe incentives increased uptake of the vaccination by 10%, a surplus that the intervention maintained throughout completion of the program, without reducing attrition between the first and third vaccinations. This finding could be taken as an indication of the superior effectiveness of the initial incentive, which may have been sufficient in maintaining the higher uptake rates without the subsequent incentives. It is not possible, however, to infer exactly how removal of the additional incentives would have affected the results. The relative effectiveness of the incentives for each HPV vaccination should be examined in future research.One potential advantage of financial incentives is that they may be more effective in motivating behavior change in the most socially deprived . ContrarAlthough the intervention in the present study was effective in promoting the HPV vaccination, the highest uptake rates observed were 28.4% for the first vaccination and 22.4% for the third (by first-time invitees in the intervention group). These figures are well below the 80% uptake target set by the NHS. They are also considerably lower than the attendance rates for the catch-up program in the BEN Primary Care Trust, where this trial was run  was not large enough to unduly influence their decision to get vaccinated had they held strong views against doing so. However, some limitations associated with the measurement of informed choice in this study reduce the certainty of the conclusions that can be drawn. First, as mentioned previously, we used a simplified version of a measure of informed choice originally developed and validated for use in the context of screening. The resulting measure may not have been sensitive enough to adequately assess informed choice in relation to HPV vaccination uptake. Further research is needed to validate the measure for use in this specific context. Second, the reliability of the measure was relatively low (Cronbach’s α = 0.63). Although related concerns might be partially alleviated by the questionable stability and appropriateness of the alpha coefficient for two-item scales , interprWhile the incentive scheme increased uptake of HPV vaccinations, it is unknown whether such effects can be achieved in more cost-effective and acceptable ways. Other HPV-vaccination programs, such as those rolled out in schools , have acDaily Mail online wrote, “HMV Voucher Bribe for Teenage Girls to Have Cervical Jabs: Fury at ‘Promiscuity Scheme’ as NHS Faces Cuts” (Even if cost effective, the use of incentives for increasing HPV vaccinations will depend on their acceptability to policymakers, health professionals, and the public. The use of financial incentives for health promotion attracts negative views , which, es Cuts” , and wides Cuts” .7The combination of financial incentives and reminder text messages increased uptake and completion of HPV vaccinations. Even with this intervention, however, the vaccination rates were considerably lower than the national target of 80%. Further research could attempt to elucidate the conditions under which the impact of incentives could be improved. However, it seems unlikely that the use of incentives alone will be sufficient to achieve effective HPV vaccination coverage targets in the United Kingdom. This, in addition to the cost effectiveness and acceptability issues surrounding the use of incentives in this context, perhaps highlight the need to consider alternative ways of achieving such targets.


To identify human papilloma viruses (HPV) in atheromatous coronary arteries.Atheromatous arterial disease is primarily an initial inflammatory response to unknown stimuli. The crucial question is “what causes the initial inflammation in atheromatous disease?” HPV infections may be relevant as US women with vaginal, high risk for cancer, HPV infections, are at up to threefold increased risk of cardiovascular disease as compared with vaginal HPV-negative women. These studies did not include analyses of HPV in atheromatous coronary arteries.Atheromatous coronary arteries were identified and collected from 20 deceased donors. Polymerase Chain Reaction techniques were used to identify HPV gene sequences. Immunohistochemistry methods were used to identify HPV E7 proteins.p = 0.024). The HPV E7 proteins were expressed in smooth muscle cells and plasma cells, foam cells, and macrophages located in the atheromatous plaque. HPV E7 proteins were not expressed in infiltrating lymph cells.HPV types 16 and 18 were identified in 11 (55%) of 20 specimens. HPV E7 protein was identified in 10 (50%) of 20 specimens. Positive and negative HPV identification and HPV E7 expression in coronary smooth muscle cells were significantly correlated (cc = 0.503, HPV gene sequences were identified in 55% of atheromatous coronary arteries and may have a role in coronary artery disease. Atheromatous arterial disease begins as an inflammatory disease , 2. The Human papilloma virus (HPV) infections may be relevant as Kuo and Fujise have demonstrated that US women with vaginal, high risk for cancer, HPV infections are at approximately threefold increased risk of cardiovascular disease as compared with vaginal HPV-negative women . This stHowever, Kuo and Fujise study did not include analyses of HPV in atheromatous coronary arteries. As we have not identified previous studies of HPV in coronary artery disease, we have conducted a preliminary study of HPV in coronary arteries from 20 deceased donors, all of whom died because of a myocardial infarction. We show that HPV gene sequences appear to be present in up to 55% of atheromatous coronary arteries.Archival formalin-fixed atheromatous coronary arteries were selected from 10 donors aged below 35 years, and 10 donors aged 35 years and over, from the Department of Forensic Medicine, Sydney, NSW, Australia. Younger and older age groups were selected as there may have been differences in the prevalence of HPVs identified in their coronary arteries. The cause of death for all 20 donors was a myocardial infarction due to atheromatous disease (as determined by Forensic Pathologists).We sought to identify HPV by polymerase chain reaction techniques (PCR). It is important to note that HPV gene sequences in coronary artery material can be best identified by nested PCR, a method, which involves two sets of primers, used in two successive runs of the PCR.Three HPV-positive and three HPV-negative specimens were selected from the 20 coronary artery specimens and were analyzed by PCR methods in an independent laboratory  using methods as per Antonsson et al. . PCR wasRB by HPV E7 protein. Positive outcomes of immunohistochemistry (IHC) analyses for these HPV proteins can offer supportive, but not conclusive, evidence with which to identify biologically active HPVs. Antibodies for the specific identification of HPV E7 proteins have recently been developed and made available on a commercial basis  was performed during the DNA extraction procedure. The gDNA extracts were quantified with a NanoDrop spectrophotometer (Thermo Scientific). The DNA quality was tested by the amplification of a 268-bp fragment of the β-globin gene using primers G073 (5’-GAAGAGCCAAGGACAGGTAC-3’) and G074 (5’-CAACTTCATCCACGTTCACC-3’). The HPV genotypes were identified by BLAST via the US National Center for Biotechnology Information.Round 1. My11 (5′GCACAGGGYCAYAAYAATGG3′) to modified Gp6 (5′AATCATATTCCTCTTCATGTC3′).Round 2. Gp5 (5′TATTTGTTACTGTKGTWGATAC3′) to Gp6 Negative controls [no DNA (water) and a reagent blank] were used in parallel with all PCR analyses. Positive control (HPV18) was DNA extracted from HeLa cells.Cycling conditions using Hot Start Taq Master Mix kit from QIAGEN: 95°C 15 min 1 cycle followed by 30 s 95°C, 30 s 55°C, 30 s, and 72°C 35 cycles.Human papilloma virus identification was considered positive if sequences were identified at least twice in DNA extracted from the same specimens.Identification of EBV gene sequences by PCR was conducted on the same genomic DNA extracts as for HPV. The primers were as described in Glenn et al. .Standard manual IHC methods were used. The antibodies were HPV E7 monoclonal “Cervimax” – Valdospan GmbH. Austria .The E7 antibody was optimized at 1/100 dilution. E7 proteins may be identified by Cervimax from a wide range of HPV types including HPVs 18, 39, 45, 59 of species 7, and HPV 16, 31, 33, 35, 52, 58 of species 9.Human papilloma virus types 16 and 18 E7 proteins are detectable in both cell nuclei and cytoplasm. The outcomes were assessed by the intensity of staining on a scale of 0 and + to +++. Cervical cancer tissues were used as positive controls and normal breast tissues were used as negative controls. IHC with omission of the E7 antibody was negative.p value of equal or less than 0.05 was considered to be statistically significant.The correlations between the identification of HPV gene sequences and the expression of HPV E7 proteins were assessed with two-tailed Spearman’s non-parametric correlation statistic using the SPSS statistical package. A two-tailed As shown in Table A cross-sectional image of HPV type 16-positive atheromatous coronary artery disease is shown in Figure Human papilloma virus type 18 was identified in two of the three HPV-positive coronary artery specimens  by the independent laboratory.EBV gene sequences were identified in 6 (46%) of 13 coronary artery specimens (sound outcomes were not achieved in 7 specimens). This data is not shown in Table p = 0.024, two-tailed Spearman non-parametric correlation).Human papilloma virus E7 was identified in 10 (50%) of 20 specimens as shown in Table As shown in Figure Positive HPV E7 protein expression was observed in smooth muscle cells in the walls of coronary arteries (not in the atheromatous plaque).It is difficult to obtain normal coronary arteries to act as negative controls for HPV E7 (Cervimax) antibodies. For this reason, we have shown images in Figures In this study, both HPV types 16 and 18 gene sequences have been identified by PCR analyses in up to 55% of atheromatous coronary arteries. HPV E7 proteins were identified in 50% of the 20 coronary artery specimens. There was a consistent correlation between the identification of HPV gene sequences and the expression of HPV E7 proteins in coronary artery smooth muscle cells. However, the mere presence of HPV in coronary artery atheromatous plaques does not infer or conclude any mechanistic role of HPV in atherosclerosis.Epstein–Barr virus sequences were identified in 46% coronary artery specimens (data not shown). This observation corroborates the previous identification of EBV in coronary artery disease .An important limitation of this study is the lack of controls. This was due to the high prevalence of atheroma in coronary arteries of potential control donors who died in motor vehicle accidents.Polymerase chain reaction amplification techniques are notoriously liable to contamination. However, false-positive PCR outcomes are unlikely as two HPV types were identified. If contamination had occurred, HPVs of the same types and sequences in a majority of specimens would have been observed. In addition, HPV type 18 was identified in the same specimens by an independent laboratory using different PCR methods and primers. This is an indication that the findings are valid. The positive identification of HPV E7 proteins also offers supportive, but not conclusive, evidence of the validity of the outcomes.The correlation between the identification of HPV gene sequences and the expression of HPV E7 proteins in coronary artery smooth muscle cells is of particular interest. This correlation is specific to smooth muscle cells and not to other types of cells in atheromatous coronary arteries. It is of particular interest that positive HPV E7 staining was observed in smooth muscle cells within the walls of coronary arteries in addition to within atheromatous plaques. This suggests that HPVs may have influences within the wall structure of blood vessels. Bonin et al.  have shoThe concept that HPV and other infections may have an initiating role in atheromatous coronary artery disease is biologically plausible. This view is based on the following evidence: (i) initially atheromatous arterial disease is primarily an inflammatory phenomena , (ii) HPThere are many factors, which contribute to atheromatous vascular disease including low density lipoproteins, cigarette smoking, hypertension, diabetes mellitus, and genetic susceptibility. The possibility that infections may have a role in atheromatous vascular disease is highlighted by the long-term pattern of deaths due to myocardial infarction. Deaths due to acute myocardial infarction among Western men and women rose dramatically  between 1920 and 1950 and then fell equally dramatically to the 1980s . These dOther viruses may alter lipid metabolism in coronary cells. For instance, atherosclerotic lesions are induced by infections with Marek’s disease herpes virus in chickens , herpes It is commonly accepted that HPV infections are predominantly transmitted during sexual activities and that they are confined to epithelial cells. There is evidence that neither of the notions is wholly true. HPVs have been repeatedly identified in white blood cells in circulating blood of normal subjects and patients with HPV-associated cervical cancer . HPVs haWhen considered together with these many prior observations, plus the epidemiological evidence that women with HPV genital infections are at up to threefold increased risk of heart attacks or stroke, the notion that HPV infections may have an active role in atheromatous vascular disease is of considerable interest. However, the data do not exclude the possibility that HPVs in atheroma and coronary artery smooth muscle cells are merely opportunistic parasites and not causal.Our preliminary investigations need to be confirmed, extended, and evidence of HPV biological activity in coronary arteries needs to be developed.All authors contributed substantially to the present work, revised the work critically, approved the final paper, and agreed to be accountable for all aspects of the work.This study was formally approved by the Human Research Ethics Committee of the Sydney Local Health District number 13/0048 and by the University of New South Wales Human Research Ethics Committee number HREC HC11421.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


INK4a. Whether HPV-triggered carcinogenesis in pSCC has an impact on tumour aggressiveness, however, is still subject to research.Up to 50% of penile squamous cell carcinomas (pSCC) develop in the context of high-risk human papillomavirus (HR-HPV) infection. Most of these tumours have been reported to show basaloid differentiation and overexpression of tumour suppressor protein p16INK4a immunohistochemistry and DNA extraction followed by HPV subtyping using a PCR-based approach. The results were correlated with histopathological and clinical parameters.In tissue specimens from 58 patients with surgically treated pSCC between 1995 and 2012, we performed p16INK4a staining patterns was observed in 58.6% of samples regardless of histologic subtype. Sensitivity of basaloid subtype to predict HR-HPV positivity was poor (11.8%). In contrast, sensitivity and specificity of p16INK4a staining to predict presence of HR-HPV DNA was 100% and 57%, respectively. By focussing on those samples with intense nuclear staining pattern for p16INK4a, specificity could be improved to 83%. Both expression of p16INK4a and presence of HR-HPV DNA, but not histologic grade, were inversely associated with pSCC tumour invasion . However, none of these correlated with nodal involvement or distant metastasis. In contrast to pathological tumour stage, the HR-HPV status, histologic grade, and p16INK4a positivity failed to predict cancer-specific survival.90.4% of tumours were of conventional (keratinizing) subtype. HR-HPV DNA was detected in 29.3%, and a variety of p16INK4a, rather than histologic subtype, as a good predictor for presence of HR-HPV DNA in pSCC. HR-HPV / p16INK4a positivity, independent of histological tumour grade, indicates a less aggressive local behaviour; however, its value as an independent prognostic indicator remains to be determined. Since local invasion can be judged without p16INK4a/HPV-detection on microscopic evaluation, our study argues against routine testing in the setting of pSCC.Our results confirm intense nuclear positivity for p16 INK4a is understood as an attempt to stop uncontrolled cell proliferation in response to HPV infection [INK4a immunohistochemistry is therefore used as a surrogate marker for high-risk HPV in cancers of the cervix uteri and the head and neck region [in situ despite diffuse intense confluent staining pattern for p16INK4a [INK4a and presence of HPV DNA and correlated the results with tumour- and patient-specific characteristics as well as cancer-specific survival. We evaluated the value of histologic subtype and various p16INK4a staining patterns as indicators for the presence of HR-HPV DNA in tumour tissue, and showed for the first time that presence of HR-HPV DNA in pSCC is inversely associated with local tumour invasion. Furthermore, we analysed the impact of HPV-driven tumourigenesis on cancer-specific survival in pSCC patients.Squamous cell carcinoma of the penis (pSCC) is associated with high morbidity and mortality and causes severe psychological impact ,2. High-nfection . P16INK4k region ,9. In blp16INK4a . Chaux ep16INK4a . Concernp16INK4a . On the p16INK4a ,14. Therp16INK4a -17. In tAll specimens were surgically removed for therapeutic purposes and subsequent histologic examination and were thoroughly pseudonymized for the use in this study. Therefore, individual written informed consent was not mandatory. The study was approved by the University of Ulm ethics committee .Tissue samples from 58 patients were included in the study; for all patients, biological parameters and tumour characteristics were available. Patients underwent surgery for penile cancer between 1995 and 2012 at the Ulm University Medical Centre (n = 37) or at the Bundeswehrkrankenhaus Ulm (n = 21). All specimens had been submitted for routine histologic examination to the Institutes of Pathology of either the University of Ulm or the Bundeswehrkrankenhaus Ulm. Clinico-pathological characteristics are summarized in Table INK4a was performed on a BenchMark Autostainer  according to the manufacturer’s protocol using a prediluted mouse monoclonal antibody . Microscopic slide evaluation/image acquisition was performed using a Leica DM6000B light microscope  and the Diskus Mikroskopische Diskussion image acquisition software . P16INK4a staining patterns were classified in the following categories: a) intense confluent or b) focally scattered nuclear and/or cytoplasmic staining pattern [INK4a staining patterns were confirmed by an experienced pathologist (PM).Immunohistochemistry for p16 pattern . HistoloHPV genotyping was performed as previously described . In shorINK4a positivity, HPV status, presence/absence of tumour invasion, presence/absence of lymph node involvement or distant metastasis, tumour differentiation grade) were tested for significance using the Chi-Square/Fisher’s exact test . The difference in disease-specific survival between low-grade/high-grade, p16INK4a+/p16INK4a- and HPV+/HPV- groups was assessed using LogRank test . A two-tailed p-value of <0.05 was regarded as statistically significant.Correlations between dichotomous variables  age of the 58 patients included in the study was 64.5 (64.0) years  tumour samples, with 30/53 (56.6%) tumours of the keratinizing subtype showing some degree of positivity. However, there was a wide variety in p16INK4a expression patterns .Altogether, positive immunostaining for p16s Figure : intenses Figure B and C ws Figure D and E wGenomic subtyping following DNA extraction confirmed presence of HPV DNA in 18 (31.0%) of 58 samples, 16 of which were classified as HPV-16 . This predictor was markedly improved by focusing on cases with intense nuclear and confluent staining patterns in all tumour cells . For verified nodal involvement or distant metastasis, there was no significant association with HPV status, p16INK4a positivity or grade of differentiation of the primary tumour .Basaloid histologic subtype of pSCC was not significantly associated with presence of HR-HPV (HPV-16 or HPV-45) in our study cohort  were also not significantly associated with cancer-specific survival .Follow-up data was obtainable for 35 patients and reached up to 204 months. No patient in the HPV-positive group, but 6 patients in the HPV-negative group died from the disease . Among the study cohort, 91% of pSCCs were classified as conventional-type (keratinizing pSCC) because various degrees of keratinization could be observed in these cases. This is a higher proportion than has been described in earlier studies, while the rate of basaloid-type tumours was comparable to data from literature [INK4a, a tumour suppressor protein that is regarded as a surrogate marker for HPV-associated tumours in other organs [INK4a immunostaining correlated significantly with the presence of HR-HPV DNA with good sensitivity (100%), but lacked specificity (60%) when all staining patterns were considered . Considering only those specimens with intense nuclear positivity for p16INK4a in all tumour cells improved the specificity for the presence of HR-HPV DNA to 85% . P16INK4a was present in all HPV-positive cases; however, lack of p16INK4a immunostaining in HPV-associated tumours due to loss of heterozygosity near the CDKN2A locus and/or hypermethylation of the CDKN2A promoter has been described previously [INK4a expression in corresponding metastases, but a 2014 study showed identical immunohistochemical or HPV in situ hybridization profiles between primary pSCCs and their corresponding metastases [INK4a positivity was significantly associated with non-invasive tumour growth (pTis/pTa stage). This finding is in contrast to a proposed proinvasive role for HPV oncoproteins that has been recently described in head and neck as well as cervical cancer, but is supported by data for penile cancer published by other authors [INK4a staining or histologic grade in our sample set; similar results have been previously described [INK4a positivity. This is in line with some previous studies that failed to confirm a proposed association between histopathologic grade and lymph node metastasis or overall survival in pSCC, while it has to be stated as a clear limitation of our study that follow-up data was only obtainable for 35 patients (60.3%) [The role of human high-risk papillomavirus infection as a causative agent in the development of penile squamous cell carcinoma is well established . Howeverreported ,21-23. Taluation ,25. Therterature ,16,26. Hterature . Therefoterature ,29. Posir organs ,30, was eviously . It is ttastases . However authors ,16,33-35 authors . For nodescribed . Further (60.3%) -40. Cons (60.3%) ), we thi (60.3%) . For theINK4a immunostaining for detection of HR-HPV DNA and propose that ubiquitous nuclear expression of p16INK4a is a strong predictor for the presence of HR-HPV in penile squamous cell carcinoma. We further show that, in contrast to histologic grading, presence of HR-HPV DNA is associated with noninvasive growth of the tumour. However, since there was no significant impact of HPV-driven tumourigenesis on metastasis and cancer-specific survival in pSCC, routine p16INK4a immunostaining or HPV genotyping in pSCC cannot be recommended.In this study, we evaluate histologic subtyping as well as p16