In addition, the past 20–40 years in many Western Countries, the incidence of HPV-positive TSCC and BOTSCC has risen, and >70% are men. This has resulted in a relative increase of patients with HPV-positive TSCC and BOTSCC that may not need the intensified chemo-radiotherapy (with many more severe debilitating side effects) often given today to patients with head and neck cancer. However, before tapering therapy, one needs to enable selection of patients for such treatment, by identifying clinical and molecular markers that together with HPV-positive status will better predict patient prognosis and response to therapy. To conclude, there is a new increasing group of patients with HPV-positive TSCC and BOTSCC with good clinical outcome, where options for better-tailored therapy are needed. For prevention, it would be of benefit to vaccinate both girls and boys against HPV16 infection. For potential future screening the ways to do so need optimizing.In 2007, human papillomavirus (HPV) type 16 was recognized as a risk factor by the International Agency for Research on Cancer, for oropharyngeal squamous cell carcinoma (OSCC), where tonsillar and base of tongue cancer (TSCC and BOTSCC) dominate. Furthermore, patients with HPV-positive TSCC and BOTSCC, had a much better clinical outcome than those with corresponding HPV-negative cancer and other head and neck cancer. More specifically, survival was around 80% for HPV-positive TSCC and BOTSCC  This urvival) ,19,20,21urvival) ,9,19. HeThere are >170 HPV types, with the majority found in the skin (cutaneous HPV types), but many also found in mucous tissues  and where clearly the vast majority cause only asymptomatic infections ,23. A phMucosal HPV types can be divided into high-risk (HR) types that have a clear and well-recognized potential to cause cancer, or low-risk types (LR) that are very rarely observed in cancer ,23. The Cutaneous HPV types are best known to cause skin warts, but multiple skin cancers may emerge from verruca-like papillomatous lesions in Epidermodyplasia vercucciformis (EV) patients that are especially sensitive to infections with e.g., HPV5 and 8 ,5,23. WhINK4a [INK4a was in the past used as a surrogate marker for presence of HPV in OSCC [INK4a or the presence of HPV DNA alone, are regarded as sufficient to point that a tumor is caused by HPV [INK4a overexpression is almost as sensitive as using the golden standard, i.e. analyzing for presence of HPV E6 and E7 mRNA [HPVs have double stranded circular DNA genomes of around 7.9 kb. The genome, including a non-coding control region (NCCR), an early and a late coding region, is enclosed together with histones within a 52–55 nm virion ,24. The INK4a ,27. Overd by HPV . However E7 mRNA . The L1  E7 mRNA ,23,29. T E7 mRNA ,31. Thes E7 mRNA ,33,34,35OSCC includes not only TSCC and BOTSCC, which accounting for 80% of the cases, but also cancer of the walls of the pharynx and the soft palate . Howevervs. 40% five-year survival) [The most prominent difference between HPV-positive and HPV-negative TSCC and BOTSCC is that patients with HPV-positive tumors have a much better clinical outcome than those with HPV-negative tumors and other head and neck cancer, including OSCC other than TSCC and BOTSCC  ,4,7,8,9.urvival) ,36.Patients with HPV-positive TSCC and BOTSCC as compared to those with HPV-negative tumors are also often somewhat younger, more often non-smokers, have smaller tumors, but have a higher tumor stage, since many patients also have nodal disease ,7,8,9,37Ink4a overexpressed in most cases, in contrast to that observed in HPV-negative TSCC and OSCC [Most HPV-positive TSCC and BOTSCC independent of episomal/and or integrated HPV genomes, exhibit E6 and E7 mRNA expression; with p53 expression more often, being normal and with 16and OSCC ,38,39,40and OSCC ,42. HPV and OSCC ,41,42.Today the golden standard of HPV-positive status in TSCC/BOTSCC/OSCC is the presence of E6 and E7 mRNA expression by RT-PCR, suggested to be associated with functional HPV expression . NotablyFor example, between 1970 and 2002, a three-fold increase in both the incidence and prevalence of HPV (both in women and men) was observed in TSCC in Stockholm, Sweden and this lead to the hypothesis that HPV was responsible for the increase in incidence of TSCC . This waParallel to the Stockholm studies, an emerging epidemic of HPV associated OSCC was suggested in the US, and in 2011 in the U.S. a rise in the incidence of HPV-positive OSCC and a decline in HPV-negative OPCC was also reported for the past decades ,16,21. FAs already mentioned, the increase in incidence of TSCC, BOTSCC and OSCC has been suggested due to a rise in HPV-positive cases and changes in life style, and there is a significant correlation between HPV-positive OSCC, early sex debut, and numbers of oral or vaginal partners . HoweverHPV-positive TSCC and BOTSCC contribute to an increasing proportion of HNSCC in recent years . Furtheri.e., package years, was also important [It has been shown that for TSCC and BOTSCC, the presence of HPV DNA/RNA and p16 overexpression are very good prognostic markers especially when combined with accurate data on that the patient being a never smoker ,28,37. IAdditional biomarkers have been investigated. It has been reported that absent/low expression of MHC class I, CD44, CD98, LMP7, or LMP10 intensity staining, or absence of HLA-A*02, or high LRIG1 expression improved prediction of clinical outcome for patients with HPV-positive TSCC and BOTSCC ,60,61,62That having high CD8+ TIL counts was favorable was expected, since an efficient immune response may result in a favorable clinical outcome of HPV-positive TSCC and BOTSCC ,64. MoreOther biomarkers that have been investigated are miRNAs and data are accumulating, but stringent concordance between different studies has not been shown so far ,66,67.Clearly, additional molecular knowledge and ways to combine different markers, both clinical and molecular for prediction of clinical outcome in patients with HPV-positive TSCC and BOTSCC would be of great value.HPV16 is the most common HPV type in the oral cavity in non-vaccinated individuals ,69,70,71In HPV vaccinated groups, oral HPV prevalence, especially HPV16, seems to be lower than in non-vaccinated groups suggesting a vaccination effect ,33,34,35For non-vaccinated individuals, other approaches may be of interest and screening may not be optimal for several reasons especially since the incidence of HPV-positive TSCC and BOTSCC is still relatively low ,14,49. FNevertheless, in the proportion of patients with HPV-positive TSCC and BOTSCC, it has been shown that the viral load of HPV16 in mouthwashes is considerably higher than that obtained in healthy youth and often comparable to that obtained in the cervix . WhetherHPV-positive TSCC and BOTSCC have better clinical outcome than corresponding HPV-negative cancers and are increasing in incidence. Preventing spread of HPV16 infection by vaccination, and deescalating intensive therapy by using additional predictive markers to identify and select HPV-positive TSCC and BOTSCC patients eligible for randomized trials with lessened therapy are issues of importance.


To generate knowledge about potential improvements to human papillomavirus (HPV) vaccination information and organization strategies, we assessed how aspects of HPV vaccination are associated with parents’ preferences for their daughters’ uptake, and which trade-offs parents are willing to make between these aspects.A discrete choice experiment (DCE) was conducted among parents with a daughter aged 10–12 years. Panel mixed logit regression models were used to determine parents’ preferences for vaccination. Trade-offs were quantified between four vaccination programme aspects: degree of protection against cervical cancer, duration of protection, risk of serious side-effects, and age of vaccination.Total response rate was 302/983 (31%). All aspects influenced respondents’ preferences for HPV vaccination (p < 0.05). Respondents preferred vaccination at age 14 years instead of at a younger age. Respondents were willing to trade-off 11% of the degree of protection to obtain life-time protection instead of 25 years. To obtain a vaccination with a risk of serious side-effects of 1/750,000 instead of 1/150,000, respondents were willing to trade-off 21%.Uptake may rise if the age ranges for free HPV vaccinations are broadened. Based on the trade-offs parents were willing to make, we conclude that uptake would increase if new evidence indicated outcomes are better than are currently understood, particularly for degree and duration of protection. Human Papillomavirus (HPV) infection is a necessary factor in the development of cervical cancer . VaccineThe attitude of parents and girls towards HPV vaccination and consequently its uptake, may be influenced by their perception of the advantages and disadvantages of the vaccine . An advaIn DCEs, it is assumed that a medical intervention, such as a vaccination programme, can be described by its characteristics  , and by The selection of attributes and their levels was based on data from the literature ; focus g4) hypothetical HPV vaccination alternatives. Using an efficient design by maximizing D-efficiency , 54 choice sets were constructed to be able to estimate all main effects and all two-way interactions between attributes. Choice sets consisted of two HPV vaccination alternatives and a ‘no HPV vaccination’ option to allow respondents to ‘opt out’ .We assessed respondents’ understanding of the DCE task by including a dominant choice set as a rationality test. In this choice set the HPV vaccine was given at the age of 12 years in both alternatives, while one alternative was characterized by logically preferable levels on all other attributes. Convergent validity was checked with a ranking task, i.e. ranking the four attributes of HPV vaccination from most important to least important. To gain more insight into respondents’ understanding of the DCE task, i.e. comparing risks and percentages, we included the Subjective Numeracy Scale (SNS), a scale that correlates well with objective measures of numeracy skills ,31. HighThe questionnaire was pilot tested to check for face validity and for problems in interpretation (n = 16). This resulted in an improved explanation of the risk of serious side-effects. Approval for the study was obtained from the Medical Ethics Committee, Erasmus MC, University Medical Center Rotterdam (MEC 2008–206).Questionnaires and information letters were sent to primary schools between March and June 2009 to be distributed to 10 to 12-year-old girls to give to their parents. Parents could return the questionnaire in a postage-paid envelope that was included in the mailing package.The DCE was analysed by taking each choice among the three options as an observation, i.e. two ‘no’ and one ‘yes’ responses. The observations were analysed by panel mixed logit regression models to take heterogeneity as well as correlation between the choice sets completed by each individual into account . After t1-β7 are coefficients of the attributes indicating the relative weight individuals place on a certain attribute (level). When considering an HPV vaccination that generates a 50%, 70% or 90% protection rate, the coefficient β1EFFECTIVENESS should be multiplied five, seven or nine times, respectively. The statistical significance of a coefficient  indicates that respondents differentiated between one attribute (or attribute level) and another in making stated choices about HPV vaccination programmes. A priori, we expected all attributes to be significant. The sign of a coefficient reflects whether the attribute has a positive or negative effect on the preference score of HPV vaccination. We expected that only the attribute ‘risk of serious side-effects’ would have a negative effect. The value of each coefficient represents the relative importance respondents assign to an attribute (level).V is the observable utility that is composed of the preference scores (β-coefficients) for the individual and the characteristics of the HPV vaccination alternative. βSensitivity analyses were conducted to explore the impact of excluding respondents who failed the rationality test by excluding their data from the sample and re-running the analysis ,33. AlsoThe trade-offs respondents were willing to make between the HPV vaccination attributes were calculated by the ratios of the coefficients of the different attributes with the degree of protection as the denominator. Choice probabilities for HPV vaccination uptake were also calculated to provide a way to convey DCE results to decision makers that is easier to interpret. The probability that an individual will say “yes” to an HPV vaccination programme is equal to:where V is defined as in Equation 1. We calculated the choice probability (i.e. the mean uptake) of a base-case compared to no vaccination (V (no vaccination) = 0)). Our base-case represents an HPV vaccination programme at the age of 12 years, a 1/150,000 risk of serious side-effects, a duration of protection of 6 years, and a 70% degree of protection. This base-case was chosen to correspond with i) the Dutch situation (vaccination at the age of 12 years) and ii) an HPV vaccination programme that contained most plausible levels based on literature. Noteworthy, in the calculation of the mean uptake all heterogeneity of the respondents was taken into account as the mean uptake is not just equal to the uptake of someone with average coefficient values of the levels. We presented these results in a “tornado” graph to illustrate the marginal effect on uptake of varying one attribute level at a time from the base-case, holding all other attributes constant  . In total, 294 out of these 302 parents (97%) completed the DCE task and were included for further analyses. The mean age of the parents was 42.7 (SD = 3.4) years. 90% of the respondents were female and about half had an intermediate educational level (54%) and had a religious affiliation (56%)  who ‘failed’ the rationality test had no relevant impact on the size or relative importance of the attributes. Adding two-way interactions did not significantly improve the fit of the model (data not shown).The results of the ranking task showed that the most important attributes were: the degree of protection ; the risk of serious side-effects ; and the duration of protection . These results are in accordance with the DCE results (i.e. the order of importance is the same as the order of the coefficients), supporting a convergent validity of the DCE results.Parents’ scores on the Subjective Numeracy Scale ranged from 1.5 to 6.0 with a median of 4.6  and inter quartile range (IQR) of 1.2 (data negatively skewed). The Cronbach alpha coefficient was 0.88, suggesting very good internal consistency reliability.Parents were willing to trade-off the degree of protection against cervical cancer in order to gain improvement in the levels of the other attributes. They were willing to trade-off 11% of the degree of protection to obtain life-time protection instead of 25 years. To obtain an HPV vaccination with a risk of serious side-effects of 1/750,000 instead of 1/150,000, parents were willing to trade-off 21%. To get a vaccination at age 14 years instead of 9 years, parents were willing to trade-off 10% , the expected uptake based on parents’ preferences was 63.3%. Especially an increase in the duration of protection from 6 years to lifetime would result in a relatively large increase in the expected uptake (12.2%). On the other hand, an increased risk of serious side-effects from 1/150,000 to 1/30,000 would result in a decrease in the expected uptake (13.4%) (Figure This study shows that the degree of protection against cervical cancer, the duration of protection, the risk of serious side-effects, and the age of vaccination, significantly influenced parents’ preferences for HPV vaccination. Parents preferred vaccination at age 14 years over age 9 years. Although parents preferred a higher degree of protection against cervical cancer, they were willing to trade-off some degree of protection in order to gain improvement in the levels of the other attributes.Our finding that the duration and the degree of protection were relevant for parents’ preferences for HPV vaccination was also found in a DCE study among mothers . Our stuThe expected uptake of our base-case HPV vaccination programme was 63% based on parents’ preferences. This rate is higher than the actual uptake of 52% in the Netherlands in 2009 at the time our study was conducted . A possiOur study has some limitations. First, the majority of responding parents were mothers, although the questionnaire was addressed to both parents. This seems common in studies assessing parental attitudes regarding HPV vaccination ,39,40. WIn conclusion, this study shows that parents’ preferences for HPV vaccination were influenced by the degree of protection against cervical cancer, the duration of protection, the risk of serious side-effects, and the age of vaccination. Uptake may rise if the age ranges within which a girl is entitled to free HPV vaccinations are broadened. Based on the trade-offs parents were willing to make, we conclude that uptake would increase if new evidence indicated outcomes are better than are currently understood, particularly for degree and duration of protection.The authors declare that they have no conflict of interests.IJK conceived the idea for the study, designed the protocol, and supervised the execution of the study; EWBG, RH, IJK, MB, TJMH, and HR designed the questionnaire; RH performed the retrieval of the sample and was responsible for all mailings; RH and EWBG were responsible for the database design and data entry; EWBG performed the statistical design and analyses; RH drafted the report. All authors revised the article critically and approved the final version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/454/prepub


Many veterans engaged in care with the Veterans Administration (VA) health system are also enrolled in Medicare and/or Medicaid and may receive care both inside and outside of the VA. Use of dual health systems has been associated with worse outcomes. Veterans with HIV may have different rates of Medicare and Medicaid enrollment and may be at greater risk of poor outcomes related to non-VA use. This study compares the frequency and factors associated with Medicare and/or Medicaid enrollment and non-VA use in an HIV-infected and uninfected population of veterans.We used data from the VA and Center for Medicare & Medicaid Services from 2004 and 2005 to determine the frequency of Medicare and/or Medicaid enrollment among a cohort of HIV-infected and uninfected veterans engaged in VA care. We then restricted the cohort to veterans enrolled in fee-for-service (FFS) Medicare and/or Medicaid with at least one hospitalization and identified characteristics associated with non-VA hospital admissions.HIV-infected veterans had higher rates of Medicare and/or Medicaid enrollment than uninfected veterans , though the opposite was true when our sample was limited to veterans 65 years and older . Among veterans enrolled in the VA and FFS Medicare and/or Medicaid, veterans with HIV had greater illness severity and more frequent hospitalizations, but were less likely to be hospitalized outside the VA . HIV infection was associated with lower odds of outside hospitalization .Veterans with HIV have higher rates of Medicare and/or Medicaid enrollment, but lower odds of non-VA hospitalization. The VA integrated model of HIV care may discourage outside use among HIV-infected veterans. A significant proportion of veterans enrolled with the Veterans Administration (VA) healthcare system are also enrolled in Medicare and/or Medicaid (“CMS enrollment”). An expansion of Medicaid coverage under the Patient Protection and Affordable Care Act of 2010 (ACA) will likely increase the proportion of CMS enrolled veterans .Previous work has described CMS enrollment and identified factors that influence CMS use among VA enrolled veterans, particularly veterans 65 years and older that have age qualified for Medicare -9. Less HIV-infected veterans are an aging cohort vulnerable to adverse outcomes because of the complexity of their care needs, including multimorbidity, polypharmacy, mental health issues, and substance use -14. The The objectives of this paper were to: 1) determine the frequency of Medicare and/or Medicaid enrollment among HIV-infected veterans engaged in VA care, compared with uninfected veterans; 2) determine the frequency of non-VA hospital admissions among male, CMS enrolled HIV-infected and uninfected veterans with any hospitalizations; and 3) identify characteristics that predict non-VA hospital admission among male HIV-infected veterans enrolled in Medicare and/or Medicaid, compared with uninfected veterans. We hypothesized that CMS enrolled HIV-infected veterans would be less likely to use non-VA care than their uninfected counterparts.This retrospective cohort study included two populations of veterans in VA care in the United States during 2004 and 2005: 1) HIV-infected veterans; and 2) age, race and facility-matched uninfected comparators.We used data from the Veterans Aging Cohort Study (VACS), a prospective, observational cohort study of HIV infected veterans and age, race and site matched controls. VACS is made up of eight urban sites, but also includes a virtual cohort (VACS-VC), a nationally representative sample of HIV-infected veterans and matched uninfected controls identified through the VA electronic medical record (EMR) system and followed since 1996 ,18. DemoWe started with a cohort that included all veterans enrolled in the VACS-VC prior to January 1, 2003  Medicare and/or Medicaid that received inpatient care in 2004 and 2005 (cohort 2). Our dependent variable was a dichotomous indicator of any non-VA admissions, (vs. only VA inpatient admissions). Hospital admissions were identified by combining VA EMR data with CMS FFS reimbursement files.We considered a number of independent variables we believed could influence use of non-VA inpatient care, including demographics, access to care, illness severity, and admission to a medical or surgical intensive care unit (ICU). Age and race data were taken from VA administrative databases. Age was constructed as a dichotomous variable, under age 65 and 65 years and older. Race was divided into three categorical variables: White, African American, and Other.Patients’ access to care was measured by three variables: enrollment in Medicare, Medicaid or both programs; distance to nearest VA tertiary care center treated as a categorical variable: 0 to 9.9 miles, 10-29.9 miles, 30-59.9 miles, 60-119.9 miles, and greater than 120 miles [We used the CMS hierarchical condition categories (HCC) model to calculate risk scores  and usedWe generated categorical variables for number of inpatient admissions (one vs. two or more) and the admission diagnosis (ICU vs. non-ICU admission) because we believe both could influence the likelihood of a veteran having a non-VA admission. Veterans with more frequent admissions have more “opportunities” to be hospitalized in a non-VA facility. Similarly, patients in critical condition, requiring admission to ICUs, may be more likely to be routed to a non-VA hospital. Though these variables do not reflect patient behavior, they may influence the likelihood of an admission outside the VA and, thus, we controlled for these factors in our model.We first examined veterans in the VACS-VC (cohort 1) to determine the frequency of CMS enrollment among veterans engaged in care. We stratified our analysis by age and HIV status, and used student t-tests to compare the frequency of CMS enrollment between HIV-infected and uninfected veterans. Next, we described the cohort of male, veterans enrolled in FFS Medicare and/or Medicaid with at least one inpatient admission (cohort 2) using the independent variables listed above. We stratified cohort 2 by HIV status and used chi-squared tests to compare the two sub-populations. Finally, we explored the association between these independent variables and non-VA hospital admissions using univariate and multivariable logistic regression. We created a multivariate model that included all patients in cohort 2, as well as separate models stratified by HIV status. All analyses were conducted using SAS version 9.2.Overall, HIV-infected veterans engaged in VA care were more likely than uninfected veterans to be enrolled in Medicare and/or Medicaid  (Table Among veterans enrolled in FFS Medicare and/or Medicaid with at least one inpatient admission cohort 2) the frequency of HIV-infected veterans with CMS use was lower than the frequency among uninfected veterans   Table . In addiThe association between HIV status and decreased odds of non-VA use remained in the multivariable model ; however, there was no longer an association between higher HCC risk score and increased non-VA use Table . We posiAfter stratifying by HIV statuses, the predictors of CMS use were similar in both the HIV-infected and uninfected models Table .In our sample of veterans engaged in VA care, we found that HIV-infected veterans were more likely than their uninfected counterparts to be enrolled in Medicare and/or Medicaid, but this difference varied by age. HIV-infected veterans under age 65 were more likely to be CMS enrolled, while HIV-infected veterans 65 years and older were much less likely to be CMS enrolled. Confirming our initial hypothesis, we also found that male, HIV-infected veterans enrolled in FFS Medicare and/or Medicaid were less likely to have an outside hospitalization. This was despite the fact that these veterans were generally sicker, more likely to have multiple admissions, and more likely to be enrolled in FFS Medicaid – independent risk factors for greater non-VA use.We suspect that HIV-infected veterans were less likely to use non-VA hospitals because they feel more tied to the VA – possibly due to the integrated care and the different providers they see in VA infectious disease clinics or the desire for continuity with established caregivers for what is historically a stigmatized disease. The prescription drug coverage afforded veterans – and the high costs of antiretroviral therapy - may also play a significant role. While every state provides some coverage for antiretroviral drugs, the coverage varies substantially. Very few states cover as many of these drugs as are covered nationally within VA. This conclusion is also supported from patient self report data in VACS 8 in which 96% of those on ART reported getting all their antiretroviral medications through VA .Our findings on frequency of CMS enrollment among veterans were not completely consistent with previous literature. Overall, we found lower rates of Medicare enrollment, but higher rates of enrollment in Medicaid . This diOur findings regarding predictors of non-VA hospitalization among CMS enrolled veterans engaged in VA care were consistent with previous literature. In general, these studies have found older age, increased distance to the VA, greater cost-sharing and worse health to be associated with non-VA use ,8,22,23.Our results should be viewed in light of several limitations – most importantly, our limited access to data on private insurance enrollment and utilization in the VA population. As a result of the lack of claims data for patients hospitalized under private insurance, some patients classified as having only VA admissions may actually have unaccounted for admissions to non-VA hospitals. Moreover, CMS enrollment with private insurance may lead to significantly lower costs for veterans, influencing their choice of health care setting. The VACS does collect some survey data on private insurance enrollment. Among veterans in VACS8, 15% of HIV-infected veterans and 20% of uninfected veterans reported enrollment in private insurance . Given the lower rates of supplemental insurance in the HIV-infected population, we believe our finding that HIV-infected veterans are less likely to have non-VA admissions likely still holds true. Another limitation is that inpatient admissions are more likely to be influenced by factors beyond patient preference. We tried to control for those variables in our analysis, but using inpatient admissions as our outcome may limit the conclusions that can be drawn from our results. We chose inpatient admissions because non-VA inpatient care places HIV-infected veterans at risk for adverse events related to poor coordination of care transitions between outside hospitals and VHA HIV clinics; however, choice in outpatient setting may be a better reflection of patient preferences and will be the subject of future studies. We also did not take into account pathways to CMS enrollment. Veterans that were enrolled in Medicare or Medicaid prior to engagement with the VA system may be more likely to have non-VA admissions; the opposite may be true of veterans that were seeking care with the VA prior to enrolling with Medicare and/or Medicaid. In addition, though most HIV infected people with Medicare qualify through disability, it is unknown if this is also true among veterans.The heterogeneity of Medicare and/or Medicaid from state to state further limits our ability to generalize the data. Medicare Advantage plans, i.e. managed care plans provided by private insurers, are highly variable by design, and Medicaid benefits and eligibility vary from state to state. Moreover, to date only 26 of the 50 states have chosen to accept the ACA provision to expand Medicaid, making comparisons between states more difficult in the post-ACA era. Finally, our findings are limited by the age of our data. We restricted our analysis to 2004 and 2005, the latest years we had complete data available. However, we believe our analysis uses more recent data than similar studies analyzing CMS enrollment and CMS use.Many veterans engaged in care in the VA are also enrolled in Medicare and/or Medicaid, including a significant proportion of veterans under age 65. Like the general population, the rates of CMS enrollment are higher among HIV infected veterans. However, among veterans enrolled in both VA and CMS care, HIV-infected veterans have lower odds of non-VA hospitalization despite having greater illness severity and more frequent hospitalizations than uninfected veterans. The VA’s integrated model of care used to treat HIV/AIDS may explain why HIV-infected veterans were more likely to seek care within the VA.The impact of CMS use on veterans with HIV and other complex medical conditions requires further investigation. HIV-infected veterans have complicated medical histories and are likely to experience inefficient care and poor outcomes related to non-VA use. Research that provides evidence of the cost and health implications of CMS use may guide the VA in how aggressively to pursue and retain these patients in the VA system.Future research should also explore why veterans with HIV were less likely than uninfected veterans to use non-VA care. If specific aspects of VA’s integrated care programs for veterans with HIV, such as intensive case management, are found to contribute to lower likelihood of non-VA admission, then this would inform strategies to limit non-VA use among other veteran populations at high risk from fragmented care, such as veterans with other major chronic diseases or illnesses.CMS enrollment is a valuable resource for veterans who face higher copayments or live long distances from VA medical centers; however, among some veterans, CMS enrollment may lead to inefficient care and poor health outcomes. As health coverage is further expanded efforts should be made to ensure that additional coverage is reaching the populations that can benefit the most. The VA and federal government should shape their policies to incentivize use of a single health system and develop procedures to ensure comprehensive exchange of information between health systems.


Conventional cytology (Pap) and visual inspection after the application of acetic acid (VIA) are currently used in primary screening in Peru. Studies suggest that the quality of VIA is highly variable. Over 36 000 women were screened with Pap and VIA in the TATI (Tamizaje y Tratamiento Inmediato de Lesiones Cervico-uterinas) project conducted in Amazonian Peru. Within a nested study to compare several screening techniques (C-TATI), a total of 5435 women were additionally screened with liquid-based cytology (LBC) and high-risk human papillomavirus testing (HR-HPV). We investigate the variation of positivity rates of VIA, Pap, LBC and HR-HPV in C-TATI and of VIA in the full TATI intervention.At the screening visit, midwives collected three cervical samples for Pap, LBC and HC2 before performing VIA. The dispersion factor “D”  was used to measure the variability of tests results. Within C-TATI, the variability of positivity rates of VIA, Pap, LBC and HR-HPV was also graphically assessed with box- and scatter plots by midwife and month of screening. Funnel plots and smoothed scatter plots were used to correlate the variation of VIA by the number of examinations performed by each midwife over the full TATI intervention.p-values<0.001). In C-TATI, VIA results varied the most while those of HR-HPV varied the least . No evidence for correlation between the number of VIAs done per midwife and the variability of VIA results was observed.Consistently over TATI, VIA results were highly variable, independently of the examiner, the time when the test was performed and the number of tests the examiner performed (D>6, The lack of over-dispersion for HR-HPV detection suggests that the variable VIA results do not reflect true variation in underlying disease, but a lack of consistency in human judgement. Cervical cancer is the fourth most common female cancer in the world . CervicaIt has been established that infection with high-risk types of human papillomavirus (HPV) is a necessary cause for cervical cancer . Both hiThe question of how best to screen populations with difficult or no access to health care remains unanswered. Visual inspection after the application of acetic acid (VIA) followed by treatment with cryotherapy has been proposed as an alternative screening approach in these settings. However, results from studies evaluating the performance of VIA have been inconsistent. In particular, several studies conducted in India and Africa have reported better VIA results than those of Latin America –10. The The methods of TATI have been described elsewhere , 12, 13.TATI was conducted over 36 months. During the first three months, the logistics of the screening processes were piloted (months-3 to 0). Over the following 11 months, a comparative study  in which 5435 women were additionally screened with liquid-base cytology (LBC) and high-risk HPV testing (HR-HPV) done by Hybrid Capture II (hc2). Simultaneously and up until the end of TATI, women were screened with only Pap and VIA .Pap and LBC reported as ASCUS or worse  were considered positive and the standard threshold of 1 (RLU/co) of hc2 was used to identify HR-HPV positive samples. No HPV genotyping was done in this study. Cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was the main disease outcome of the study.Initial training was offered to 19 midwives and 12 general doctors on VIA , 13. TheX with a chi-square distribution with “n” d.f. is “n”, the expected value of D under the null hypothesis of equal underlying properties in each strata is approximately 1, i.e. E(D) = E(X)/n = 1. Thus, D values close to zero will represent minimal dispersion (less than random variation) and highest values of D, further above unity, will represent over dispersion.We used the dispersion factor (D) to measure the variability of the screening tests results by a number of factors: age (in five-year bands), midwife who performed VIA and month of screening, overall and separately by sub-study (C-TATI vs. R-TATI). D was defined as the ratio of the Pearson chi-square value of a 2 x m table  divided by the degrees of freedom (d.f.= n = m-1). Since the expected value of a random variable Positivity rates (PR) of VIA, Pap ASCUS, LBC ASCUS and HR-HPV were calculated overall, by age, by month of screening and by midwife who collected the samples and performed VIA. The PR of each midwife (42 different VIA PRs) participating in TATI was calculated including the results of all examinations performed throughout the intervention and grouped into Group 1: VIA performed by 19 midwives who participated in C-TATI (19 VIA PRs) and Group 2: VIA performed by 23 midwives who participated in R-TATI but not in C-TATI (23 VIA PRs). As the 19 C-TATI midwives in Group 1 screened women in C-TATI and in R-TATI in parallel , their PRs were also calculated separately for examinations done in C-TATI (19 PRs) and R-TATI (19 PRs). Next, the overall PR within each group was calculated summing up the results of all VIAs performed in the group with corresponding binomial 95% confidence intervals (95%CI). A test for trends was used to compare VIA PRs over time  in R-TATI.We graphically assessed the variation of PRs with: 1) box plots to compare the variation of PRs of screening tests in C-TATI, 2) error-bar plots to summarise the difference between VIA PRs performed by the same midwife during the first year of intervention in C-TATI and R-TATI, 3) funnel plots to evaluate the extent to which the variability of VIA results could be explained by the number of examinations performed by each midwife and 4) smoothed scatter plots to evaluate whether the variability of VIA results could be explained by a learning curve and if so to identify a minimal number of VIA examinations required to obtain less variable results .Both funnel plots and smoothed scatter plots allow visualisation of the variation of VIA PRs taking into account the number of VIAs performed. Funnel plots were graphed in two steps: 1) scatter plots of VIA PRs by the number of VIAs performed, and 2) funnels with 95%CIs (control limits) were constructed around the overall VIA PR. In these graphs, the areas within the control limits represent the amount of variation expected. Thus, the higher the number of PRs outside the control limits, the more over-dispersed the data are; and the most closely clustered the PRs are to the overall rate, the less random variation. A running line was used to smooth the scatter plots for each midwife and obtain the smoothed scatter plots.All statistical analyses were done using the Stata software, version 12.The TATI project was approved by the Ethical Committee of the Ministry of Health of Peru and the Ethics Committee of the Pan American Health Organization. Written informed consent was obtained from all participants by trained midwives.After excluding 812 VIAs performed by doctors, a total of 35 947 VIAs were performed by 42 midwives over 36 months. During the pilot phase of the intervention, 2108 women were screened with Pap and VIA. Over the next 11 months, 19 midwives participating in C-TATI performed 12 515 VIA examinations, 5401 within C-TATI and 7114 within R-TATI; while 568 VIAs were performed by nine of the 23 midwives who only participated in R-TATI. Once C-TATI finalised, VIA was performed in 20 756 women (61% of these examinations were done by the 19 C-TATI midwives). From here on, results are based on 33 839 women who were screened by a midwife after the pilot phase .p-values for trends were highly significant, p-values<0.001). In contrast, HR-HPV testing was the most homogeneous test . PRs of LBC ASCUS+ also varied significantly  while those of Pap ASCUS+ varied little . After taking into account the low rate of disease detected by Pap  than LBC IRs and PRs of LBC and HR-HPV (data not shown). We also repeated the analysis on positivity rates of VIA and Pap in R-TATI. The results were similar, VIA PRs were more heterogeneous than Pap smear PRs, however, the results of both tests were highly variable (data not shown).In C-TATI (n = 5401), the overall PRs were 24.2% for VIA, 2.1% for Pap ASCUS+, 17.9% for LBC ASCUS+ and 12.6% for HR-HPV testing. Each of these tests were positive in 36 (43%), 32 (39%), 64 (77%) and 79 (95%) of the 83 CIN2+ detected cases. The PRs of VIA and HR-HPV testing decreased with increasing age while those for Pap and LBC increased with increasing age, mostly due to an increasing trend of moderate dysplasia and worse abnormalities by age , although both dispersion factors were highly significant. The results did not change after excluding midwives who performed less than 100 or more than 2000 VIA examinations. Surprisingly, the PR of midwives in Group 1 was significantly higher when they screened women in C-TATI  than when they screened women in R-TATI , and higher than the overall PR of all midwives together . In view of these results, we compared PRs in C-TATI and R-TATI of the 19 midwives in Group 1 by plotting the difference between the two positivity rates (C-TATI—R-TATI) with 95%CIs. The difference was negative (decrease of 0.4%) only for one midwife while the rest had higher PRs in C-TATI than in R-TATI . Thus, midwives assigned more frequently a positive result within C-TATI than within R-TATI (A summary of VIA PRs per midwife by group of midwives is presented in n R-TATI ..p-for-trend<0.001). However, all Ds were once again highly significant. It is worth noting that five midwives in Group 2 performed less than 50 VIAs in the first year, hence the range of VIA PRs varied between 0% and 67% (D = 8.6). After excluding extreme values, the results did not change .p-values<0.001 for all possible combinations).In summary, independently of whether: i) VIA was performed in C-TATI or in R-TATI, ii) VIA was performed by midwives in Group 1 or Group 2, or iii) VIA was performed in the first, second or third year; VIA positivity rates were significantly and consistently heterogeneous  but to the subjective nature of the test.Surprisingly, despite the fact that the 19 C-TATI midwives screened women in C-TATI and in R-TATI in parallel, the overall PR of VIA performed by them in C-TATI (24.2%) was twice the PR when they screened women in R-TATI 13.7%). Indeed, 18 of 19 VIA PRs in C-TATI were higher than in R-TATI during the first 11 months of the intervention. One potential explanation for these differences could be bleeding of the cervix associated to additional sampling (LBC and HPV after Pap) in C-TATI. The bleeding and the high prevalence of vaginal infections may have interfered with the visualisation of the transformation zone, affecting VIA performance, despite this, VIA results in C-TATI were similar to those of other studies %. Indeed–19.The minimal number of VIAs and the average monthly rate of VIAs that a provider needs to achieve VIA proficiency have not yet been determined . On firsSimilarly to previous studies, the high variability of VIA results per midwife was not explained by lack of adequate and uniform training , 20–23. Our study has some limitations. First, we were not able to contrast positivity rates with disease ascertainment in R-TATI because only Pap and VIA were used to refer women and undoubtedly several cases of CIN2+ were missed. However, we performed this comparison within C-TATI and we showed that the high variation of VIA results was not due to disease variation but to human judgement. Second, midwives started performing VIA at different time points in TATI and only four midwives performed VIA each month of TATI, possibly affecting the likelihood of achieving proficiency. However, we evaluated the variation of VIA results performed in a month per midwife among midwives who at least performed 500 examinations, and found no consistent pattern among the proportion of VIA positive results of midwives. Clearly, the strongest evidence would have come from having multiple midwives examining each woman blinded to each other’s opinion or by assigning women to midwives at random. Neither of such options was practical, and so we have relied on analysis of data that could be subject to confounding. Nevertheless, our study is the first to compare the VIA results of 42 midwives who altogether performed more than 36 000 VIAs within their routine clinical activities in a low-resource setting, and the results are robust to the method of analysis and are likely to reflect true variation.Despite inconsistent results on its performance, VIA is usually the main screening technique in use in low-resource settings, partly because it is the only simple low-cost test available and partly because VIA results are known immediately allowing see-and-treat schemes. New HR-HPV low-cost tests may soon become available and HPV-and-treat or HPV-VIA-and-treat schemes are already being considered as potential alternatives to VIA-and-cryotherapy. The results presented here will contribute to the evidence needed by decision makers when choosing cervical screening algorithms appropriate for their local needs.S1 FigC- TATI = Comparative screening study (months 1–11). R-TATI = rest of the TATI intervention (months 1–33). Difference between positivity rates of VIA (C-TATI—R-TATI) of each midwife is represented by a capital letter. The vertical dotted line represents the mean difference .(TIF)Click here for additional data file.S1 TableC-TATI = Comparative screening study (months 1–11). CIN2+ = Cervical intraepithelial neoplasia grade 2 or worse. VIA = Visual inspection after the application of acetic acid. Pap ASCUS = Pap result ASCUS or worse. LBC ASCUS = Liquid-based cytology result ASCUS or worse. HR-HPV = High-risk human papillomavirus testing.(DOCX)Click here for additional data file.


DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type of head and neck cancers namely oropharyngeal squamous cell carcinoma. Our understanding of HPVs role in cancer development comes from extensive studies done on cervical cancer and it has just been assumed that HPV plays an identical role in the development of all other cancers arising in the presence of HPV sequences, although this has not been proven. Most invasive cervical cancers have the HPV genome integrated into one or more sites within the human genome. One powerful tool to examine all the sites of HPV integration in a cancer but that also provides a comprehensive view of genomic alterations in that cancer is the use of next generation sequencing of mate-pair libraries produced from the DNA isolated. We will describe how this powerful technology can provide important information about the genomic organization within an individual cancer genome, and how this has demonstrated that HPVs role in oropharyngeal squamous cell carcinoma is distinct from that in cervical cancer. We will also describe why the sequencing of mate-pair libraries could be a powerful clinical tool for the management of patients with a DNA viral etiology and how this could quickly transform the care of these patients. DNA viruses are known to play very important roles contributing to the development of a number of different cancers and it has been estimated that 15% of all human cancers may be attributed to viral infections ,3,4,5. PThe different mechanisms whereby these DNA viruses are responsible for the development of different cancers have not been fully elucidated. However, considerably more is known about how HPV is involved in the development of cervical cancer. HPV infection into cervical epithelium results in cellular immortalization but not transformation, thus, additional alterations are required for the development of invasive cancer ,10,11. IThe detection of viral integration sites in the host genome has been a challenge. The traditional methods used for detecting virus integrations are always labor extensive and suffer from a lack of accuracy and sensitivity. With the dramatic increases in sequence output provided by next generation sequencing it is now possible to identify and characterize most viral integrations and the resulting changes at and around integration sites with great specificity. In this review, we will mainly focus on the discussion of HPV infection and integrations in the development of cervical cancer and one sub-type of head and neck cancers, oropharyngeal squamous cell carcinomas (OPSCCs). This will serve as an example that virus integration events including the sites of the virus integration both within the virus and also the sites of integration in the human genome actually play very important roles in cancer development. Indeed, the information revealed by virus integration also has been reported as being related to the diseases outcome. We will discuss how one specific application of next generation sequencing based upon paired end sequencing of mate-pair libraries offers a powerful tool for the characterization of the HPV-driven OPSCCs. In addition we will describe how this could be a very powerful clinical tool which could transform management of patients with all cancers with a DNA viral etiology.Cervical Cancer is the second most common cancer worldwide and it has been estimated that it is responsible for 600,000 incident cases and 300,000 deaths each year . PersistpRB) [Studies in cervical cancer have shown that the viral early genes E1 and E2 were the preferential sites for integration and the E2 region was found to be more commonly disrupted or deleted than any other sites . The E2 pRB) , thus, tpRB) ,33. InitIn situ hybridization is another common technique used to determine the physical status of HPV by characterizing if there is hybridization to just a single position in interphase nuclei or at multiple positions across the nucleus. The observation of diffuse signals would indicate the presence of copies of episomal HPV while the observation of punctate nuclear signals would suggest that HPV is integrated into the cell genome [There are different approaches that can be used to determine the physical status of HPV in an individual cervical cancer. One indirect approach is to measure the expression of the E2 transcript by real time PCR assuming that a unique region of the E2 open reading frame (ORF) is most often deleted during HPV 16 integration. Hence, by measuring the expression of the E2 transcripts and comparing it to the expression of the E6 or E7 transcripts, using real-time RT-PCR it has been suggested that the virus was integrated if E6/E7 expression was high, but E2 expression was low . Howeverl genome . Howeverc-myc oncogene [There are other techniques that have been utilized that are able to characterize the sites of HPV integration both within the HPV genome and the precise integration sites in the human genome. These include Amplification of Papillomavirus Oncogene Transcripts (APOT) , Restriconcogene . These roncogene . HoweverHPV is also known to be associated with a spectrum of other anogenital cancers. It has been estimated that HPV is involved in over 80% of anal cancers and approximately 40% to 50% of penile, vulvar and vaginal cancers . PersistWhile HPV is found to be involved in the development of other anogenital cancers, not many studies have been done to investigate the physical status or the sites of HPV integrations in these cancers. Limited research examining vulva intraepithelial neoplasia and vaginal cancer by DIPS and APOT have suggested that HPV integrations are indeed common in these cancers and that there is active transcription of the HPV genome. Preliminary studies have also suggested that HPV integration frequently occurred within the common fragile sites ,48. HoweIn the United States over the past two decades, there have been dramatic increases in the incidence in OPSCC although the overall incidence of other sub-types of head and neck cancer have been dropping due to decreases in tobacco consumption. OPSCC is a subtype of Head and Neck cancer derived from the tonsil and the base of the tongue, as well as the cancers from the walls of the pharynx and the soft palate . It has At the Mayo Clinic over the past several decades the frequency of OPSCCs that are HPV positive has increased from less than 20% to over 80%. However, there is far greater complexity in the characterization of viral-driven OPSCCs as compared to similar studies in cervical cancer. The first is that many OPSCC patients have a history of smoking and drinking. How these risk factors interact with HPV to cause many OPSCCs is at present unclear. Recent studies have demonstrated that smokers are much more likely to be HPV-positive , but wheA greater complexity is the fact that not all OPSCCs that contain HPV sequences are the same. Some patients contain HPV sequences but very low expression of HPV-specific transcripts. These are thought to represent “latent” HPV infections . WhetherWhether or not an OPSCC patient has HPV sequences present and active transcription of those sequences is turning out to be very important clinically and biologically. In spite of the fact that tumors in patients with HPV sequences present tend to be larger and of a later stage, patients who are HPV positive are found to have a better overall clinical outcome than those whose tumors are HPV negative . This maAll the work in characterizing HPVs role in the development of cervical cancer has led many to assume that this virus must play a similar or identical role in all other cancers where it is found. However, the studies in characterizing the physical status of HPV in OPSCC in a large number of these cancers are limited. Studies from two groups have examined the E2/E6 expression ratio by real-time RT-PCR and based on this assay one group determined that 48% of the cancers they tested had HPV integration while the other found that 78% of their cancers had HPV integrations ,56. in situ hybridization assay and the detection of punctate nuclear signals, Mooren et al. [Utilizing the fluorescence n et al.  reportedPreviously, we reported that we could not detect any HPV integration from 40 HPV16 positive OPSCC by using restriction site oligonucleotide PCR (RSO-PCR) , while tThe development of next generation sequencing based upon massively parallel sequencing has transformed our ability to characterize genome changes in cancer ,62,63 anc-myc oncogene on chromosome 8q24.21 [c-myc oncogene (within one of the CFSs that flank c-myc), and functional studies demonstrated that this integration was responsible for increased expression of c-myc. This is most likely the reason that the cervical cancer that was responsible for the death of Henrietta Lacks was so aggressive [Whole genome sequencing is known to be a powerful technique to identify viral integration sites and the alterations around those sites caused by the integration event. This was aptly demonstrated by the full sequence characterization of the HeLa genome, a cell line established from the cervical cancer patient, Henrietta Lacks. The whole genome sequence of the HeLa cell line revealed that this HPV18 positive cervical cancer had HPV integrated into the hot-spot region for HPV18 integrations which we previously had identified in the vicinity of the  8q24.21 . The HPVgressive . This deFHIT, LRP1B and DLG2 were the sites of HPV integrations in multiple cervical cancers and the protein expression of FHIT and LRP1B was down regulated when HPV integrated in their introns [FHIT is a known and very important tumor suppressor gene that resides within the most highly unstable CFS region in the human genome [LRP1B and DLG2 are very attractive tumor suppressor candidate genes [Recent studies using whole-genome sequencing analyzing cervical intraepithelial neoplasias, cervical carcinomas and cervical cancer-derived cell lines provided further support that the unstable CFS regions are indeed hot spots for HPV integrations in the human genome. Three very large CFS genes  introns . FHIT isn genome ,68. Bothte genes ,70,71. TWhole genome sequencing is one strategy for studying HPV integrations but a number of other investigators have instead focused their efforts on just the coding portion of the genome and have therefore been doing whole exome sequencing instead . This provides a much more tractable characterization of alterations within cancer genomes and solves many of the informatics and cost problems associated with whole genome sequencing. Unfortunately, this approach does not provide as comprehensive information about changes in copy number throughout the genome, nor does it provide information about HPV integration events, unless they occur within the relatively small proportion of the genome that codes for protein. We recently performed whole exome sequencing on cervical adenocarcinomas derived from Hong Kong women. These studies revealed that 13 of the 15 cancers characterized appeared to have HPV integration events near coding genes , but theAnother alternative to whole genome sequencing for characterizing HPV integrations is to analyze whole transcriptome sequencing . The advantage of this is that it not only identifies the level of transcription of human genes, but also those of the HPV genome. If the HPV integration has resulted in the production of a fused transcript it too can be detected from this data. et al. [et al. [et al. [RAD51B and ERBB2.Khoury et al. , analyzeet al.  given th [et al. , were re [et al.  also repOf all these massively parallel sequencing techniques used, the most powerful technique to characterize all HPV integrations is WGS. Exome sequencing and RNA seq will only detect a sub-set of integrations that occur near exons or cause exon-HPV transcript fusions. However, there are a number of limitations to whole genome sequencing. The first is that the full cost of whole genome sequencing is considerably greater than just the cost of generating the sequence. While the cost of WGS has dropped considerably in the past few years that is only a fraction of the total cost of generating the complete sequence of a normal or a cancer genome. An important cost is for alignment the short read sequences into a fully integrated genome sequence. In addition, it is frequently necessary to validate putative alterations detected in a cancer genome with PCR and subsequent sequencing which can greatly increase the cost of analyzing a single cancer genome. Long term storage of the sequences generated  can be quite expensive as well. Finally, interpreting WGS and determining which of the alterations detected are clinically important is still in its infancy.The limitations of whole genome sequencing have precluded it from yet becoming an important clinical tool for the characterization of cancer genomes. There is, however, a powerful and much cheaper alternative to whole genome sequencing which can provide a great amount of information about genome-wide changes in viral driven cancers namely the construction of mate-pair libraries. By producing larger sized inserts and then circularizing them to capture just the ends of the large inserts one can generate libraries ready for sequencing which can then be paired-end sequenced. This then generates sequence from the two ends of the much larger original DNA fragments isolated.The Biomarker Discovery Program of the Mayo Clinic Center for Individualized Medicine, under the direction of George Vasmatzis has pioneered the use of this strategy to characterize cancers. They have further developed a powerful algorithm to characterize the generated mate-pair sequence data. This is based upon a binary mapping algorithm which greatly simplifies aligning of DNA sequences. Vasmatzis and co-workers have used their algorithm and mate-pair sequencing to characterize many different cancer genomes ,77,78 anMate-pair sequencing can also provide a powerful alternative for whole genome sequencing as the longer DNA fragments can be used to bridge together DNA fragments even in the presence of highly repetitive DNA sequences which are rife in human and many other complex genomes. Companies, such as Lucigen, have been working to generate considerably longer mate-pair libraries and these have proven to be powerful tools for fully assembling the sequence derived from complex genomes ,80. The We have used mate pair sequencing to study OPSCC and have demonstrated that this alternative approach is less costly and can serve as a very powerful tool to detect genome structural variation, HPV integration events as well as the HPV copy number with high sensitivity and specificity. In our study, we performed mate pair sequencing on DNA isolated from both tumor and adjacent normal tissues from 20 OPSCC patients, with 14 of them being HPV 16 positive and six being HPV 16 negative . Over 20The mate pair sequencing also detected bridged coverage of various HPV genomes present in each of the OPSCC samples, irrespective of whether or not HPV was integrated in that tumor. When the HPV bridged coverage is very high, it generally correlates very well with HPV copy number. By comparing the HPV DNA bridged coverage with the HPV E6 and E7 transcriptional oncogene expression, apart from one exception, the bridged coverage of HPV DNA sequence was consistent with the level of HPV E6 and E7 transcription in 12 of 13 samples . Our findings suggested that HPV could be playing different roles in the development of different OPSCCs and many of these are quite distinct from its well characterized role in cervical cancer. Thus additional studies are needed to determine the different roles that HPV plays in the development of OPSCC. Most importantly, based on our results, most HPV positive patients have just episomal HPV sequences present. How having just HPV present in the episomal form and sometimes with a low copy of HPV contributes to carcinogenesis of OPSCC will need further investigation.P53 related genes  and precise breakpoints were successfully confirmed by PCR and Sanger sequencing. Additional studies from 190 PTCL tissue samples revealed that 5.8% (11 out of 190) of the cases had TP63 rearrangements and PTCL patients who carried the TP63 rearrangements had significantly worse survival compared with patients without the TP63 rearrangements [Although this is the first example for the virus integration detection using the mate pair approach, the principles of the artificial algorithm mapping demonstrated that this approach could be used for the detection of other DNA virus integration events in other cancers. In addition, the high sensitivity and specificity of this technique for detecting structural variations and the gene fusions which has already been demonstrated in both hematologic malignancies and solid tumors. In an analysis of peripheral T-cell lymphomas (PTCL), Vasmatzis and Feldman’s group identified 13 recurrent abnormalities including inter and intra chromosome rearrangements in which five of them occur within ngements . In addingements ,82. All In our study, in addition to HPV integration events, the data generated from mate pair sequencing also detected inter and intra chromosome translocations in the OPSCC tumor samples. We have successfully validated the chromosome translocations and the novel fusion genes were indeed present in the tumor sample but not in the matched normal samples using PCR and Sanger Sequencing (data not published). In OPSCC, the amount of genome rearrangement rate is not as high as found in other cancers, such as breast. Currently we are investigating if there is any relationship between the virus integration and the genome translocation in the OPSCC, and how these events are related to patients’ clinical outcome, as well as if tumors with greater genomic instability tend to be more likely to have viruses integrated into the genome.Thus, from what we have observed from our study in the HPV related OPSCC, the sequencing of mate pair libraries has generated information regarding the virus integration events, copy number variations, as well as the genome translocation information. All these will provide insight not only to potential mechanisms involved in the cancer development, but also to the biomarkers discovery for disease diagnosis, prognosis and potential therapeutic targets.Mate-Pair sequencing is thus a powerful tool for the characterization of the physical status of HPV, or other DNA viral genomes, in cancer. Not only could it help us to determine the sites of HPV integration and how that integration disrupts the human genome at those integrations but it also provides information about the copy number of HPV in each cancer. In addition, the mate-pair sequencing also provides information about changes in copy-number throughout the genome. Thus, we could determine with very high resolution regions of chromosomal amplification as well as regions of deletion.versus less aggressive cancers.It is well known that cancers that have more genomic instability generally have a worse overall clinical outcome ,84. ThusAnother thing that is obtained from limited sequencing of the mate-pair libraries is the identification of cancer-specific alterations. These could be HPV integration events  but could also be inter- and intra-chromosomal translocations. These could serve as powerful biomarkers for monitoring the clinical course of treatment for that cancer patient.The ability of limited sequencing of mate-pair libraries to identify cancer-specific alterations may actually turn out to be the most powerful information provided by this approach. It is well known that circulating tumor-free DNA can be detected in the blood of patients with all different types of cancer. The amount of this circulating DNA is also indicative of the amount of the primary tumor present for solid tumors . FinallyPatients with OPSCC are generally first surgically treated and then this is followed up with both chemotherapy and radiotherapy treatments. These subsequent procedures are costly and can severely compromise the quality of life for patients. It is for this reason that clinicians are looking for ways to identify less aggressive cancers which could be treated but with a de-escalation of therapies. Thus, the finding that patients whose tumors are HPV positive have a much better clinical outcome has led to the suggestion that these are precisely the patients who could have less aggressive therapies. Unfortunately, this is not a perfect strategy as many HPV positive patients do have recurrence. However, the ability to quantitatively measure circulating tumor-free DNA could be a powerful tool to determine those patients whose surgical procedure has resulted in the removal of all tumor.In addition, having the means to determine when tumors are beginning to recur could provide a powerful tool for clinicians to monitor an individual patients’ response to therapy. The relatively non-invasive detection of the concentration of tumor-free DNA in the blood of patients could prove to be a powerful tool to monitor in real-time how patients are responding to therapies. In the case of chemotherapy this could also be used to quickly take patients off of chemotherapeutic agents that are not working and substituting others that might prove more effective.However, the problem with the detection of circulating tumor-free DNA is complicated by the fact that the concentration of tumor DNA could be a very small fraction of the total DNA present in blood. The identification of cancer-specific alterations by mate-pair sequencing  provides cancer-specific markers that could be both detected and quantified.While this could be done by various real-time PCR techniques, a more powerful tool with much greater sensitivity is the use of digital droplet PCR . Variouset al. [Work on a variety of different cancers has therefore begun to determine the feasibility of this approach for monitoring the clinical treatment of cancers. A recent comparison of conventional quantitative PCR and droplet digital PCR for the measurement of clinically significant EGFR mutation found that while quantitative PCR could detect samples where the mutant molecules were present at just 5% or even down to 1% , that the digital PCR method could reliably detect 0.5% and down to 0.1% mutation rates . A similet al.  demonstret al. .Therefore, this approach could be a very powerful tool to help to monitor patients with OPSCC. Mate-pair sequencing of the primary tumor, obtained at the time of surgery could identify cancer-specific alterations which could then be detected with PCR primers specific for those alterations (again focusing on cancer breakpoints which can be readily observed from limited mate-pair sequencing). PCR primers produced could then be used with digital droplet PCR to quantitate the amount of circulating tumor-free DNA from the blood of that patient which was obtained just prior to surgery. This will quantify the amount of tumor prior to surgery. Then, blood obtained from those patients several days to a week post-surgery could be used to determine the effectiveness of that surgery to eradicate all, or most, of the tumor. For those patients where there is almost no residual cancer DNA, it may not be necessary to follow-up with chemotherapy or radiotherapy and those patients could be monitored with blood draws every six months to determine if there is any cancer recurrence. For those patients that do have residual tumor DNA detected, they could then proceed to subsequent clinical treatments which could also be monitored with the liquid biopsy to determine their effectiveness. This could therefore prove to be a powerful clinical tool for monitoring patients through their treatments.In conclusion the power of limited sequencing of mate-pair libraries produced from DNA isolated from tumors with a viral etiology can already be a very useful tool to aid clinicians in evaluating and treating these patients. For patients with cervical cancer the ability of this approach to detect all the sites of viral integration could help to differentiate patients with potentially less aggressive cancers from those with more aggressive cancers . For patients with OPSCC this approach can determine which patients have viral integrations and where those integrations occur. Thus, this could also identify patients whose tumors might be more aggressive based upon the sites of integration. For the majority of OPSCC patients, who will not have viral integrations, this approach will still determine the copy number of episomal HPV sequences present which could also have clinical significance. It is, at present, unclear for the other anogenital cancers with an HPV etiology what role HPV plays in these cancers, but this will soon be addressed using next generation sequencing technologies. There are other DNA viruses that an involved in the development of cancer, such as hepatitis B in hepatocellular carcinoma. HBV tends to have many more viral integrations in each hepatocellular carcinoma than HPV does in cervical cancer , but thiIn addition to determining the physical status of viruses in each cancer, limited mate-pair sequencing can also provide a high resolution cytogenetic characterization of each cancer. Thus, it can identify regions of chromosomal amplification and regions of deletions. Further studies correlating different specific alterations with clinical outcome could reveal a number of these alterations that do have clinical significance, thus, in the future, this could provide very important information about each specific cancer and its potential to be more or less aggressive.Finally, and perhaps most powerful of all, the partial sequencing of mate-pair libraries can identify alterations present in each cancer. In our work with mate-pair sequencing of OPSCCs we have found that each OPSCC has multiple alterations that are readily detected. This could include human-HPV junctions in those cancers that do have integration, but also includes several inter-chromosomal translocations and multiple intra-chromosomal alterations as a result of either insertions or deletions. These alterations are cancer-specific  thus are powerful markers for the detection of circulating tumor-free DNA. Then the power of digital droplet PCR to determine the concentration of this specific tumor DNA in the blood from those patients provides a powerful liquid biopsy to monitor the clinical course of that cancer in that patient. This will quickly transform how clinicians can reliably and accurately follow the clinical treatment of their patients.


Head and neck cancers (HNCs) are a highly heterogeneous group of tumours that are associated with diverse clinical outcomes. Recent evidence has demonstrated that human papillomavirus (HPV) is involved in up to 25% of HNCs; particularly in the oropharyngeal carcinoma (OPC) subtype where it can account for up to 60% of such cases. HPVs are double-stranded DNA viruses that infect epithelial cells; numerous HPV subtypes, including 16, 18, 31, 33, and 35, drive epithelial cell transformation and tumourigenesis. HPV positive (HPV+) HNC represents a distinct molecular and clinical entity from HPV negative (HPV−) disease; the biological basis for which remains to be fully elucidated. HPV positivity is strongly correlated with a significantly superior outcome; indicating that such tumours should have a distinct management approach. This review focuses on the recent scientific and clinical investigation of HPV+ HNC. In particular, we discuss the importance of molecular and clinical evidence for defining the role of HPV in HNC, and the clinical impact of HPV status as a biomarker for HNC. Head and neck cancer (HNC) comprises a diverse group of tumours, with an incidence of over 500,000 cases annually worldwide . The mosHPV is a non-enveloped, double-stranded DNA virus encoding a total of 8–9 proteins in approximately 8000 base pairs , with thThe HPV capsid proteins L1 and L2 are responsible for tissue-specific infection. Replication is controlled by the E1 and E2 proteins, which regulate transcription of other viral genes . A depicHPV+ HNC represents a distinct disease from HPV− tumours. Patients with HPV+ tumours present at a younger age (median = 57 vs. 64 years for HPV+ vs. HPV− OPC) , and areDespite a more aggressive clinical presentation, HPV status is the best independent predictor of survival in these patients ,33,34. Ap53 mutations, widespread copy-number loss, and promoter hyper-methylation and mutation of CDKN2A, leading to a loss of p16 expression [TRAF3 loss, PIK3CA activating mutations, and E2F1 amplification; while HPV− HNSCC commonly harbour 11q amplifications, and mutations in CASP8 and HRAS [TP63 and SOX2, and the PIK3CA oncogene [In both HPV+ and HPV− HNC, the p53 and pRb pathways are frequently altered; however, the mechanism of inactivation is distinct. As described above, the E6 and E7 viral proteins functionally inactivate p53 and pRb in HPV+ HNC. However, the majority of HPV− tumours have pression ,40. Furtpression ,42,43. Hand HRAS . Howeveroncogene . In addioncogene . Transcroncogene , as welloncogene .HPV testing has recently been included in the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) guidelines as a standard pathological assessment for OPC . HoweverMicroRNAs (miRNA) are small endogenous non-coding RNAs that are approximately 22 nucleotides in length . DysreguMiRNAs also hold potential as biomarkers for predicting treatment response in HNC, as a number of reports have shown the association between specific miRNAs with development of DM and survival . NumerouCirculating biomarkers indicative of HNC prognosis or treatment response would enable a non-invasive method of monitoring disease status and for improved treatment stratification. Few studies have identified circulating biomarkers for HNC; however, this important field of research is rapidly growing. Tinhofer et al. examined the persistence of circulating tumour cells (CTC) isolated from peripheral blood following treatment with adjuvant chemotherapy for locally advanced HNSCC. Interestingly, in the subset of OPCs within this dataset, CTCs were not predictive of disease free survival (DFS) or overall survival (OS). However, in non-OPCs, CTC were an independent prognostic marker of poorer DFS and OS . TherefoThough outcome for HPV+ HNC patients is generally superior to HPV− patients, a subset of HPV+ patients have a poor prognosis, associated with the development of DM ,58. The Due to the distinct clinical and demographic features of HPV+ HNC as compared to HPV− HNC, HPV status has now been established as a reliable prognostic biomarker for this disease . As suchSmoking is well-implicated as a causative agent in HPV− HNC, and a history of heavy tobacco use is correlated with poor prognosis . SmokingImmune modulation also appears to play a significant role in HPV+ HNC, which exhibit higher levels of immune infiltration than HPV− tumours ; these iCurrent treatment regimens for HNC are aggressive and have significant treatment-associated toxicities, as a result of surgery, chemotherapy, and/or radiation treatment. These include both acute toxicities, such as mucositis, nausea, pain, hematologic changes, and stomatitis, among numerous other acute side effects, and late toxicities, such as chronic xerostomia, fibrosis, edema, trismus, and dysphagia . Low risAs discussed previously, HPV16 is the most prevalent HPV subtype in HNC, contributing to over 80% of HNSCC ,15. InteIn summary, HPV has become a recognized oncogenic driver in HNC, and the incidence of HPV+ HNC, particularly HPV+ OPC, is on the rise. HPV associated HNC is a distinct clinical entity, with significantly improved treatment response and survival rates in comparison to HPV− HNC. Clinical management of low risk HPV+ HNC may therefore require unique treatment approaches focused on de-intensification of current standard of care therapies. Results of new and ongoing treatment de-escalation trials in these low risk patients will be crucial for establishing whether the current rates of DFS and OS are maintained while treatment-associated toxicities are reduced.HPV16 is the subtype most frequently associated with HNC, and current clinically relevant detection methods focus on identifying the presence of HPV16 by PCR or ISH, or detection of p16 protein expression in tumour samples by IHC. However, HPV+ tumours involving other HPV subtypes generally exhibit reduced survival rates, though these rates may be somewhat superior to HPV− disease. Thus, a subset of HPV+ tumours may not benefit from treatment de-intensification strategies, highlighting the importance of informed clinical trial design. Additionally, diagnostic and prognostic biomarkers, including HPV-associated miRNAs and circulating monocytes or lymphocytes, may prove to yield strong clinical utility, and warrant further investigation and clinical validation.


Temporally unpredictable stimuli influence murine and human behaviour, as previously demonstrated for sequences of simple sounds with regular or irregular onset. It is unknown whether this influence is mediated by an evaluation of the unpredictable sound sequences themselves, or by an interaction with task context. Here, we find that humans evaluate unrelated neutral pictures as more negative when these are presented together with a temporally unpredictable sound sequence, compared to a predictable sequence. The same is observed for evaluation of neutral, angry and fearful face photographs. Control experiments suggest this effect is specific to interspersed presentation of negative and neutral visual stimuli. Unpredictable sounds presented on their own were evaluated as more activating, but not more aversive, and were preferred over predictable sounds. When presented alone, these sound sequences also did not elicit tonic autonomic arousal or negative mood change. We discuss how these findings might account for previous data on the effects of unpredictable sounds, in humans and rodents. Forecasting the state of the world is fundamental for all organisms. Because environments differ in the degree to which events are predictable, humans and other animals can maximise predictability by, for example, moving from one environment to another. A large literature shows that organisms actively prefer predictable over unpredictable salient events such as punishments and rewards , 2. SuchMuch experimental evidence on uncertainty avoidance relies on the use of unpredictable salient events such that unpredictability has rarely been assessed per se. In fact, it has been argued that a preference for predictable salient events does not reflect a propensity to reduce uncertainty, but instead a propensity to maximise periods of safety. Such safety is lacking from environments characterised by unpredictable salient events . This exWe previously investigated responses to unpredictability per se by exposing mice and humans to a stream of temporally predictable (i. e. regular onsets), or unpredictable (i. e. random onsets), simple beep sounds , where eAll participants were adults who gave written informed consent. The study (including the form of consent) was approved by local research ethics committees . All participants were debriefed to the aims of the study.www.csounds.com) and played over headphones with a loudness of ~80 dB (experiments 1–4) or ~85 dB (experiments 5–6). Experiments 5–6 were performed before experiments 1–4; in experiments 1–4 we increased onset jitter and slightly decreased loudness to maximise the experience of unpredictability, and minimise any possible aversiveness due to the beeps themselves. Experiments 1–4 realised a between-subjects design in which each participant heard one sound sequence for the entire duration of the experiment. In experiment 6, each participant heard all sound sequences in balanced order in a within-subjects design, while participants chose which sound sequence they heard in experiment 5.In all experiments, we used a predictable sequence of neutral beep sounds  presented with an average repetition frequency of 5 Hz, i.e. an average stimulus onset asynchrony of 200 ms. For the unpredictable sequence, onset deviation was randomly drawn from a uniform distribution over ± 60 ms (experiment 1–4), ± 20 ms (experiment 5), or ± 40 ms (experiment 6). Sound sequences were produced using CSounds (http://pspm.sourceforge.net) [We recorded skin conductance responses (SCR) on thenar/hypothenar surface of the non-dominant hand using Ag/AgCl cup electrodes and 0.5%-NaCl electrode paste , 22. SCRrge.net) . Tonic srge.net) , 25, andrge.net) , 26. Everge.net) .a priori contrast of interest for all experiments. In experiments 1–2, and 6, possible effects of mere auditory stimulation were analysed in an a priori contrast of predictable vs. silence. For analysis of phasic sympathetic arousal and picture ratings, experiments 1, 3–4 had an additional within-subjects factor picture valence. Experiment 5 was a forced-choice paradigm where we tested deviations from a uniform distribution of responses. We analysed time spent with unpredictable sound sequence, sound sequence that participants spent more time with (termed preferred sequence), and the ratings. Deviations from a uniform distribution were tested in SPSS with one-sample t-tests (continuous measures) and binomial tests (frequencies). In exploratory analyses, we examined the relation of state and trait anxiety on continuous response measures from experiment 5 in two separate simple regression models with anxiety as predictor. The impact on binary response measures was analysed with generalised linear models with anxiety as linear predictor, and logit link function–equivalent to logistic regression.Estimates of tonic arousal from experiments 1, 2, 4 and 6, as well as phasic sympathetic arousal and picture ratings from experiments 1–4, were analysed using ANOVAs (or ANCOVAs) in SPSS. We were interested in the difference between predictable and unpredictable stimuli such that this constituted our Experiment 1 followed a factorial design with the between-subjects factor sound type (silence/predictable/unpredictable) and the within-subject factor picture type . We recruited 60 participants ; one dataset was excluded due to technical malfunction. The experiment lasted about 20 minutes. During the first 2 minutes we obtained baseline skin conductance recordings for estimation of tonic arousal. Afterwards, participants watched, in randomised order, the 45 least arousing neutral  and 45 most arousing aversive pictures  from the International Affective Picture Set  for 1 sAcross all conditions, aversive pictures were more often rated negative than neutral pictures Tables –3, Fig 1Aversive pictures elicited stronger stimulus-evoked SCRs than neutral pictures across all conditions. SCRs to both aversive and neutral pictures were stronger in the unpredictable than in the predictable condition. This main effect survived correction  for baseline arousal, state anxiety, and trait anxiety. Tonic baseline arousal as indexed by SF, and reaction times (RT), were not influenced by the sounds. Thus there was no significant increase in tonic sympathetic arousal when the unpredictable sequence was presented alone. Phasic stimulus-associated sympathetic arousal was markedly increased in the unpredictable condition while stimulus ratings were more negative during the unpredictable sequence but only for neutral stimuli.The combination of negative and neutral pictures was chosen in light of a previous investigation into unpredictable sound sequences . ContradWe designed this experiment to replicate an alteration of neutral picture ratings found in experiment 1 while removing any impact of arousing visual context. Thus, we only assessed neutral pictures on their own, without negative stimuli interspersed.Experiment 2 realised a one-way factorial design with the between-subjects factor sound type (silence/predictable/unpredictable). We recruited 61 participants . The experiment included only the 45 neutral pictures from experiment 1 with the same event timing and responses, and was divided into 3 blocks.Contrary to our expectations, we found that sound sequence had no impact on ratings, RT, tonic sympathetic arousal quantified as SF, or phasic stimulus-associated SCR. This suggests that the context of highly arousing IAPS pictures is relevant for the impact of the sound sequence on neutral picture appraisal in experiment 1, and also for the impact on phasic arousal.The discrepancy in neutral picture ratings between experiments 1–2 may suggest that an arousing context is required to reveal an impact of sound unpredictability on ratings of neutral images. To address whether this is due to arousal associated with the visual context, or whether it requires a negatively valenced context, we revisited this question using negative, neutral and positive IAPS stimuli in experiment 3.This experiment was similar to the first, with an additional third condition of positive arousing images, and thus followed a 2 (sound type: predictable/unpredictable) × 3  factorial design. Forty participants  watched the 16 least arousing neutral, most arousing aversive and most arousing positive images  from the IAPS, for 1 s each, in randomised order, and in one single block. ISI was 4.4 s. Responses were the same as in experiment 1. Two datasets were excluded: one due to technical malfunction, one due to lack of compliance with the instruction to rate the images by a key press.2 = .967; p < .001), RT  = 6.3; η2 = .149; p < .01) and SCR  = 8.8; η2 = .197; p < .001). Compared to neutral images, positive images were more often rated as positive  = 418.0; ηpositive , and assThe lack of a sound sequences effect or interaction with valence suggests that the combination of negative and neutral pictures in experiment 1, rather than a generally arousing context, was crucial to the impact of unpredictability.Using a different picture context than in experiment 1, experiments 2–3 provided null results. Hence, we sought to confirm results from experiment 1, again using a context of negative and neutral images. At the same time, to facilitate interpretation of our results with respect to a previous experiment employing a dot probe task with faces , we usedSimilar to experiment 3, in this experiment we implemented a 2 (sound type: predictable/unpredictable) × 3  factorial design. Forty-two participants  watched, in randomised order, photographs from 38 actors of the Karolinska Directed Emotional Faces set  with angOverall, angry faces were rated negative more often than fearful faces, while fearful faces were rated negative more often than neutral faces , and initially confined our analysis to experiments 1–3 as these involved the same type of neutral (IAPS) pictures.2 = .061; p < .05), and a main effect of experiment  = 48.7; η2 = .465; p < .001). This confirms differences between experiments concerning the impact of sound sequence on ratings of neutral images. In particular, the impact of unpredictable sounds on neutral picture ratings in experiment 1 was larger than in experiment 2  or experiment 3 (p = .09). Also, neutral pictures in experiment 2 were rated more negative than in experiment 1 (p < .001) or experiment 3 (p < .001).For picture ratings, this analysis revealed a significant interaction of the experiment and sound factors  = 3.7; η2 = .037;p < .05), and also differed between experiments  = 4.6; η2 = .075; p < .05). SCR were similar between experiment 1 and 2 (p = .47) but smaller in experiment 3 compared to experiment 2 (p < .005).There was no effect of sound or experiment on RTs. SCRs to neutral stimuli were significantly higher in the unpredictable than predictable condition  = 4.3; ηAll these effects were replicated when including neutral faces from experiment 4 together with the neutral IAPS pictures from experiments 1–3, again confirming the difference between experiments with respect to the effect of sound, and the overall phasic SCR increase in the unpredictable condition.In summary, this analysis corroborated a significant impact of visual context on the impact of the unpredictable sound sequence. As a side finding, ratings of neutral pictures were also impacted by visual context; in particular they were rated more negative when presented on their own. This may be due to the fact that in the other two experiments, the contrast with the aversive images may have influenced their ratings. It will be interesting to investigate whether this would also occur if pictures were chosen from a continuum between neutral and negative.Given an evaluation bias of visual stimuli in the context of the unpredictable sound sequence in experiment 1 and 4, we investigated how subjects evaluated the sound sequences themselves, both in terms of "wanting" and "liking" .regular and irregular as descriptions of the two sequences.During 5 minutes, 72 participants  heard predictable or unpredictable sound sequences over headphones and could change between the two sequences ad libitum with a mouse click. The starting sequence was counterbalanced. After the experiment, participants were asked to rate which sequence they found more unpleasant, interesting, and activating, on a scale with 5 levels, anchored with 0: 150 s; M (unpredictable) = 153.2 s; SEM = 9.9 s; t(71) = 0.32; p = .74), but that more than half of the sample spent more time with the unpredictable compared to predictable sound sequence . In terms of "liking", subjective ratings for each dimension ranged from -2 (predictable) to 2 (unpredictable), and were tested against zero. The sequences were rated as similarly unpleasant  = 0.88; p = .38), and the unpredictable sound sequence was rated as more interesting  = 9.37; p < .001) and more activating  = 3.17; p < .01). There was no influence of state or trait anxiety on preference, or on the time spent with the regular sequence. Participants switched on the average 18.9 (SD = 2.1) times between the sequences; the number of switches was not related to any of the other dependent measures, or to state/trait anxiety levels.In terms of "wanting", we analysed time spent with the sounds. Under the null hypothesis that there is no difference between the sequences, each participant would on average spent half the time with the unpredictable sequence, and half of the participants would spend more time with the unpredictable than with the predictable sequence. We found that the average time spent with the sounds did not differ from a uniform distribution  to 6 (very strongly). This instrument has been validated in the context of state anxiety [Experiment 6 followed a one-way factorial design with a within-subjects factor sound type (silence/predictable/unpredictable). Twenty-four participants  listened to 5 minutes of silence, predictable, and unpredictable sound sequences  in counterbalanced order. As self-report measures, we used the multidimensional mood checklist , based  anxiety , 34.ECG was analysed by automatically counting R-spikes per each 2-minute epoch with visual control for artefacts . Heart rate quantified over short periods of time can be seen as a measure of combined autonomic input to heart  and has p > .20). There were no subjective mood changes on negative dimensions induced by the unpredictable sound sequence . On positive dimensions, relaxedness was slightly reduced by the unpredictable sound sequence  = 5.81; p < .05, not corrected for multiple comparison). The predictable (control) sound sequence induced a heart rate increase from the silence (baseline) condition  = 7.21; p < .05) and an increase in feelings of anger/aggression  = 7.16; p < .05) while having no effect on other measures. Even at the trend-level, there was no difference on these particular measures between the predictable and unpredictable sequence.Even without correction for multiple comparisons, the unpredictable sound sequence had no effect on estimates of tonic sympathetic arousal  sympathetic arousal is unaltered by the sound sequence in all experiments. Altogether, this suggests that anxiogenic properties of the sound sequences in humans are negligible when they are presented on their own.The fact that evaluation of pictures is only changed when neutral and negative stimuli are presented in the same set  suggests that picture context is crucial for the expression of an impact of sound predictability. This finding lends itself to a number of interpretations. First, it is possible that the unpredictable sound sequence increases uncertainty in the evaluation of the pictures. Prior expectations, shaped by the visual context, might then exert a stronger influence on ratings, biasing them towards more negative in a context with negative pictures. We note that ratings of the different picture categories do not simply become more similar to each other. Although such a hypothesis might be construed from the cross-over interaction of picture type and sound sequence in experiment 1, all pictures including the (mildly) negative ones were rated more negative in experiment 4. As a second explanation for our finding, hearing an unpredictable sound sequence might increase arousal, and this could be misattributed to the pictures. This explanation mainly fits the data for neutral pictures. In a context of negative images, the possibility of having seen a negatively valenced picture constitutes a likely source of arousal. In a state of increased phasic arousal, a neutral picture might then be perceived and rated as negatively valenced. This should not occur in a context of positive and negative, or neutral images alone. The findings with respect to neutral images are in line with this interpretation, and phasic arousal is indeed increased in response to neutral pictures by the presence of unpredictable vs. predictable sounds across experiments 1 to 4. However, this explanation appears to be restricted to neutral images and therefore remains speculative. A possible way of testing this hypothesis would be to present only neutral and highly arousing positive pictures. According to the arousal hypotheses, ratings of the neutral pictures should then be rendered more positive. As a note of caution, these interpretations rely on a post-hoc analysis of responses to neutral picture across the different experiments. It would be desirable to compare the impact of image context on evaluation of neutral images within one experimental design.We found that an unpredictable sound sequence is not evaluated as more aversive by itself. In fact, subjects actively preferred this sound sequence over a predictable control sequence, showing increased "wanting". Explicit evaluation of the two sound sequences in "liking" ratings indicated no difference in aversiveness, although the unpredictable sequences were rated as more interesting and activating. Finally, we found no evidence of anxiogenic properties for any of the sequences in subjective feeling. As a caveat, it is well known that when engaged in a task, unrelated unpredictable events are more distracting than predictable ones . It is pAltered appraisal of context events can explain our previous findings about the effects of unpredictable neutral sounds in humans . An incrA final limitation that we need to acknowledge is that the rating scale used to assess picture evaluation was simple. This choice of a binary rating scale was motivated by the need to minimise possible distracting influences of sounds on the rating process. A more elaborate rating procedure might be harnessed to reveal more subtle effects particularly in the evaluation of highly arousing pictures.In conclusion, we demonstrate that a sequence of unpredictable neutral events alters the evaluation of unrelated neutral pictures, and face photographs, but only when presented in a negatively valenced visual context. At the same time, this unpredictable sequence had no effect on tonic sympathetic arousal, negative subjective feeling, or subjective preference, when presented alone. Instead, participants were more likely to spend more time with the unpredictable than with the predictable sequence. This suggests that unpredictable sequence is neither anxiogenic nor aversive. This opens a new perspective on unpredictability and might help understanding human behaviour in ever-changing natural environments.