Korean women with a history of never smoking and with adenocarcinoma showed an increasing trend in lung cancer occurrence during 2002 to 2012. The two modifiable factors of never-smoker lung cancer in women are hormone and oncogenic virus infection. Based on previous studies, hormone replacement therapy (HRT) and human papillomavirus (HPV) infection might afford protection or be a risk factor, respectively. It is necessary to perform a pooled analysis of cohort studies to evaluate HRT and never-smoker lung cancer in women and a systematic review of case-control studies to determine the association between HPV infection and never-smoker lung cancer. Smokingity rate . In addiity rate , the incKnown risk factors of NSLCa include environmental exposures such as second-hand smoke, radon, asbestos, and cooking fumes; genetic susceptibility; hormones; and oncogenic virus ,6,8. Of Since cooking fumes or second-hand smoke exposure in childhood has already occurred, they are not modifiable; hence, the effect of a preventive project is limited. In addition, based on genomic analysis of NSLCa, Krishnan et al.  claimed As for female hormones, Siegfried  summarizAfter the claim by Syrjänen  in 1979 Of the risk factors for lung cancer in never-smoker women, the grounds of association between HRT history and HPV infection histories, which are modifiable factors, have been reviewed in this study. It is necessary to conduct pooled analysis on cohort studies for HRT history and a SR on case-control studies for HPV infection history. If the findings from these studies show that HRT history is a protective factor against lung cancer incidence and HPV infection history is a risk factor, it would be possible to prepare preventive measures that are more effective.


Most women in developing countries have never attended cervical screening programmes and often little information exists on type-specific human papillomavirus (HPV) prevalence among these populations. Self-sampling for HPV testing (self-HPV) using a dry swab may be useful for establishing a screening program and evaluating HPV prevalence. Our aim was to evaluate self-HPV using a dry swab stored at room temperature.This community-based study in Madagascar consisted of 449 women aged 30–65. Eligible women were provided a dry swab to perform self-HPV. HPV analysis was accomplished by two different real-time PCR tests using the same extracted DNA from the samples.n = 152). Distribution of 19 hrHPV and 9 low-risk HPV (lrHPV) types revealed most frequently 53 and 68 among hrHPV and HPV 54, HPV 70 and HPV 42 among lrHPV. Agreement between the two PCR methods for any of the 14 high-risk HPV (hrHPV) strains detected was 89.9 % . In 385 (85.7 %) samples the DNA load of ß-globin demonstrated a signal with medium or high level copies. Conversely, in 28 (60.9 %) invalid samples the signal was undetectable. The HPV-DNA load signal was predominantly of intermediate level .Overall, 52 (11.6 %) specimens were invalid for HPV detection. The delay between sampling and laboratory processing of DNA extraction considerably increased invalid results. Overall HPV prevalence of 14 hrHPV types detected by the two PCR tests was found to be 38.2 % (Self-HPV using a dry swab stored at room temperature could be a useful method for HPV screening and for conducting population-based surveys on HPV prevalence in resource-poor settings. The high sensitivity of clinically validated HPV tests together with the limitations associated with a Pap test , led to Several studies have shown that Self-HPV is acceptable to women –7 and coSelf-HPV implementation in LMIC requires a careful analysis of logistics. The choice of HPV test and collection device is of paramount importance. Optimization of transport and storage still remains a challenging issue. Most of the collection devices are stored in specimen transport medium or liquid-based cytology media, which preserve HPV-DNA at room temperature but are expensive and unavailable in a resource-poor context. Additionally, they are toxic and flammable and spillage and leakage can happen during collection and transit.Compared with liquid media, dry swabs have the advantage of being less expensive and not requiring a special media, so the sample can be safely transported at ambient temperature. Studies demonstrated a good agreement (70–92 %) for HPV detection between dry and wet swabs –15.The goal of this study was to determine, within field conditions of CC screening in Madagascar, the validity of HPV detection in self-obtained samples using a dry swab stored at room temperature. Additionally, we analysed HPV prevalence and genotype distribution among the screened population.The study was conducted by the University of Geneva in collaboration with Madagascar’s Health and Family Planning Ministry and the Saint-Damien Healthcare Centre in Ambanja, a mainly rural area with 125,056 inhabitants.All participants signed an informed consent form before inclusion. The study was conducted from February to March 2015 in the Saint-Damien Healthcare Centre. Ethical approval of the study was obtained from the Malgasch National Commission for the Ethics of Science and Technology, as well as from the Ethical Cantonal Board of Geneva, Switzerland (CER: 14–071).We recruited 449 women aged between 30 and 65 years. Exclusion criteria were former conisation or hysterectomy and pregnancy beyond 20 weeks.Participants received educational intervention on cancer and HPV infection. It was followed by information on how to use the self-HPV, written in the local dialect language. At the same time the participants received a sterile, cotton-tipped swab in a dry, labelled tube. Self-HPV was always performed without supervision. Then participants were invited to answer a questionnaire regarding socio-demographic information. HPV tests were analysed in Switzerland. HPV test analysis was done in a minimum time of 15 days after the sampling.HPV-negative women were informed that they were not at risk for CC and that they would not require a test within the next 5 years. HrHPV-positive women were invited for colposcopic examination. A biopsy on acetowhite lesions coupled with endocervical curettage was performed, or a biopsy at the 6 o’clock position and endocervical curettage if colposcopy was normal. Treatment and follow-up was proposed according to the histological diagnosis. In case of an invalid result, women were asked to repeat their self-sampling. Histology was also analysed in Switzerland.Upon arrival to the laboratory, the swabs were suspended in 4.3 mL of cobas® PCR media and were pulse vortexed for 3 × 10 s. A volume of 400 μL of each sample was used for DNA extraction and the rest of the sample was stored at 4 °C. DNA extraction was carried out using the cobas® HPV Test (Roche Molecular Systems). Nucleic extracts were then stored at −20 °C.HPV analysis was accomplished by two different real-time PCR tests: the cobas® HPV Test (cobas) and the Anyplex™ II HPV28 (H28) test , using the same extracted DNA from samples.Amplification and detection were first carried out with cobas , which detects 12 pooled hrHPV genotypes  and concurrently provides separate results for HPV-16 and HPV-18. The detection is based on amplification of the L1 gene and TaqMan probes [Amplification and detection using the stored DNA extracts were then performed with the H28 test using the CFX96™ real-time thermocycler . Data recording and interpretation were automated. Details of the procedure and evaluation were described by Estrade et al. , Kim et H28 is a semi-quantitative real-time multiplex PCR assay for screening and HPV genotyping. This test uses Dual Priming Oligonucleotides (DPO™) and Tagging Oligonucleotide Cleavage and Extension (TOCE™) technologies and allows to simultaneously detect 19 high-risk HPVs  and 9 low-risk HPVs . ß-globin is also detected for internal control of assay validity.2 copies/reaction), to intermediate , to high  (17).Knowledge of the step at which the melting curve becomes positive allows for semi-quantification of the DNA load of ß-globin gene and HPV genomes, from low .Inter-rater agreement statistics and kappa coefficient with 95 % confidence intervals, percent total agreement and percent positive agreement were calculated for the paired results obtained by the cobas and H28 tests. The calculation was restricted to 14 hrHPV.The trend of association between HPV results and DNA load was evaluated with the chi-square test. We also evaluated the effect of transport time on sample degradation by applying a Kaplan–Meier failure estimate.The HPV prevalence was calculated from the number of positive cases divided from the number of tested specimens by both PCR methods. HPV positivity was distributed by age-group.HPV type distribution in single and multiple infections of 19 hrHPVs and 9 lrHPVs was explored using the H28 test.The study included 449 self-obtained swab specimens from women whose median age was 43 (IQR: 36–51). Overall, 52 (11.6 %) specimens were invalid for HPV detection, 44 (9.8 %) were invalid for cobas, 46 (10.0 %) were invalid for H28 and 38 (73.1 %) were concordantly classified as invalid: 6 were invalid by H28 but negative by cobas, 5 were invalid by cobas but negative by H28, 1 was invalid by cobas but positive by H28 and 2 were invalid by H28 but positive by cobas (data not shown).The overall prevalence for the 14 hrHPV types detected by one of the two methods was 38.2 % (152/397); 19.7 % (30/152) of samples were positive for HPV-16/18, while 88.2 % (134/152) were positive for the pooled 12 hrHPV types. The overall prevalence for 19 hrHPV and 9 lrHPV was 51.8 % (209/403); the prevalence for hrHPV only was 38.6 % (156/403).Overall, 96 HPV-positive patients came to the colposcopy clinic. There were 6 (6.3 %) cervical intraepithelial neoplasia grade 1 (CIN1), 4 (4.2 %) CIN grade 2 (CIN2), 4 (4.2 %) CIN grade 3 (CIN3) and 2 (2.1 %) squamous cell carcinomas. The overall CIN2 or more severe (CIN2+) prevalence was 10.4 %.The agreement was 89.9 % for any HPV type, 97.5 % for HPV-16/18 and 90.7 % for the category with HPV pooled types. The proportion of positive agreement was 84.8, 80.0 and 84.0 % for any hrHPV, HPV-16/18 and pooled HPVs, respectively .Overall, in 85.7 % of the samples the DNA load of ß-globin demonstrated a signal with medium- or high-level copies. In 60.9 % of invalid samples the signal was undetectable. For 7 HPV-positive cases the ß-globin was undetectable Table . There wp = 0.177). The mean delay between sampling and laboratory processing was 22.7 ± 0.35 days for valid samples and 24.3 days for invalid samples (p = 0.129). In Fig. The delay between sampling and laboratory processing increased invalid results from 5.7 % after 2 weeks to 9.4 % after 3 weeks and 12.6 % after ≥4 weeks (p = 0.206). The strength of the HPV-DNA load was not related to the strength of the ß-globin DNA load (p = 0.154). There were no significant differences in HPV-DNA load between pre- and postmenopausal women (p = 0.806) (data not shown).Analysis of the relationship between HPV positive test results and DNA load Table  showed tThe overall prevalence for 14 hrHPV types was found to be 32.1 % with cobas and 29.5 % with H28. Stratification by age demonstrates a decrease from the age group of 30–35 years to the age group 36–45 years. Then a rebound is observed in the age group of 46–55 years, followed by a decrease in the age group of 56–65 years .Overall, 31.6 % of the HPV infections were single whereas 68.4 % were multiple. Sixty-one percent of women’s infections revealed hrHPV, particularly HPV-53 and 68, followed by HPV-73, HPV-52, HPV-35, HPV-16, HPV-33, HPV-31 and HPV-18 in decreasing order. Infections with low risk types disclosed particularly HPV-54, HPV-70 and HPV-42, followed by HPV-44, HPV-40 and HPV-6 in decreasing order. There were no significant differences in the distribution of mono-infections vs. multi-infections for hr- and lrHPVs . Agreement between the two real-time PCR assays for the detection of any HPV among the 14 hrHPV types was 89.9 % (kappa = 0.77), despite different technologies and cut-off algorithms. A similarly-strong agreement was observed after the hierarchical categorization of hrHPV types into two groups, according to cancer risk (HPV-16/18 and pool of others).p = 0.206) and the strength of HPV-DNA load was not related to the strength of the ß-globin DNA load (p = 0.154). Interestingly, in 7 samples with undetectable ß-globin, PCR analysis with H28 revealed positive HPV-DNA. This finding may corroborate the hypothesis that HPV-DNA present predominantly as episomal copies in dry samples is less affected from degradation [We investigated the quality of the samples by analysing the relationship between HPV test results and the DNA load. The human reference gene ß-globin, a marker for cellularity, and the HPV-DNA for 19 hrHPV and 9 lrHPV were semi-quantified. Our findings are in line with other studies indicating that the quality of a dry specimen is sufficient for HPV detection  but the radation .Our data show that menopausal status does not affect the quality of samples. Nevertheless, it should be noted that besides DNA degradation because of the transportation time, undetectable DNA may also be the result of an inadequate sampling or the presence of PCR inhibitors .This study provides for the first time information on age-specific HPV prevalence in screened women in Madagascar. Cobas and H28 were in agreement for the overall prevalence rates found for 14 hrHPV types. The pattern of HPV prevalence for the restricted age-range of our study appeared similar to some surveyed African countries, but different to others . The obsThe high HPV prevalence found among the screened Malagasy women is consistent with published meta-analyses for Eastern Africa. Systematic reviews reported a HPV prevalence of 33.6 %  and 42.2In this study we used simple cotton swab for sample collection. Flocked swabs were found to have higher sensitivity to detect HPV infection compared to Dacron swabs , which wWe do recognize that the approach for HPV testing and subsequent follow-up applied in this study is unrealistic for a routine CC screening program in Madagascar. Besides invalid samples, a long delay between screening and reporting the results may lead to increased dropout of HPV-positive women who need to be recalled for management. A solution is the use of emergent rapid PCR-based methods for detecting HPV-DNA, with minimal requirements for laboratory equipment, enabling primary screening and treatment in a single visit. Another limitation in our analysis is the lack of histopathological results for all tested women, which would be useful as gold-standard for both PCR methods.Because estimates of HPV prevalence and type-specific distribution in Madagascar are necessary for orienting CC prevention and monitoring the impact of vaccination, our study needs to be completed with estimates of a wider age range (15–60 years).In conclusion, our study provides evidence that self-HPV with dry swabs stored at room temperature is a valid alternative for screening with HPV testing in low resource settings or remote areas, though HPV analysis should be performed ideally within 2 weeks. Moreover, our findings suggest that unsupervised self-sampling with dry swabs in conjunction with a PCR-based method is feasible in conducting population-based surveys on HPV prevalence, which are limited or missing in many LMIC.Ethical approval of the study was obtained from the Malgasch National Commission for the Ethics of Science and Technology, as well as from the Ethical Cantonal Board of Geneva, Switzerland .


The change in transcription pattern induced by post-transcriptional RNA splicing is an important mechanism in the regulation of the early gene expression of human papilloma virus (HPV). The present study was conducted to establish a method to specifically amplify HPV-16 E6-associated transcripts. The E6-related transcripts from 63 HPV-16-positive cervical tumor tissue samples were amplified, consisting of eight cases of low-risk intraepithelial lesions, 38 cases of high-risk intraepithelial lesions and 17 cases of cervical cancer (CxCa). The appropriate amplified segments were recovered following agarose gel electrophoresis, and subjected to further sequencing and sequence alignment analysis. Six groups of E6 transcription patterns were identified from HPV-16-positive cervical tumor tissue, including five newly-discovered transcripts. Different HPV-16 E6-associated transcription patterns were detected during the development of CxCa. Over the course of the progression of the low-grade squamous intraepithelial lesions to CxCa, the specific HPV-16 E6-associated transcription patterns and the dominant transcripts were all different. As indicated by this study, the transcription pattern of the E6 early gene of HPV-16 was closely associated with the stages of cervical carcinogenesis, and may also be involved in the development of CxCa. CxCa is the second most common form of malignant tumor that results in female mortalities worldwide. Persistent infection with high-risk human papilloma viruses (HPVs), particularly HPV-16 and HPV-18, is a key factor in the development of CxCa ,3. The eBicistronic or polycistronic transcription, with two or more open reading frames (ORFs), is a feature of HPVs. Regulatory elements (including E2-binding elements or those that bind to activator protein-1 and octamer-binding protein-1 transcription factors), early promoters , early splice sites and early polyadenylation signal sites , are allWhile HPV-16 early gene transcription patterns have been reported in recent years, HPV-16 E6 transcription patterns, during the course of CxCa genesis have not been systematically analyzed. Therefore, the present study used the amplification of papillomavirus oncogene transcripts (APOT) to determine the profiles of E6-associated early gene transcripts and comprehensively investigate the variation of transcriptional patterns in eight low-grade squamous intraepithelial lesions (LSILs), 24 high-grade squamous intraepithelial lesions (HSILs) and eight CxCa HPV-16-positive cervical biopsy samples.ex-vivo procedures, the specimens were preserved in liquid nitrogen until further experimentation. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University . All participating patients provided informed consent.Samples from 63 hospitalized or outpatient subjects, with HPV-16-positive cervical malignancies, were collected from the Second Affiliated Hospital of Wenzhou Medical University  between December 2010 and April 2012. These samples included eight cases of LSILs, 38 cases of HSIL consisting of 22 cervical intra-epithelial neoplasia (CIN) II cases and 16 CIN III cases, and 17 CxCa cases. The patient age range was 25–59 years, with a median age of 43 years. HPV genotyping and pathological confirmation was conducted for all specimens during the collection. The patients did not receive any radiotherapy or chemotherapy prior to the surgery. In addition, eight control cervical tissue samples negative for HPV and with normal cytology were obtained from patients who underwent a hysterectomy owing to benign gynecological diseases. Subsequent to The cryopreserved cervical tissues were crushed to a powder in liquid nitrogen, and the RNA was extracted with TRIzol reagent according to the manufacturer’s instructions . To remove the residual contaminating DNA, the RNA preparation was treated with RNase-free DNase I . The purified RNA was dissolved in RNase-free water  and stored at −80°C. The concentration and purity of total RNA were quantified by an ultraviolet spectrophotometer  at 260 and 280 nm, respectively, and the integrity of the RNA was examined by 1% agarose gel electrophoresis. Only RNA samples with an A260/A280 ratio of 1.8–2.0 and high integrity were used for further experiments.17 RT primer, coupled to a linker sequence (5′-GAC TCGAGTCGACATCGATTTTTTTTTTTTTTTTT’3)  was reverse transcribed using an oligo (dT)TTTTT’3) . ReverseThe final PCR products were electrophoresed in 2.5% agarose gels, and the DNA was transferred to a nitrocellulose membrane  and fixed for 2 h at 80°C prior to mixing with hybridization solution. Following pre-hybridization for 1 h, the membranes were incubated overnight at 46°C in hybridization solution, with a 5′-biotin-labeled HPV-16 E6-specific probe (5′-CTGCGACGTGAGGTATAT GACTTTG-3′) at a final concentration of 100 ng/ml . Following hybridization, the membranes were washed twice with pre-warmed 2X saline sodium citrate (SSC) solution containing 0.1% sodium dodecyl sulfate (SDS) at 50°C for 20 min, and twice with pre-warmed 0.2X SSC solution containing 0.1% SDS at 53°C for 15 min. Detection of the probe signal was performed using a chemiluminescence detection system  according to the manufacturer’s instructions, followed by film exposure.pEASY™-blunt zero cloning vector , and the sequencing of amplified sequences was conducted using an ABI3730 XL Genetic Analyzer , according to standard procedures. The alignment analysis of the sequencing results was conducted using the BLASTn service supplied by the National Center for Biotechnology Information .The APOT amplification products were visualized by 2.5% agarose gel electrophoresis. The PCR-amplified fragments of interest were separated and purified from the gel using an agarose gel DNA extraction kit, according to the manufacturer’s instructions . The corresponding amplimers were cloned into a The basis of an APOT assay is the 3′ rapid amplification of cDNA ends, which enables amplification and cloning of the region between a single short sequence in the cDNA molecule, and its unknown 3′ end . In ordeet al , and were amplified by PCR according to the aforementioned method. Six different types of HPV-16 E6-associated transcription patterns were identified . Among tet al . Of thesHPV-16 E6-associated transcriptional patterns were systematically analyzed in HPV-16-positive tissues of LSIL, HSIL and CxCa, and were found to significantly differ between the different tissues. Over the course of LSIL progression to CxCa, there was an observable difference in the expression of specific HPV-16 E6-associated transcription patterns  and 5. AThe double-stranded, circular DNA genome of HPV is ~7.9 kb in length and consists of the non-coding long control region, the early gene region  and the late gene region (encoding the capsid proteins L1 and L2) . In addiIn order to analyze the HPV-16 E6-associated transcription profile in the different stages of CxCa, an APOT assay was used in the present study to determine E6-associated transcript patterns in 63 HPV-16-positive cervical tumor tissues. In total, six E6-associated transcription patterns were identified in the HPV-16-positive cervical tumor tissues. In contrast with previous studies ,14,15, tet al (The role of HPV-16 early gene transcription patterns in the carcinogenesis and development of CxCa has not yet been established by previous studies. In the present study, distinct differences in HPV-16 early gene transcriptional patterns, at different stages of CxCa progression, were observed. All samples positive for patterns A and B originated from patients with LSIL, while all samples positive for pattern F were identified in patients with CxCa. In total, 50% of the samples from patients with LSIL and HSIL were positive for pattern C transcripts, while the majority of samples positive for patterns D and E were identified in patients with HSIL and CxCa. Van Tine et al  proposedet al . This ma


Emotion recognition is very important for social interaction. Several mental disorders influence facial emotion recognition. War veterans and their offspring are subject to an increased risk of developing psychopathology. Emotion recognition is an important aspect that needs to be addressed in this population. To our knowledge, no test exists that is validated for use with war veterans and their offspring. The current study aimed to validate the JACFEE photo set to study facial emotion recognition in war veterans and their offspring. The JACFEE photo set was presented to 135 participants, comprised of 62 male war veterans and 73 war veterans’ offspring. The participants identified the facial emotion presented from amongst the possible seven emotions that were tested for: anger, contempt, disgust, fear, happiness, sadness, and surprise. A loglinear model was used to evaluate whether the agreement between the intended and the chosen emotions was higher than the expected. Overall agreement between chosen and intended emotions was 76.3% (Cohen kappa = 0.72). The agreement ranged from 63% (sadness expressions) to 91% (happiness expressions). The reliability by emotion ranged from 0.617 to 0.843 and the overall JACFEE photo set Cronbach alpha was 0.911. The offspring showed higher agreement when compared with the veterans , which confirms the construct validity of the test. The JACFEE set of photos showed good validity and reliability indices, which makes it an adequate instrument for researching emotion recognition ability in the study sample of war veterans and their respective offspring. Emotions are inherent to human nature. They exist because we need to recognize and respond to significant events that are related to survival and/or the maintenance of well-being, and thus they serve several functions . EmotionBasic emotions are considered to be universal, because they have been shown to be categorized by different cultures and ethnicities , 6. EmotBasic emotions have been shown to have specific autonomic nervous system responses . For exaFacial expressions are an important component of emotional behavior and are specific to basic emotions . Facial Facial emotion recognition is known to diminish with age –20, and Many clinical situations limit facial emotion recognition, such as psychiatric disorders and some neurologic disorders. Depression and bipolar disorder are associated with moderate and stable deficiency in facial emotion perception, moderated by self-reported depression, age at time of testing, and years of education . Facial Several methodologies have been used to study facial emotion recognition. In the literature we can find many photo sets and videos for this purpose. Some sets include only one ethnicity, whilst others include hundreds of stimuli , and othOne of the most important and frequently used methodologies is the set of photos developed by Matsumoto and Ekman—the Japanese and Caucasian Facial Expressions of Emotion (JACFEE) . It is cJACFEE photos have been used in several research fields, such as: Basic emotion research neurobiology in normal subjects , 41; psyWar veterans are a unique population, as they have been exposed to traumatic events and are at risk of developing psychiatric disorders , such asPortugal was involved in colonial wars from 1961 to 1974, during which 1 million Portuguese solders were involved. This has resulted in psychopathology, both in veterans  and alsoTo our knowledge, there are no reliability or validity studies of veterans’ populations and their offspring for tests of facial emotion recognition. A valid and reliable test for veterans and their offspring is needed, which can be used in future studies that attempt to control for hereditary problems that influence facial emotional recognition, or studies that want to show that problems influencing facial emotional recognition are transferred across generations. To carry out this type of studies we need a test which is valid and reliable in both groups. Our aim was to study the JACFEE photo set reliability/validity for a sample of veterans from the Portuguese colonial wars and also that of their offspring. We show that the JACFEE set of photos has good validity and reliability indices in the study sample of war veterans and their respective offspring.This study was approved by the Ethics Committee of the author’s university . All participants received a written and verbal description of the study and gave their written informed consent.Data are presented as the mean (standard deviation). Sixty two white veterans [mean age = 65.31 (3.71) years] of the Portuguese colonial wars were enrolled for the study. The sampling procedure used two ways of selecting participants: 75.8% from an outpatient clinic of the Portuguese Disabled Veterans Association (ADFA), and 24.2% from three lists of veterans’ companies from war time. Half of the veterans (51.61%) were disabled, and all were outpatients. Women did not participate in the Portuguese colonial wars as combatants. Offspring  from 46 veterans were also included . The mean age of the total sample (n = 135) was 49.27 (15.41) years’ old, and the median education in years (inter-quartile range) was 9 (4–15). Veterans’ median education was 4 (4–9.5) years, and offspring’s median education was 12 (9–15) years. All participants were of European descent.In a university setting, participants identified the emotion expressed in 56 photos from the JACFEE set , balanceWe used relative and absolute frequency of chosen emotions per intended emotional expression. The Cronbach alpha was used to evaluate the internal consistency. See A generalized linear Poisson model with log link was used to evaluate whether the agreement between the intended and chosen emotions was higher than the expected, assuming independence between chosen and intended emotions . FurtherFor each image, we calculated how many participants chose the target emotion See . The oveA close look at The frequencies of the categories in cases where the chosen expression equals the intended expression (agreement categories) was 21.4 (95% CI: 20.2 to 22.7) times higher in comparison to the categories where intended expression was different from chosen expression (disagreement categories), assuming the independence between both factors . Table 5In this study, the 56 photos from the JACFEE set  were valThere were systematic patterns of disagreement for fear, surprise, content and sadness. These patterns are similar to those found by Goeleven et al  and LangSince facial emotion recognition can be considered as a multiple effect indicator construct model, reliability analysis through Cronbach alpha is adequate . For theWe found that offspring had significantly higher frequencies in agreement categories when compared to the veterans. To our knowledge, no studies exist which compare the performance of parents with their adult offspring in facial emotion recognition. However offspring are expected to have better performances. This is probably accounted for by the lower age, higher education, and the inclusion of women in the offspring group, which are factors that have all been reported to positively influence facial emotion recognition ability –19, 21. Limitations: the participants were forced to choose between limited emotional term options, which might have biased the choice that they really would have made. However, this methodology is in accordance with the one used to validate this photo set and it relies on the rigorous construction of the set, and on the universality of these basic emotions . Other fSome researchers still use the earlier Ekman and Friesen’s Pictures of Facial Affect , 26, butIn conclusion, for the first time, this research replicated in a sample of war veterans and their respective offspring the results of previous studies which aimed to validate instruments for evaluation of facial emotion recognition in non-clinical samples. The JACFEE set of photos showed good validity and reliability indices, and seems to be a valid instrument for investigating the recognition of facial emotions in populations of war veterans and their offspring. We think that now this test can be used in future studies that aim to control for hereditary problems that influence facial emotional recognition, or those that want to show that problems influencing facial emotional recognition are transferred across generations. We are now going to use this same test to characterize the influence of war-related PTSD on facial emotion recognition ability, and also the role of inheritance with regards to the interaction between these variables. Emerging questions of trauma inheritance and how it relates to psychopathology and social interaction need to be further investigated. The JACFEE photo set seems to be an adequate instrument for this purpose.


Parity is well established as a risk factor for cervical cancer. It is not clear, however, how pregnancy influences the natural history of HPV infection and cervical neoplasia. Our objective was to study the risk of HPV infection and cervical squamous intraepithelial lesions (SIL) after pregnancy.We used the Ludwig-McGill cohort study which includes 2462 women recruited in Sao Paulo, Brazil in 1993–97 and followed for up to 10 years. Cellular specimens were collected every 4–6 months for Pap cytology and HPV detection and genotyping by a polymerase chain reaction protocol. Study nurses recorded pregnancy occurrence during follow-up. HPV and Pap results from pregnant women were available before and after, but not during pregnancy. The associations between pregnancy and post-partum HPV infection/SIL were studied using generalized estimating equation models with logistic link. Adjusted odds ratios (OR) were estimated with empirical adjustment for confounding.We recorded 122 women with a history of pregnancy during follow-up. Of these, 29 reintegrated the cohort study after delivery. No association between HPV and pregnancy was found. A single SIL case (high grade SIL) occurred post-partum. Likewise, there was no association between pregnancy and risk of low grade SIL or any-grade SIL at the next visit  after controlling for confounders.No associations were found between pregnancy and HPV or LSIL. The single observed case of HSIL post-partum was more than would be expected based on the rate of these abnormalities among non-pregnant women. As this association was found with only one case, caution is required in the interpretation of these results. The central causal role of the so-called high oncogenic–risk (HR)-human papillomavirus (HPV) genotypes, such as HPV-16 in cervical carcinogenesis, has been established . HPV infThe women included in this study were enrolled into the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and precursor lesions of cervical cancer. A detailed description of the design and methods of the study has been published previously . BrieflyThe study enrolled 2528 women, corresponding to a 70 % response rate and subsequently, 66 ineligible women were excluded. Follow-up for the remaining 2462 women consisted of 1 visit every 4 months for the first year and 2 visits per year thereafter. Cervical specimens were taken for Papanicolaou (Pap) cytology and HPV testing at every visit with a fixed-sampling area Accelon device. For the first 4 visits and for each annual visit thereafter, subjects answered a nurse-administered questionnaire designed to collect information on socio-demographic, lifestyle, sexual, reproductive and contraceptive characteristics. Pregnancy status was registered by study nurses along with date and procedures of visits, reasons of dropouts and reintegration, and other study-relevant incidents. Pregnant women were excluded from the study during pregnancy and offered to be reintegrated in the study after a minimum of 45 days following delivery, if there was no clinical impediment and if they consented. HPV and Pap results from pregnant women were available before and after, but not during pregnancy.An Accelon biosampler  was used to collect ecto- and endocervical samples for each visit and a Pap smear was prepared on a glass slide and fixed with 95 % ethanol. The sampler containing the residual exfoliated cells was immersed in a tube containing Tris-EDTA buffer pH 7.4) and agitated to release the cells. The tubes containing cell suspensions were frozen until testing. Samples were then sent to the Ludwig Institute for storage and testing. Pap smears were sent to the study centre in Canada for cytology reading by one of the co-authors (AF). Cytopathology reports were based on the 1991 Bethesda system for cytological diagnoses [.4 and agThe Accelon device containing ecto- and endocervical cells was placed in a tube containing Tris-EDTA buffer (pH 7.4). DNA was extracted, purified by spin column chromatography, and amplified by polymerase chain reaction (PCR), using the MY09/11 and PGMY protocols , 9 for dWe explored the relationships between pregnancy and outcomes such as HPV infection and cervical squamous intraepithelial lesions (SIL) using generalized estimating equation (GEE) via a logistic regression link, which take into account the clustering within each individual caused by the repeated-measurements design. GEE models were based on an exchangeable correlation pattern. We studied the association between pregnancy and the presence of HPV, LSIL and HSIL. Post-partum visits were compared to other usual visits for the presence of HPV or SIL. We explored the relationship between HPV infection and pregnancy by considering grouped HPV genotypes per their oncogenic potential (high-risk and low-risk as described above), per their phylogenetic relationship within the genus Alpha-papillomavirus   and grouWe estimated crude and adjusted odd ratios (ORs) with respective 95 % confidence intervals (CI). For adjusted estimates, we controlled for empirical confounders using a 5 % change-in-estimate method (variables that changed the estimates by +/− 5 % were included in the model) considering the following variables: age at enrolment (linear), race , marital status , income (quartiles of income), smoking , age at first sexual intercourse , number of previous pregnancies , Pap testing before enrollment in the cohort  and lifetime number of sexual partners . For the analysis concerning the association between SIL and pregnancy, we also considered HPV status  as a potential confounder or mediator.A total of 2475 women were included and provided altogether 24,558 visits. The mean and median follow-up time were 5.1 (SD = 3.2 years) and 6.4 (inter-quartile range = 1.0-7.5) years, respectively. The mean age at enrolment was 32.7 years  and most women were white (64 %). We recorded 122 women with a history of pregnancy during follow-up. After giving birth, 29 (23.8 %) women reentered the study. The mean time between visits for reintegration into the cohort was 3.9 years (SD = 1.6).Table Table In this longitudinal study, we analysed the risk of HPV infection and cytological abnormality following pregnancy. No association was detected between pregnancy and LSIL, nor for post-partum HPV infection. Although, the single observed case of HSIL post-partum was more than would be expected based on the rate of these abnormalities among non-pregnant women, this association was found with only one case, which calls for caution in interpreting these findings.There are plausible mechanisms whereby pregnancy may influence the natural history of HPV infection and progression to clinical lesions. Increased levels of estrogen or growth hormone associated with pregnancy such as human chorionic gonadotropin (hCG) may increase HPV molecular activity , 15. StuOur finding of no increased risk of HPV detection with pregnancy concurs with other reports –27. OnlyIt is not clear however, whether or not the risk of progression of HPV infection to cervical neoplasia (preinvasive and invasive cancer) is higher when detected during pregnancy. Studies on the progression of biopsy proven cervical neoplasia during pregnancy are rare, since biopsies tend to be avoided during pregnancy. When Pap results are used to estimate risk, reports provide a wide range of estimates: 10–70 % of cytological abnormalities found during pregnancy regress, 25–89 % persist without progression and 3–30 % progress –46. StudThis study has limitations. It is possible that residual confounding may explain our findings. For example, lack of control for women’s partners’ sexual behaviour could create confounding if pregnancy is associated with a higher likelihood of partners’ extra-conjugal sexual relationships. However, this potential confounding bias is unlikely to have occurred, as no association was found for HPV infection or LSIL. We also used cytology and not histology results in this analysis. However, Morimura et al. (2002)  has showOne of the strengths of our study was the use of a longitudinal design approach including prospective collection of data before and after pregnancy. This limits the potential for bias that would have occurred if pregnant women were more likely to be screened compared to non-pregnant women because all pregnant women in our study were similarly followed-up in the cohort prior to becoming pregnant. We also applied a conservative control for confounding including sexual behaviour and variables that were different between pregnant and non pregnant women such as age.In conclusion, we found no association between pregnancy and HPV infection and cervical lesions but admittedly, our cohort included a limited number of pregnant women. A single SIL case (an HSIL) occurred post-partum which calls for caution in interpretation.


Delivering training to participating sites is an essential part of successful trial setup especially when new techniques, treatments or technologies are involved. RAIDER is a multicentre (> 30 NHS Trusts) phase II trial of complex radiotherapy for muscle invasive bladder cancer. The radiotherapy techniques were developed at a single UK NHS site and in addition to written materials ideally require demonstration using the relevant radiotherapy planning and imaging software, only available at the experienced site. Rather than arranging a series of visits from key participating site personnel  and research radiographers), a virtual meeting plan was developed.Cisco’s WebEx was selected as it allowed screen sharing and live demonstration of radiotherapy contouring techniques. Sites were provided with guidelines for using WebEx and given a practice session to check connectivity. The 2 hour interactive meeting included an introduction to the trial, technical details of treatment delivery and opportunities to ask questions. Feedback was requested from attendees.132 attendees from 27 sites participated, including 18 PIs. 26/27 sites provided feedback with 20 stating that the virtual meeting allowed more staff to attend compared to a face-to-face meeting; 17/26 preferred the virtual meeting format. The usefulness of the meeting was rated 8.6/10 on average.The virtual meeting format improved attendance rates, was considered beneficial by attendees and facilitated recording for future use by staff or sites unable to attend. The approach was a useful training tool for RAIDER and will be adopted by other ICR-CTSU trials.


HPV infects multiple sites in the epithelium, including the genitals and oral cavity. The relation between genital and oral infections and serum antibodies can help explain the natural history and epidemiology of HPV.We analyzed HPV data from NHANES derived from self-collected vaginal swabs , oral rinses , and serum .Type-concordance of cervicogenital and oral infections in women was found to vary widely by age. Prevalence of oral infections with type-concordant antibodies was low but varied by sex: 0.2 % (95 % CI 0.0–0.8) for women vs 0.8 % (95 % CI 0.4–1.3) for men. Vaccination was associated with a reduced risk of cervicogenital infection for vaccine genotypes among ages 14–17 (0.2 (95 % CI 0.1–0.8)) and 18–24 (0.2 (95 % CI 0.1–0.3). Seroprevalence trends in women showed a dramatic increase for recent birth cohorts, likely due to vaccination. By contrast, trends for men remained relatively constant. Age-specific cervicogenital prevalence showed a consistent peak in the late teens and twenties. Relative cervicogenital prevalence has largely been decreasing since the 1940–50 birth cohort.There are complex patterns in HPV prevalence trends and type-concordance across infection sites and serum antibodies. A multisite sampling scheme is needed to better understand the epidemiology and natural history of HPV.The online version of this article (doi:10.1186/s12879-015-1314-0) contains supplementary material, which is available to authorized users. The human papillomavirus (HPV) infects multiple mucosal sites in the epithelium and is the etiological agent for over 90 % of anogenital cancers and an increasing fraction of oropharyngeal cancers . AlthougThere are many strains of HPV, and these are typically classified according to their oncogenic risk. Genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 (Group 1) are known to cause cancer, genotype 68 (Group 2A) probably causes cancer, and genotypes 26, 30, 34, 53, 66, 67, 69, 70, 73, 82, 85, 97 (Group 2B) are possibiliy carcinogenic , but somVaccines have been developed to target certain strains of HPV. Three vaccines are currently approved by the FDA: GlaxoSmithKline Biologicals’s bivalent  Cervarix, and Merck’s quadrivalent 6, 11, 16, 18) Gardasil and Merck’s nonavalent  Gardasil 9 , 16, 18 . Vaccinen=3) to be especially informative  andAlthough discussion of HPV in the public health context is often limited to mucosal types, other strains, including those that cause common and plantar warts, infect cutaneous tissue. It is possible that this tropism, that is preference for certain kinds of tissue, may be relevant within the mucosal types, with certain strains preferring genital tissue and others preferring oral tissue. It is difficult to determine whether some strains—such as types 16, 62, 84—are commonly found at both sites because of a lack of tropism, or simply because they are relatively prevalent. However, the pattern of prevalence for type 44 is consistent with a tropic preference for oral tissue. More work will be needed to assess whether this and other patterns—such as the higher prevalence of types 53, 54, and 61 among those with cervicogenital infections than among those with oral infections—can be attributed to tropism or are a reflection of cohort/timing patterns. Tropism may be important when considering an evolutionary perspective; changing sexual behaviors –39 may hAmong those women who are seropositive for 6, 11, 16 or 18, prevalence of type-concordant cervicogenital infections is low and decreases with age, as might be expected since older individuals are more likely to have already seroconverted during previous infections. For racial demographics, seroprevalence more closely follows the cerivcogenital rather than oral profile. This finding may simply be a result of cervicogenital infections being much more common than oral ones for women, but it also raises the possibility that seroconversion may occur more readily following cervical rather than oral infection. Combined with the different prevalence of serotype-concordance for men and women (Fig. Seroprevalence increases dramatically for 14–17 and 18–24 year old women over the period studied. Since the aim of the vaccine is seroconversion, it is likely that this increase is largely caused by the introduction of the vaccine Fig. . FurtherNon-Hispanic black men and women have consistently high prevalence at genital and oral sites and seroprevalence, which is consistent with previous work , 32. TheOne strength of this study is the large sample size in the NHANES survey, which allowed analysis at a relatively fine demographic stratification. NHANES is the only population data source for oral infections and also allows for analysis of multiple sites in one individual. Additionally, the use of APC models allows for the analysis of temporal trends in the data separate from the age effects. Limitations of the NHANES data set include the relatively limited time span, especially for oral infections, the limited number of serotypes, and the lack of genital prevalence for men and anal prevalence for men and women. Although, to our knowledge, no studies have investigated seroconversion in women following anal infections, anal infections may play an important role in seroconversion in men , 17, 42.This study expands on the analysis by Steinau et al.  to proviThis study also provides further evidence that the probability of serconversion from an infection may differ depending on which mucosal epithelium is infected. Seroconversion from cervicogenital infection appears to be common and may provide some measure of defense against oral infections, based on the lack sero-oral concordance in women. Previous studies show that men are less likely to seroconvert from genital infections than women, suggesting that they may thus be more vulnerable than women to oral infection. Further, there is some indication that some genotypes are strongly tropic, preferring to infect one site over another.NHANES data is publically available, although data on minors is restricted by the NCHS Research Data Center.


INK4A expression has been used as a surrogate marker for human papillomavirus (HPV) infection in cervical cancer and head and neck cancer. p53 has also been reported as a feasible marker to identify HPV-positive oropharyngeal carcinoma and penile lesions. This study aimed to investigate p16INK4A and p53 expression levels and their correlation with HPV status and clinical parameters in Kazakh patients with esophageal squamous cell carcinoma.p16INK4A and p53 were evaluated in 163 cases of esophageal squamous cell carcinoma in Kazakh patients. The presence of HPV DNA was detected by polymerase chain reaction.Immunohistochemical expression of p16INK4A-positive expression was detected in 19.0 % of patients, and its expression was significantly correlated with a lower frequency of lymph node metastasis (p = 0.038). By contrast no significant association was found between p16INK4A-positive expression and HPV status . p16INK4A-positive expression did not affect the odds of tumors being HPV positive . The sensitivity of p16INK4A-positive expression as an HPV marker was 0.164, with a specificity of 0.788 and a positive predictive value of 0.391. p53-positive expression was present in 88.3 % of all cases. Although no significant correlation with available clinical parameters was found, a significantly inverse correlation was observed between p53 expression and HPV status . Moreover, p53-positive expression decreased the odds of tumors being HPV positive (OR = 0.292 with 95 % CI = 0.086–0.990). The sensitivity of p53-negative expression as an HPV marker was 0.179, with a specificity of 0.940 and a positive predictive value of 0.714. The overall HPV prevalence was high (45.5 %) in Kazakh patients, with no significant association between HPV positivity and available clinical parameters or combined p16INK4A/p53 expression.p16INK4A-positive expression was associated with lymph node metastasis. Results indicate that p53-negative expression and not p16INK4A-positive expression may be used as a marker for HPV status in ESCC; however, this finding requires further studies for validation.p16 China is one of the geographical regions with the highest incidence of esophageal cancer. Of the two main histological types, the squamous cell carcinoma (ESCC) and the adenocarcinoma (EAC), the former is the predominant type in China accounting for more than 80 % of cases [Esophageal cancer (EC), is one of the most common malignancies and the sixth most frequent cause of cancer-related death worldwide with a global number of 400,000 deaths in 2012 (Human papillomavirus (HPV) are small circular non-enveloped double-stranded DNA viruses exhibiting strict epithelial tropism and infecting either mucosa (α-papillomaviruses) or skin (β- and γ-papillomaviruses) . SeveralINK4A and its degradation mediated by HPV E7 causes the abnormal p16INK4A and p53 expression. Surrogate markers of HPV infection are essential for cancer screening given the low cost and high sensitivity of immunohistochemistry (IHC). Studies have shown that p16INK4A expression measured by IHC correlates with the presence of HPV DNA and thus can be used as a surrogate marker of HPV infection in squamous cell carcinoma (SCC) of the cervix, vagina, and oropharynx [HPV E6 and E7 oncoproteins are able to inactivate p53 and pRb oncosuppressors, respectively, interfering with cell cycle control. The oncosuppressor pRB is a negative regulator of the cyclin-dependent kinase inhibitor p16opharynx –17. p53 opharynx  and peniopharynx . HoweverINK4A and p53 expression levels and their correlation with HPV status and clinical parameters in Kazakh patients to raise the possibility of using them as surrogate markers of HPV infection in ESCC.This study aimed to investigate p16All formalin-fixed paraffin-embedded tissue (FFPE) blocks from 163 Kazakh patients who underwent esophagectomy without prior chemotherapy or radiotherapy were obtained from the First Affiliated Hospital of Shihezi University School of Medicine, the People’s Hospital of Xinjiang Uyghur Autonomous Region, and Xinjiang Yili Prefecture Friendship Hospital in Northwestern China from 1984 to 2013. Detailed clinical data  of all tAll tissues were sectioned and stained with hematoxylin and eosin. Furthermore, the morphologically representative tissue areas of each sample identified from these stained slides were marked. Subsequently, the fields corresponding to these selected regions were located in the paraffin block for tissue microarray (TMA) construction. 1.0 mm diameter tissue cylinders were punched from these areas of each donor tissue block and brought into a recipient paraffin block using a homemade semi-automated tissue arrayer . The region of each tissue cylinder was reviewed to guarantee that at least 70 % represented the typical region of interest in that sample. Finally, 5 μm-thick serial sections were prepared from the TMA blocks for immunohistochemical staining.INK4A and p53 were performed using an automated immunostainer . Slides were de-paraffinized using a bond dewax solution  and rehydrated in absolute alcohol. Afterward, bond epitope retrieval solution 2  was used for epitope retrieval, and the slides were incubated for 20 min at 100 °C and 12 min at room temperature. The slides were washed with bond wash solution  for 3 min. Endogenous peroxidase activity was abolished by incubating the slides for 5 min in a peroxidase-blocking solution. A total of 150 μl of the primary antibody against p16INK4A  or p53  was dropped onto each slide, followed by incubation for 15 min. After posting primary and polymery for 8 min each, the slides were incubated in DAB buffer for 5 min and then washed with distilled water. Subsequently, hematoxylin was added onto each slide followed by incubation for 5 min. The slides were dehydrated in graded alcohol to xylene and mounted on an anti-fade mounting medium with mounting glass. p16INK4A-positive cervical cancer and p53-positive esophageal cancer tissues were used as positive controls. The data of p16INK4A immunohistochemistry were available in 158 patients, whereas p53 expression was evaluable in 163 patients. Other samples were not evaluated because of the lack of carcinoma in the residual tissue material.Immunohistochemical staining of p16INK4A was considered positive if strong and diffuse staining was present in >50 % of the tumor cells [All the immunostained slides were independently evaluated by two experienced pathologists. Cases in which the two pathologists disagreed on the immunostaining results, a third pathologist was consulted to analyze the staining. The expressions of these two markers were scored based on cytoplasmic/nuclear staining intensity and percentage of positively stained cells. The staining intensity was categorized as follows: 0, negative; 1, buff; 2, yellow; and 3, brown. The percentages of positive stained cells were scored as follows: 0 (<5 % positive cells), 1 (6 %–25 % positive cells), 2 (26 %–50 % positive cells), 3 (51 %–75 % positive cells), or 4 (≥76 % positive cells). p16or cells , 21.The percentages of positive stained cells and the staining intensities were further multiplied to generate the immunoreactivity score for each case and evaluate p53 expression. Four categories of expression were listed as follow: − (a score of 0–1), + (a score of 2–4), ++ (a score of 5–8), and +++ (a score of 9–12) . p53 wasThe FFPE samples were collected in 5 μm thick sections with 10–15 slides per sample for genomic DNA extraction using QIAamp DNA FFPE Tissue Kit  according to the manufacturer’s instructions. The methods used were as previously described  to minimwww.ncbi.nlm.nih.gov/BLAST) to confirm the HPV type detected by PCR.HPV DNA was detected as previously described . Non-degINK4A, p53 expression, HPV status, and clinical parameters were analyzed using χ2-test or Fisher’s exact test. Spearman’s rank correlation coefficients were analyzed to investigate the possible correlations between HPV status and p16INK4A and p53 expression levels. All statistical tests were two-sided and p-values considered significant when p < 0.05.All statistical analyses were performed using SPSS Statistics 17.0 software. Associations among p16INK4A expression through immunohistochemistry, with 30 (19.0 %) and 128 (81.0 %) showing positive and negative expressions, respectively . Patients with p16INK4A-positive expression had significantly less lymph node metastasis (35.7 % versus 57.5 %) and were diagnosed at less advanced TNM stage, although the difference was not significant (p = 0.147). No correlation was found between p16INK4A expression and gender, age at diagnosis, histopathological grade, and invasion depth . p16INK4A-positive expression did not affect the odds of tumors being HPV positive . The sensitivity of p16INK4A-positive expression as an HPV marker was 0.164, with specificity of 0.788 and positive predictive value of 0.391.Although p16p = 0.171 and 0.070, respectively). No correlation was detected between p53 expression and other clinical parameters  and 19 (11.7 %) exhibited p53 positive and negative expression, respectively . The prevalence of HPV DNA in p53-negative specimens was significantly increased compared with that in p53-positive tumors (71.4 % versus 42.2 %). p53-positive expression decreased the odds of tumors being HPV positive (OR = 0.292 with 95 % CI = 0.086–0.990). The sensitivity of p53-negative expression as an HPV marker was 0.179, with specificity of 0.940 and positive predictive value of 0.714.A significantly inverse correlation between p53 expression and HPV status was found  were found HPV-positive and 67 (54.5 %) HPV-negative  but not with p16INK4A expression (p = 0.987). Similar results were obtained considering all HPV infections. p16INK4A and p53 expression patterns exhibited a slight inverse correlation, but this correlation was not statistically significant , and are frequently associated with lower-grade TNM stage (p = 0.147), which are in accordance with previous studies [INK4A positivity has been detected in 16.4 % of HPV-positive patients with ESCC, which is lower than previously published data reporting a range of prevalence between 20 % and 86.2 % [INK4A overexpression and HPV DNA positivity was previously found in HPV-related oropharynx carcinoma [INK4A over expression is not associated with HPV status . This finding is consistent which data described in a meta-analysis [INK4A overexpression. In the present study, a cut-off value of 50 %, which has been validated to correlate with the presence of HPV in oropharyngeal SCC [INK4A staining. The discrepant results may also be attributed to the variation in HPV prevalence because of different geographic areas and ethnicity of patients [INK4A staining for HPV status, an aberrant p16INK4A expression such as p16INK4A (+)/HPV(–) and p16INK4A (–)/HPV(+) cases in various cancers exists [INK4A expression were HPV-negative indicating that non-HPV factors also lead to p16 overexpression in ESCC. The diametrical expression of p16INK4A may be caused by different genetic alterations. For example, 11q is frequently detected to be gained in HPV-negative oropharyngeal SCC, wherein Ets (a protein that can raise the p16INK4A level) is located [INK4A overexpression have also been described in non-HPV-driven tumors [INK4A expression in HPV-negative tumors needs to be further investigated to obtain additional information in ESCC etiology, especially in low-incidence HPV geographic regions.The use of p16nt years , 28–30. l origin . In our  studies , 26, 32.d 86.2 % , 34. In arcinoma , 35. Thiarcinoma . Howeveranalysis  and recegeal SCC , was utis p = 0.0, and arepatients . In addis exists , 40, 41. located , 42. Rb1n tumors . Therefop = 0.171) and had poorer differentiation levels (p = 0.070) than those with p53-negative expression, although these differences were not significant. Similar to previously reported data, [p = 0.039, OR = 0.292 with 95 % CI = 0.086–0.990), which is in accordance with a previous study [Acting as a transcription factor in cell cycle regulation, genomic stability and apoptosis, p53 protein displays the highest correlation with a number of cancers , 45. p53ed data, , 48, theus study . HPV-assus study , whereasus study . Thus, pINK4A and p53 co-expression did not statistically differ. Likewise, p16INK4A and p53 expression levels have been analyzed in primary adenocarcinoma of the urinary bladder on 36 samples. A slight inverse correlation between p16INK4A and p53 expression was observed but without statistical significance [HPV positivity was detected in 45.5 % of ESCC patients in this study, which is similar with previously reported data in Kazakhs –12, withificance , which iINK4A-positive expression should not be interpreted as a reliable surrogate marker for HPV infection in Kazakhs with ESCC but can indicate lower risk of lymph node metastasis. Our findings show that p53 expression may be a useful biomarker for diagnosis and prognosis of HPV-positive ESCC. Nonetheless, further studies should be performed to investigate toinvestigate the molecular alterations of these cell-cycle related proteins to elucidate ESCC pathogenesis.In summary, p16


In the present research (N = 48), we found that the perceptual difference between white–gold perceivers  and blue–black perceivers  decreased significantly when the illumination information provided by the original digital photo was reduced by means of image scrambling (Experiment 1). This indicates that the illumination information is one potentially important factor contributing to the color ambiguity of #TheDress—possibly by amplification of a slight principal difference in psychophysics of color perception which the two observer groups showed for abstract uniformly colored fields displaying a white–blue ambiguity (Experiment 2).In early 2015, a public debate about a perceptual phenomenon that impressively demonstrated the subjective nature of human perception was running round the globe: the debate about  It had been initialized by the photo of a lace dress posted on Tumblr  that evoked ambiguous color perceptions in its observers. The debate mainly went: It’s white with golden lace!—No, it’s blue and black!—No, no, now it’s this, then it’s that, I can switch! … The observation that a stimulus can induce intra- or interindividually varying, sometimes even switching perceptions is not new, indeed. Perceptual multistability elicited by ambiguous figures such as the Necker cube is a classic example in this regard, and the evocation of perceptual challenge by means of ambiguity is even a core aesthetic principle, for example, in modern art  with normal or corrected-to-normal vision (checked via Snellen test) and normal color vision (checked via a short version of the Ishihara test) participated in the study which comprised Experiments 1 and 2. Each participant completed both experiments within one session. In order to prevent the psychophysical functions assessed in Experiment 2 from being biased by previous exposure to the photo of #TheDress which was shown in Experiment 1, the order the experiments were actually executed in was inverse to the numbering we use here: first Experiment 2, then Experiment 1. On basis of the ratings participants gave for the original digital photo of #TheDress in Experiment 1, they were post-hoc classified as white–gold perceivers and blue–black perceivers, respectively. The rating scale ranged from 0  to 100 (very blue), and participants’ classification was executed on basis of a mean score of 50—actually the result of classification would have been exactly the same if we had used a median split instead as the median was 52.5, so very close to the mean score of 50. By chance, it turned out that half of the sample was to be classified as white–gold perceivers , and the other half was to be classified as blue–black perceivers . The two groups clearly differed from each other . Participants were naive to the purpose of the experiments and were tested individually. All procedures were in accordance with the national ethical standards on human experimentation and with the Declaration of Helsinki of 1975, as revised in 2008. Informed written consent was obtained from all participants.A total of 48 volunteers ; the empirical primaries of our monitor were Red (R) = , Green (G) = , and Blue (B) =  in terms of Commission Internationale de l’Éclairage (CIE) color space xyY values. During the experiments, the participants were sitting at a constant distance of 50 cm in front of the screen, which was ensured by using a chin–forehead rest. For stimulus delivery and data collection we used the up-to-date Experiment Builder version 1.10.1241 .Both experiments were conducted in the same laboratory with constant lighting conditions . The experiments were run on a Dell Desktop PC with a true color Eizo Color Graphic CG245W TFT monitor . The monitor was linearized via the integrated calibration routine (IPS Panel) at the beginning of each session. The white point of the monitor was In Experiment 1, we tested whether illumination information provided by the photo of #TheDress had an impact on the perception of the colors of #TheDress. Our rationale was: If the color constancy explanation (see earlier text) is right, that is if interindividual differences in the use of illumination information significantly contribute to the differences in the perception of the dress, these differences will decrease when the illumination information of the image is reduced. A reduction of illumination information was implemented by means of image scrambling.a which were randomly permuted. For the present experiment, we defined the following 17 side lengths: a = 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2 pixels, and 1 pixel. The smaller the used scramble squares were , the less valid illumination information was preserved in the stimulus. So, for the scrambled version with the smallest scramble squares, illumination information was most strongly reduced as compared with the original photo . The scrambling process was performed twice for each side length. We thus obtained 2 × 17 = 34 scrambled versions. In sum, we had 1  + 1 (cropped image rectangle) + 34 (scrambled versions of the cropped image rectangle) = 36 stimuli to be used in Experiment 1. Several exemplary stimuli are presented in Note. Scrambling is only one possible procedure for systematically reducing the degree and validity of illumination information besides, for example, phase manipulation by adding noise, see We used the original digital photo of #TheDress and multiple variants of the same that varied with regards to the degree of information about illumination. Photographs usually provide some kind of information about the illumination of the original scene such as background or surround, detectable shadows, reflections, familiar objects the color of which is known, and so forth. For the present experiment, we systematically reduced illumination information by cropping and scrambling the digital photograph of #TheDress that had been posted on tumblr. More precisely, we first selected a rectangular part of the original digital photo so that there was nothing of the original background left in the resulting cropped image rectangle to prevent integrative scrambling of the background . The order of stimulus presentation was randomized. Instructions and a trial scheme for Experiment 1 are shown in Participants rated the original digital photograph of #TheDress as well as a cropped rectangular version and scrambled variants of the same with regards to their degree of being blue. The whole experiment comprised 36 trials. In each trial, one of the 36 stimuli was presented singly in the center of the screen until participants pressed the space key to indicate that they were ready to give their rating. On the following screen, participants were asked to indicate their impression of how blue the just-presented stimulus was. In order to do so, they entered an integer between 0 = While white–gold perceivers and blue–black perceivers differed markedly in their interpretations of the color of less segmented versions, their ratings became closer and closer to each other with increasing image segmentation . In factThe next question, however, is: Which factor(s) might underlie this individual difference in color constancy found for #TheDress? In Experiment 2, we investigated whether white–gold and blue–black perceivers also differ in their perceptions of simpler stimuli. To that end, we assessed the different observers’ psychophysical functions of color perception for a white–blue and a gold–black color continuum, respectively, and compared the perceptions of the two observer groups (white–gold and blue–black perceivers).1 xyY: 0.3340, 0.3456, 98.68) to blue , and one ranging from gold  to black , respectively—see General Method for monitor primaries. We divided each continuum into 51 color levels (shades) to be presented on the screen in the form of uniformly colored full-screen rectangles .We defined two color continua, one ranging from white  and to confirm their decision by pressing the enter key. The next trial started automatically . In sum, the experiment comprised 2 (parts) × 51 (stimuli) = 102 trials. Instructions and a trial scheme for the white–blue continuum are shown in The experiment comprised two parts. In the first part, participants rated all 51 color levels of the white–blue continuum; in the second part, they rated all 51 color levels of the gold–black continuum. For each continuum, the respective stimuli were presented on the screen one by one in randomized order until participants pressed the space key to indicate that they were ready to give their rating. On the following screen, participants were asked to indicate the color of the presented stimulus by entering an integer between 0 and 100 , especially in the case of color levels (shades) that were colorimetrically more white than blue. Here, blue–black perceivers reported higher amounts of blue than white–gold perceivers . This might reflect a slightly higher sensitivity for blue components or, at least, a slight bias toward interpreting white–blue ambiguities as blue rather than white.On the white–blue but not the gold–black continuum, the average psychophysical function obtained for white–gold perceivers differed slightly but systematically and significantly from the function obtained for blue–black perceivers  accessible any longer. The Gestalt itself, in this case the dress, its design and the texture of the material it is made of, however, are potential cues for inferences about color. In our study, the participants did not explicitly report changes of their percepts of #TheDress. Yet, in related studies in which we investigated further aspects of the phenomenon of #TheDress in different contexts (unpublished), we registered so-called “switchers” as well, and according to the data of The difference between the psychophysical curves of white–gold and blue–black perceivers that we found in Experiment 2 might be a first indication of more basic differences underlying the phenomenon of different percepts observed for #TheDress. An important next step would be locating the  basis of the found psychophysical results within the visual apparatus. Furthermore, the processes relating them to the differential use of illumination information remain to be identified.


Increased p16 expression is also seen in squamous neoplasms arising at other sites, including head, neck, and oropharyngeal tract. Squamous lesions are also frequently encountered at ocular surface and peri-orbital skin sites, but the prevalence of increased p16 expression in these lesions has been poorly studied.We retrospectively surveyed 13 ocular surface and 16 orbital squamous lesions biopsied at UC San Diego Healthcare System and VA San Diego Healthcare System for p16 expression by immunohistochemistry. These cases included ocular surface lesions with diagnoses of conjunctival intraepithelial neoplasm (CIN) and squamous cell carcinoma in situ. Peri-orbital eyelid biopsies included lesions with diagnoses of SCCis and invasive squamous cell carcinoma. We performed multivariate logistic regression, followed by student’s T-test or Fisher's exact test to determine if there were statistically significant associations between p16 immunoreactivity and patient age, gender, diagnosis, and ethnicity. Statistical significance was defined as p < 0.05.We found an unexpectedly large prevalence of strong nuclear and cytoplasmic p16 immunoreactivity in our cases. Almost all of the ocular surface squamous lesions were diffusely positive for p16 expression (12/13). All of the periorbital lesions showed diffuse p16 immunoreactivity (16/16). Altogether, 28/29 lesions tested showed strong and diffuse p16 expression. We found no statistically significant correlation between p16 expression and patient age, gender, ethnicity, or diagnosis. In 6 of the peri-orbital biopsies, we had sufficient tissue to assess high-risk HPV expression by in situ hybridization. Interestingly, all of these cases were negative for HPV, despite strong p16 expression.Strong p16 expression was observed in virtually all of the ocular surface and peri-orbital squamous neoplasms in our study. The relationship between p16 expression and HPV infection in ocular surface and peri-orbital sites requires further investigation. INK4A expression is seen in HPV-associated squamous lesions, and the strength and subcellular staining pattern of p16 immunohistochemical reactivity correlates closely with the degree of dysplasia, with higher-grade lesions showing intense, diffusely positive nuclear and cytoplasmic p16 expression in affected squamous cells [INK4A expression arises from neutralization of cellular p53 and pRb tumor suppressor proteins by HPV E6 and E7 oncogenes in infected cells [INK4A induction because they produce E6 and E7 viral oncogenes with lower ability to neutralize endogenous tumor suppressors [INK4A expression is commonly employed as a surrogate marker of infection by high-risk HPV subtypes.HPV is linked to development of cervical, anogenital, head/neck, and lung squamous lesions and carcinomas –5. Increus cells . Increased cells , 16. Lowpressors . TherefoMalignant squamous lesions of ocular and peri-ocular structures include conjunctival intraepithelial neoplasia (CIN) to frank invasive squamous cell carcinoma (SCC). HPV has also been detected in prior studies of ocular and peri-ocular squamous lesions and suggested to contribute to the pathogenesis of squamous lesions in the eye –21. HoweIn our study, we hypothesized that p16 immunoreactivity was significantly increased in malignant ocular squamous lesions. To examine this, we examined 29 ocular surface and periorbital squamous lesions excised at our institution that comprised malignant (CIN and SCC) ocular squamous neoplasms. We performed p16 IHC analysis on all lesions, followed by HPV ISH on a subset of p16-positive cases. We compared the histopathologic and molecular findings with clinical data to identify potential significant correlations between p16 expression in ocular squamous lesions and clinical characteristics of our patient population.We examined a total of 29 ocular surface and periorbital eyelid squamous lesion cases retrieved from the VA San Diego and UCSD Medical Center between July 2010-May 2014. These cases were routinely fixed in 10 % formalin and embedded in paraffin. H&E (hematoxylin and eosin)-stained slides were reviewed and classified by an ophthalmologic pathologist. All lesions with a diagnosis of CIN or SCC (in situ or invasive) were included in this study. The study was approved by the Institutional Review Boards at UCSD and VA San Diego.Immunohistochemical studies for p16 were performed using a Ventana Benchmark Ultra. The p16 antibody used was a monoclonal mouse antibody . Lesions were considered positive for p16 when both cytoplasmic and nuclear staining was observed in epithelial cells. Staining in dendritic cells and non-epithelial cells was disregarded.In-situ hybridization was performed on formalin-fixed, paraffin-embedded tissue by ARUP laboratories . The test was run on a Ventana Bentana Benchmark Ultra , using the INFORM HPV II FAMILY 6 (ASR) probe. A probe set for high-risk HPV [risk HPV , 18 wereStatistical analysis was performed on CINs and SCCs. The correlation between p16 immunoreactivity and patient’s age, gender, diagnosis, and ethnicity was first analyzed by Multivariate Logistic Regression using SAS 9.3, followed by individual student’s T-test (for age difference) or Fisher’s exact test . A difference was considered statistical significant if p < 0.05.A total of 29 cases were evaluated for p16 immunoreactivity Table , Table 1in-situ hybridization against HPV genotypes 6, 11, 16, and 18 was performed on 6 lesions in total that were intensely and diffusely positive for p16 by immunohistochemistry. Of six squamous cell carcinomas tested, none were positive for HPV. CIN cases had insufficient remaining tissue in the blocks to perform the HPV ISH assay due to the small size of the conjunctival biopsy.HPV We next examined if positive p16 expression correlated with pathologic type of squamous lesion, patient age (less than or older than 50), patient gender, and ethnicity  by multivariate logistic regression, Student’s t-test, and Fischer’s exact test. No significant differences were seen between the pathologic types of lesions tested and p16 positivity (p >0.05). In addition, there were no significant differences between p16 positivity and age, gender, ethnicity or diagnosis.Our study reveals a surprisingly high prevalence of p16 immunoreactivity in ocular squamous lesions from our patient population, but interestingly, these squamous lesions with strong p16 expression did not show HPV 6, 11, 16, and 18 genetic material by ISH. We discuss some potential causes for the increased p16 expression but low HPV infection observed in our study.P16 immunoreactivity is well established as a biomarker for HPV infection in sites such as cervix, and the vast majority of HPV-associated cancers show increased p16 expression , 24, 25.Our HPV ISH test was performed by ARUP labs, using a commercially available limited HPV ISH probe against HPV genotypes 6, 11, 16, and 18 that is widely used to identify HPV infection in human tissue specimens. These HPV genotypes are widely observed in cervical squamous lesions but their trophism to other squamous sites is less clear. Ateenyi-Agaba and colleagues isolated several uncharacterized HPV genotypes from their conjunctival SCC lesions . It remaHPV detection techniques vary in their sensitivity . Our stuOur studies showed that p16 expression is frequently increased in malignant squamous lesions arising at the conjunctiva or adjoining eyelid.


Anal cancer is a rare disease with an increasing incidence worldwide but, unfortunately, even today the scientific community still has a limited knowledge and limited options of treatment.More than 50% of patients with anal cancer presenting at diagnosis with locoregional disease have good chances of cure with chemoradiotherapy (CT–RT). However, once patients develop metastatic spread, the prognosis is very poor.Human papillomavirus (HPV) is present in more than 80% of anal cancers and while multiple etiologic connections between HPV infection and anal cancer have already been well elucidated, its prognostic and/or predictive role is currently under investigation, especially among immunocompetent patients affected by this disease.In a single-institutional set, we have retrospectively analysed clinical data of 50 consecutive cases homogeneously treated with CT–RT for stage I–III anal squamous cell carcinoma. We found that HPV-positive anal cancers had a statistically significant improved five-year disease-free survival (DFS) compared to HPV-negative group. These findings could be explained by an increased chemo/radiosensitivity of HPV-positive tumours. Further efforts should be directed towards a better understanding of HPV-related oncogenesis and towards designing novel tailored strategies for the management of this disease both in terms of prevention and treatment. Anal carcinoma (AC) is a rare disease in the general population, being estimated at about 1.5 cases per 100,000 people per year worldwide, but with an increasing incidence particularly in women .Several biomolecular and epidemiologic studies have clearly elucidated a strong etiologic connection between AC and high-risk genotypes of HPV , 4, and Factors increasing the risk of HPV infection and/or modulating host response and the persistence of this infection appear to affect the epidemiology of this tumour. Anal intercourse and a high lifetime number of sexual partners increase the risk of persistent HPV infection in men and women, leading eventually to malignancy. Other important risk factors include human immunodeficiency virus (HIV), immunosuppression in transplant recipients, use of immnunosuppressants, a history of other HPV-related cancers, autoimmune disorders, social deprivation, and cigarette smoking .Not surprisingly, AC is no longer a rare disease within specific epidemiologic categories, since among HIV seropositive men the incidence of AC increases to 75–135 per 100,000 and is also higher among HIV seropositive women . TherefoOn the other hand, few studies have so far evaluated the prognostic or predictive role of HPV in AC, especially among the general population and immunocompetent patients.Based on these considerations we have retrospectively reviewed the data of a series of 50 consecutive and immunocompetent cases, treated in our institution between 2000 and 2012. These patients had AJCC (American Joint Committee on Cancer) stage I–III histologically-proven anal squamous cell carcinoma, and had undergone standard treatment with concurrent chemo-radiotherapy (CT–RT). In all patients the presence of HPV infection was assessed by a polymerase chain reaction (PCR) analysis on the tumour biopsy provided at the time of diagnosis before treatment. Genomic DNA was extracted from three 10-μm-thick paraffin sections using QIAamp DNA FFPE Tissue kit , following the manufacturer’s instructions, then 200 ng of DNA were amplified using L1 primers and GP5+/GP6+ primers. For all the PCR procedures, a positive and a negative control were provided. All the positive cases were confirmed in a second round of amplification. The PCR products were detected by electrophoresis with a 2% agarose gel and stained with ethidium bromide and in the presence of HPV infection, we proceeded to do the genotyping by testing the presence of high-risk HPV16, 18, 31, 33, and 45, using specific primers. Inclusion and exclusion criteria as well as the details on concurrent CT–RT are extensively reported elsewhere .In our study we found that 84% of patients were HPV-positive and, among these HPV genotype 16 was detected in 90% of cases. HPV-positive patients had more advanced disease at diagnosis compared to HPV-negative patients in terms of nodal involvement affecting TNM clinical stage.In particular, within HPV-positive group 2.4% of patients had stage I, 23.8% stage II, 45.2% stage IIIA, 28.6% stage IIIB, and within HPV-negative group 0% of patients had stage I, 62.5% stage II, 37.5% stage IIIA, 0% stage IIIB.In this regard, in the statistical analysis, we decided to apply an adjustment for clinical stage in multivariate analysis since the HPV-positive patients presented a higher rate of nodal involvement .After a median follow-up of four years, we found that five-year DFS in HPV-positive and HPV-negative patients was 92.5% and 50% respectively (p < 0.01). Five-year overall survival (OS) in HPV-positive and HPV-negative patients was 93.3% and 66.7%, respectively (p = 0.12). In conclusion, patients with HPV-positive AC had a strong statistically significant improved DFS compared to that in HPV-negative patients; the same trend was observed for OS, but statistical significance was not reached.Despite its small sample size and retrospective nature, this study shows that HPV infection in AC is a positive prognostic factor for DFS. This could be explained by the better response of HPV-positive AC to CT–RT, as already shown in HPV-positive oropharyngeal squamous cell carcinoma , 9.Certainly a greater patient sample size and longer follow-up might also show in the near future a significant difference in OS between HPV-positive and negative AC.From our perspective, the most intriguing and challenging question is whether HPV-positive AC and HPV-negative AC are two completely different malignancies sustained by different biomolecular features and genetic alterations. Further research should be directed towards investigating which specific pathways are selectively altered in anal cancers which are HPV-positive and those which are HPV-negative, by adopting a comparative approach. This should lead to a better understanding of HPV-related oncogenesis and to the design of novel and personalised tailored strategies for the management of this disease both in terms of prevention and treatment.Preclinical models as faithful as possible to the true HPV-related tumour cell biology should be the right place from which experimental studies can embark upon their challenging road.Currently the vast majority of modern studies, including analysis of E6 and E7 mRNA , 11, PIKDespite its small sample size and retrospective nature, this study shows that HPV infection in anal cancer is a positive prognostic factor for disease-free survival. This could be explained by the better response of HPV-positive anal cancer to chemo-radiotherapy, as already shown in HPV-positive oropharyngeal squamous cell carcinoma.From our perspective, the most intriguing and challenging question is whether HPV-positive anal cancer and HPV-negative anal cancer are two completely different malignancies sustained by different biomolecular features and genetic alterations. Further research should be directed towards investigating which specific pathways are selectively altered in anal cancers which are HPV-positive and those which are HPV-negative, by adopting a comparative approach. This should lead to a better understanding of HPV-related oncogenesis and to the design of novel and personalised tailored strategies for the management of this disease both in terms of prevention and treatment.The authors declare that they have no conflicts of interest.