The tumours of head and neck district are around 3% of all malignancies and squamous cell carcinoma is the most frequent histotype, with rapid increase during the last two decades because of the increment of the infection due to human papilloma virus (HPV). Even if the gold standard for the diagnosis is histological examination, including the detection of viral DNA and transcription products, imaging plays a fundamental role in the detection and staging of HPV + tumours, in order to assess the primary tumour, to establish the extent of disease and for follow-up. The main diagnostic tools are Computed Tomography (CT), Positron Emission Tomography-Computed Tomography (PET-CT) and Magnetic Resonance Imaging (MRI), but also Ultrasound (US) and the use of innovative techniques such as Radiomics have an important role. Aim of our review is to illustrate the main imaging features of HPV + tumours of the oropharynx, in US, CT and MRI imaging. In particular, we will outline the main limitations and strengths of the various imaging techniques, the main uses in the diagnosis, staging and follow-up of disease and the fundamental differential diagnoses of this type of tumour. Finally, we will focus on the innovative technique of texture analysis, which is increasingly gaining importance as a diagnostic tool in aid of the radiologist. The tumours of head and neck district are around 3% of all malignancies and squamous cell carcinoma is the most frequent histotype –4. The iThe diagnosis of OPSCC is based on pan-endoscopy with biopsies of the primary lesion . When trOn imaging HPV + tumours differ from HPV-negative (HPV-) ones . ImagingUltrasound is used for the evaluation of suspected laterocervical lymphnodal metastasis. HPV + tumours usually show cystic and necrotic nodal metastasis, differently from HPV- ones . At compAlso, innovative new techniques are also increasingly being used in the evaluation of this type of tumour, including MRI perfusion imaging and texture analysis.The aim of this review is to illustrate the main imaging features of HPV + tumours of the oropharynx, in US, CT and MRI imaging. In particular, we will outline the main limitations and strengths of the various imaging techniques, the main uses in the diagnosis, staging and follow-up of disease and the fundamental differential diagnoses of this type of tumour. Finally, we will focus on the innovative technique of texture analysis, which is increasingly gaining importance as a diagnostic tool in aid of the radiologist.While the inherent limits of ultrasound (US) imaging don’t generally allow for the evaluation of primary site HPV-related cancer, its availability, relative low-cost and its great definition for the study of superficial structures make it ideal to investigate suspicious lymph node swelling in the head and neck district –23. ThusWhile no specific nodal station in related to HPV disease as is, it is true that metastatic drainage in the head and neck is district specific , 25, 26.Cancers of the oropharynx, hypopharynx and larynx tend to metastasize more frequently to the internal jugular chain (leves II-III-IV), while upper cervical and submandibular chains (levels Ia-Ib and IIa) are more frequently related to oral cancers , 22. WhiSize is considered an indirect indicator of malignancy.Optimal cut-off values for the minimal diameter have been identified as 10 mm cervical nodes on an axial plane . It is iIn addition, size evaluation over time can be useful for patients that have already been diagnosed with a primary head and neck tumour.Normal or inflammatory nodes usually appear as oval or flat in shape. Metastatic lymph nodes tend to be round in shape. Borders of malignant nodes are typically sharper and better defined from surrounding tissues than normal on ultrasound , 28. A pHPV-related metastatic lymph nodes tend to be hypoechoic than adjacent muscle tissue. Usually, no hyperechoic hilus can be detected even though it might still be present in the earlier phases of the process of tumoral invasion of the node. A typical sign of squamous cell HPV-related carcinoma is the development of echolucent cystic necrosis Fig. , which iInflammatory edema of surrounding tissue might be present around all metastatic nodes, regardless of HPV correlation. Lymph node matting is rare.Vascular pattern of metastatic nodes tends to be peripheral, as no clear hilar vascularization can be identified. In the presence of cystic necrosis, however, it is possible to observe avascular nodes due to vase destruction.In addition to the imaging features that might help in the evaluation of nodal metastases, it is also important to remember the role played by US in ultrasound-guided fine needle aspiration cytology (FNAC) for the definitive diagnoses. The employment of ultrasound to provide more accurate information can be used both for diagnosis and follow-up.Along standard MRI, CT imaging has become one of the fundamental imaging techniques for both diagnosis and staging of head and neck cancer, regardless of HPV status.CT plays a vital role in the definition of primary tumor in the head and neck region. To this end, CT has been employed both to better define the size and relation to contiguous tissues and structures of a suspect lesion found on clinical examination or during laryngoscopy and to study the head and neck district in search of a primary tumor of unknown origin when faced with suspicious lymph node swelling. To these ends, contrast-enhanced CT of the head and neck region is fundamental to both identify the lesion and to better evaluate its extension to surrounding tissues.While definitive diagnosis of HPV related disease can only be proven through histology, a certain amount of imaging features can help the radiologist to infer the HPV status of a tumoral mass. In addition to that, a number of prognostic biomarkers can be evaluated during staging, allowing for a better definition of oncologic risk.HPV-related cancers of the head and neck district tend to present certain features that can help the radiologist in their characterization Table .When it comes to non-contrast imaging, among morphological and topographical differences between HPV + and HPV- disease can be found the usually smaller overall size of HPV + lesions and their exophytic growth , 29.On post-contrast CT, HPV-related cancer tends to show a stronger enhancement as opposed to HPV- disease which is usually less enhanced , 31 Fig and 3. IHPV-related cancer usually presents itself as an exophytic lesion with well-defined borders and little to no invasion of the submucosa or of the adjacent tissues, such as muscle , 29–32  of adjacent structures being rarer in HPV + disease , 33.CT imaging is most commonly used to stage head and neck cancer, regardless of HPV status , 35–37.CT imaging of primary lesion can be used, with or without the involvement of MRI, to determine tumor size and invasion of adjacent tissue , 36–38. In addition to that, nodal involvement can be evaluated via CT in order to define N staging. While MRI can be used to detect metastatic lymph nodes, CT tends to be more reliable and, to this extent, better overall for N staging .Finally, the employment of High-Resolution CT (HRCT) of the chest allows for a better evaluation of possible metastases to the lung and is thus used for M staging.Positron emission tomography-computed tomography is a technique that combines the precise anatomical definition of a CT scan with information regarding the metabolic activity of primary tumor and metastatic sites. As such PET-CT plays a fundamental role in both staging and post treatment surveillance of OPSCC, regardless of HPV status .Fusion CT images produces by PET-CT may sometimes provide a better definition of primary site tumors, especially in those sites that are notoriously harder to study through other techniques  especially with smaller lesions that might have been easy to spot on clinical examination but might be too small to show significant contrast enhancement to help differentiate from surrounding tissue . This isWhen it comes to HPV-correlation, multiple studies have shown that HPV + cancers tend to present themselves with higher SUVmax values when compared to HPV- tumors , 45. As While conventional, anatomical, MRI yields results similar to CT in terms of employment in the diagnostic stage of head and neck cancer , 40, as As stated above, elements of differential diagnosis of HPV status in head and neck cancer are comparable between CT and MRI and, as a such, will only be briefly repeated Table . MRI imaNo significant difference seems to be present after contrast administration between HPV + and HPV- lesions , 53–55.When it comes to nodal involvement, the same consideration that were made for CT are valid for MRI, with metastasized lymph nodes that tend to develop cystic necrosis , to form clusters without signs of matting, with well-defined borders and little to no radiologic extra-nodal extension (rENE) , 56–58 .Due to its superior accuracy and contrast resolution, MRI is often employed for the local staging of tumoral lesions, and local extension to adjacent tissues , 52, 59.Locoregional nodal involvement can also be studied with MRI techniques with high sensitivity and specificity .In these recent years, the employment of functional MRI modalities alongside conventional morphological imaging techniques has been steadily rising in the study of head and neck cancer , 54. Dift) and apparent diffusion coefficient (ADC) when compared to HPV- tumors [When it comes to DWI, it has been reported that HPV + lesions tend to overall show lesser values in both tissue diffusion coefficient  with evaluation of D* and diffusion kurtosis imaging (DKI) with evaluation of skewness and kurtosis don’t seem to produce any significant element of differential diagnosis for HPV status , 54 thoutrans parameter tend to be associated with HPV + lesions, this association seems to be inconstant and not significative enough to provide sufficient evidence in most cases [The role played by DCE-PWI in the seems to be unclear, though higher values of Kst cases , 68–70  and the extraction of features. It can be applied on CT, MRI and also on positron emission tomography (PET-CT) –78. It cThe use of radiomics applied on CT and MRI images has becoming increasingly interesting both for diagnosis and evaluation of pre-therapy HPV + oropharyngeal cancer. In fact, lot of studies are evaluating whether these tools can be able to characterize tumour histotypes –92 and iAs already said, many studies have tried to evaluate whether radiomics features are able to differentiate malignant and benign lesion or to evaluate HPV status of malignant lesions. Regarding the differentiation of the status, a study by Choi et al.  and the Radiomics has also been used to try to differentiate OPSCC from other tumours that can affect tissues like tonsils, as for example, lymphoma as in the study by Bae et al.  on MRI iTexture analysis has been used also to discriminate normal tonsillar tissue from neoplastic one, basing the study not only on structural, morphological and dimensional parameters, but also on the heterogeneity of the structure, as in the study by Kim et al. .The evaluation of prognostic features capable of assessing patients with high risk of distant metastases in HPV + OPSCC is very important in order to establish the correct treatement. In a study by Rich et al.  they fouAlthough radiomics is not yet used in clinical practice, it may become in the future a valuable aid in the diagnosis, evaluation of HPV status and therefore aggressiveness of squamous cell tumours and, therefore, in decision-making process. There are currently limitations to the use of texture analysis. One of the challenges is the necessary implementation of standardised methods for the analysis of textural parameters, due to the large number of different procedures and software that can be used  –113. In HPV-related cancer of the oropharynx is characterized by different treatment options and prognosis compared to its HPV- counterpart. It is thus of paramount importance to identify means to distinguish between the two entities. While at present biopsy is the gold standard for diagnosis, there are a plethora of imaging criteria that can help the radiologist infer the nature of a primary lesion of the oropharynx or of a metastatic lymph node. Knowledge of these criteria is fundamental for any radiologist that deals with the head and neck district and will become more and more important as HPV-related disease continues to rise in numbers.


Background: This survey investigated the knowledge, attitudes, and behaviors towards HPV preventive measures among university students in Italy and their associated factors. Methods: The cross-sectional survey was conducted between November 2022 and April 2023. Results: Only 41.7% knew about HPV infection and the main preventive measures. Multilevel mixed-effects logistic regression analysis showed that females, those aged 25–30 years, those who have had oral sex, those who had received information about HPV infection and preventive measures from physicians, and those who needed additional information about HPV infection and preventive measures were more knowledgeable. Only 39.1% have received at least one HPV vaccination dose, whereas 29.2% and 31.7% had not been vaccinated or did not remember. Multilevel mixed-effects logistic regression analysis identified that female students enrolled in the field of health sciences, those who did not report a prior history of a sexually transmitted infection, and those with a higher knowledge about HPV infection and preventive measures were more willing to receive the HPV vaccination. Conclusions: Despite some limitations, this was the first detailed survey on this topic conducted in the post-acute phase of COVID-19 among university students in Italy. The survey underlined the need to develop and to implement comprehensive educational programs and health interventions among students, to enhance their knowledge and modify their attitudes and improve the HPV vaccine uptake. Human Papillomavirus (HPV) vaccines are the most effective method, along with other precautionary measures, to reduce the incidence of infections and prevent the associated cervical, vulvar, vaginal, oropharyngeal, anal, and penile cancers . In ItalIn line with the initiation of the vaccination against HPV, a body of literature has focused attention on the level of knowledge, attitudes, and behaviors regarding preventive measures and the vaccination. When looking at this literature, many of the studies have been conducted among adolescents ,5, parenThis survey is part of larger research activities that investigated the genotype-specific prevalence of HPV infection in southern Italy’s young population in urine and oral samples. A cross-sectional survey was conducted between November 2022 and April 2023. The target population consisted of students enrolled in a degree program in the fields of medicine, healthcare professions, pharmacy, and sports science from three public universities in various geographic locations of the southern part of Italy. In particular, the sample was selected with a two-stage cluster method. In the first stage, 25 degree courses have been selected from the lists of the three universities and in the second stage, from each course the students have been selected through a simple random sampling. The sample size of the survey was determined to be 768, assuming an expected genital HPV prevalence of 20% in the non-vaccinated population and a post-vaccination HPV 16/18 prevalence of 10%, with 95% confidence intervals and a 0.05 margin of error, and a design effect of 2. In addition, accounting for an expected response rate of 90%, the minimum number was increased to 853. Overall, a total of 1652 students were selected.The members of the research team approached the students on randomly selected days during their courses. A self-administered questionnaire was used for data collection. It required approximately 15 min to answer all the questions. At the beginning of the questionnaire, the students were informed of the main objectives of the survey, assured that it was completely anonymous, that the participation was voluntary, that the responses would be treated with complete confidentiality, that they could withdraw at any time during questionnaire completion without consequences, that they would not receive any financial compensation, that the data would be used for the objectives of the survey, and that the informed consent for their participation was implied by returning the questionnaire.Six pieces of information were collected in the questionnaire. In the first part, all participants completed questions exploring socio-demographic and general characteristics including gender, age, and family history of cancer. In the second part, ten items assessed the level of knowledge about HPV. Five of these questions were closed-ended with “Yes”, “No”, and “Do not know” answers and the other five were multiple-choice types and participants were allowed to choose more than one answer from a list of the options offered. In the third part, concerns about the perception of the risk of acquiring an HPV infection or a related disease, or a sexually transmitted disease, were assessed with three items and for these questions the responses were measured on a five-point Likert scale with 1 being “strongly disagree/not at all” and 5 being “strongly agree/very much”. Three questions about preventive measures were asked including the perceived utility of the vaccination, of the Pap test, and of the DNA HPV test. In the fourth part, participants indicated whether they had been vaccinated against HPV and those who were unvaccinated responded to a question exploring their willingness or uncertainty or unwillingness to take it, and also to indicate the most likely reason(s) for their willingness or uncertainty or unwillingness from five options. The last part included questions on the main sources of HPV-infection and related preventive measures information and participants were requested to tick any of the five options offered. Finally, they were asked whether additional information was needed.Before the questionnaire was distributed, a pilot study was conducted among a small group of 50 students in the field of health sciences to ensure the questionnaire was easy to understand and to answer.p-value ≤ 0.25 in the univariate analysis were included in three multilevel mixed-effects logistic regression models to calculate the odds ratio (OR) and the 95% confidence interval (CI) in order to verify the significant relationship between dependent variables and independent variables. With the aim of accounting for the multilevel dataset structure (procedures are “nested” within degree courses), the variable degree courses were introduced in the models as a random factor. The outcomes of interest were the following: knowledge about the HPV infection and the preventive measures  (Model 1); quite/very concerned about the risk of acquiring an HPV infection  (Model 2); and willingness to receive the HPV vaccination  (Model 3). The outcome of Model 1 assessed the level of knowledge about HPV with the ten questions and it was created by giving the respondent a score of “1” for each correct answer and “0” for each incorrect or unknown answer. The total knowledge score for each respondent was calculated and ranged from 0 to 10; those with a score above the median value of 4 was considered to be knowledgeable, and coded with “1”, and those with a score below or equal to 4 to be unknowledgeable, and coded with “0”. The following independent variables have been tested for all outcomes: gender , age , undergraduate health sciences course , sexual orientation , family history of HPV-related cancers , having smoked at least 100 cigarettes during lifetime , consuming alcohol , having had sexual intercourse during lifetime , current partners , having had oral sex during lifetime , having had a diagnosis of a sexually transmitted infection (STI) , physicians as source of information about HPV infection and preventive measures , and needing additional information about HPV infection and prevention . The variable HPV vaccination status  was included in Models 1 and 2 and knowledge of HPV infection and preventive measures  in Models 2 and 3. To assess the level of knowledge, an overall knowledge score was constructed giving 1 point for each correct answer to the ten questions about HPV and respondents with a score above the median were considered to have knowledge of HPV infection and of the main preventive measures. Multiple imputation (10 imputed datasets), by assuming that data were missing at random, with chained equations with linear regression for continuous variables and logistic regression for binary variables has been used to fill the missing values. All variables from the logistic regression analysis have been included in the imputation models. To perform multiple imputation analysis, the multilevel mixed-effects logistic regression models have been fitted to the multiple imputed datasets and the results combined using Rubin’s rules. For all analyses, two-sided tests, p-values ≤ 0.05, were considered statistically significant. The results from the multiple imputation analysis have been presented as the primary study results.STATA software version 17 was used for the statistical analysis. First, the different information was presented as mean and standard deviation for the continuous variables and frequency and proportion for the categorical variables. Second, univariate analysis using the Chi-square test or Student’s t-test was performed to assess the associations, respectively, between categorical and continuous variables and the outcomes of interest. Third, independent variables with a Of the 1652 randomly selected students, 1612 returned the questionnaire for a response rate of 97.6%. The principal characteristics of the enrolled participants are described in The median value of the knowledge score was 4 (range = 0–10) and less than half of the participants (41.7%) had a score greater than or equal to 5 and were knowledgeable about HPV infection and the main preventive measures. Multilevel mixed-effect logistic regression analysis was used to assess which of the several characteristics influenced the different outcomes of interest and the results for both the complete cases and multiple imputed data are shown in A multilevel mixed-effect logistic regression model was performed to identify factors associated with the level of knowledge about HPV among those vaccinated and the results showed that participants aged 25–30 years , those who had received information about HPV infection and preventive measures from physicians , and those who needed additional information about HPV infection and preventive measures  were more likely to be knowledgeable about HPV.The statements of the perceived risk of acquiring an HPV infection or an HPV-related cancer or a sexually transmitted disease, measured on a 5-point Likert-type scale, indicated that, respectively, 23.9%, 50.7%, and 45.3% were quite/very concerned. The multivariate logistic regression analysis revealed that three factors predicted participant’s perceived risk of HPV. Females , those knowledgeable about HPV infection and of the main preventive measures , and those who needed additional information about HPV infection and preventive measures  were more likely to be quite/very concerned about the risk of acquiring an HPV infection , those enrolled in the field of health sciences , those who did not report a prior history of a STI , and those knowledgeable of HPV infection and of the main preventive measures  were more willing to receive the HPV vaccination (Model 3 in More than two-thirds of the sample had received information about HPV infection (67.9%) and related preventive measures (66.9%). Physicians and the Internet were mentioned as the two principal sources of information, with overall values of 22% and 20.3%, respectively. More than half (54.5%) of the students expressed the need to have additional information.This is the first detailed survey on the level of knowledge, attitudes, and behaviors towards HPV infection and preventive measures in the post-acute phase of COVID-19 among university students in Italy. Several implications for public health interventions and communication strategies can be drawn from this survey.First, assessing the level of knowledge identified some major gaps among the sampled students for the different items regarding HPV. Indeed, only one-third and one-half were able to recognize, respectively, that oral and penile cancers were HPV-associated, two-thirds correctly identified the role of the HPV vaccine in preventing cervical cancer, and only 20.1% that a lower number of sexual partners helps to prevent HPV transmission. Given these gaps, improving the level of knowledge is of particular importance because the present survey reported that having a high level of general knowledge about HPV was positively associated with a favorable attitude towards vaccination and a higher likelihood of intention among the unvaccinated respondents who are at a higher risk.Second, only 39.1% have already received at least one HPV vaccination dose, whereas 29.2% and 31.7% have not been vaccinated or did not remember. This figure is close to that from Switzerland where 36.9% of university students had received at least one HPV vaccination dose , whereasThird, the willingness to receive the HPV vaccine among those unvaccinated or who did not remember was very low with a value of 59.4%, whereas 33.9% were uncertain, and 6.7% did not want to be vaccinated. This observed intention was considerably lower than the values of 69.8% among female university students in Kuwait , and 65.Fourth, the other findings of the multivariate logistic regression analysis indicated that several socio-demographic and general characteristics, such as age, gender, university course, sexual behaviors, and health status, were independently identified as significant determinants of the outcomes of interest. Understanding these characteristics is useful for identifying individuals for targeted efforts to increase, for example, the HPV vaccination uptake. This survey adds to the prevailing evidence that respondents aged 25–30 years had a higher level of knowledge. One possible explanation for these associations could be that those that are younger are less exposed to the infection risk and consequently they perceive themselves to be at lower risk while, in contrast, as age increases, the prevalence of the infection and of the associated diseases increases, making those that are older more knowledgeable, with a more positive attitude towards the importance of the vaccination. Moreover, knowledge about HPV infection and preventive measures, concern about acquiring an HPV infection and a related cancer, and willingness to receive the vaccine were observed to be significantly higher in women. In the literature, similar surveys from different countries among different groups of individuals have shown that women generally have a higher knowledge and more positive attitudes towards HPV vaccination than men ,30,31. TFifth, assessments of the principal sources used to gain information about HPV infection and related preventive measures, revealed that physicians, although only in about a fifth of the respondents, were the most widely used, followed by the internet. The findings regarding the sources of information clearly provide evidence and offer insight that physicians may directly and positively impact respondents’ knowledge and attitudes. Indeed, respondents who received information from physicians had higher HPV infection and preventive measures knowledge and positive health care attitudes. Numerous studies among diverse populations from different geographic areas, have found that healthcare professionals, especially physicians, are the preferred source of health-related information and have emphasized the importance of providing preventive campaigns, which may translate into safer and more appropriate behaviors ,36. HoweIt is necessary to acknowledge the possibility of some methodological limitations when interpreting the findings. First, the cross-sectional nature of this survey limits the ability to attribute causation to the associations that have been observed between the independent variables and the outcomes of interest. Second, the population sample was selected from two geographic areas in the Southern part of Italy and, therefore, caution is needed in generalizing these findings more broadly to all Italian students. Third, the data were gathered through a self-administered questionnaire and therefore, the recall bias of participants may have influenced the results, for example, on the sexual habits and on immunization status. Fourth, participants might have more positively answered the questions and therefore, for example, due to the sensitive nature of those regarding sexual behaviors, might be subject to social desirability bias. However, keeping the identity of the participants anonymous, we are confident that this limitation was likely to have had little influence on the results.The results highlight the need to increase students’ knowledge on HPV. This should be attempted with educational interventions, by involving both schools and universities and physicians, and also by taking advantage of other opportunities to access health services such as primary care visits and vaccination centers. Moreover, the HPV vaccination coverage and the willingness to receive it were very low. Therefore, there is ample room for healthcare policymakers and managers to implement appropriate preventive strategies. Efforts should be made to increase vaccination coverage through targeted interventions that provide a more accessible service, increase the trust in the HPV vaccine for parents and young people, and involve healthcare professionals in preventive programs.In conclusion, the present survey underlined that it is needed to develop and to implement comprehensive educational programs and health interventions as essential tools to meet the information needs of this population and that will contribute to enhancing their knowledge, modify their attitudes and improve the HPV vaccine uptake. The findings of this survey are also useful for future investigations focusing on attitudes and behaviors of healthcare professionals towards HPV infection and vaccination given their key role in increasing vaccination coverage.


Long-term follow-up of large cohorts is needed to determine the effects of HPV and screening on CIN3  and ICC .Women were recruited when attending for routine cervical screening in Greater Manchester, UK: 1987–93 for the Manchester Cohort  and 2001–03 for the ARTISTIC Cohort . Both were followed through national registration for cancer incidence and mortality to 2020.Risk patterns following HPV infection differed for CIN3 and ICC. Risk of ICC in the MC rises for 30 years following a single positive HPV test, reaching 2.5% (95% CI: 1.3–4.5%). A similar pattern was seen in the AC, but the risks of cancer were approximately halved. CIN3 was diagnosed much sooner in the AC due to more efficient cytology. More sensitive HPV testing was able to better predict future risk.The sensitivity of HPV testing and cytology influences the CIN3 detection rate. Sensitive HPV testing enables effective risk stratification. Increased risk of ICC is observed 15–30 years after HPV infection. Women testing HPV + should be followed until their infection clears. Discharging women from screening programmes whilst they remain HPV + may not be safe, even if cytology and colposcopy tests are normal. Over the past 30 years, cervical cancer incidence and mortality in the UK have declined considerably as a result of a highly effective cervical screening programme (CSP) despite increasing HPV infection rates , 2. PrimBetween 1987 and 1993, in collaboration with over 100 general practitioners and screening clinics in the Greater Manchester area, 78,062 cervical samples were collected from 61,564 women attending for routine Papanicolaou (pap) screening. There was no age restriction. Women consented to having a cervical sample taken and stored after their routine smear. Samples were stored at −30 °C [n = 6462) of the samples assayed. Samples found to be HPV-positive were dot blotted onto new membranes and hybridised with a series of biotinylated high-risk HPV (HR-HPV) type-specific probes. Further description of the sample collection methods and HPV results have been published elsewhere [To avoid the cost of testing all stored samples for HPV DNA using PCR, an age-stratified random sample of 7278 (11.8%) women aged 15–69) was selected for testing in the early 1990s. HPV L1 consensus PCR amplification was used with MY09/MY11 primers [9 was seln = 7504), inadequate Pap smear (n = 3391), abnormal smear within the previous year (n = 509), or they had ever had a diagnosis of CIN3 (n = 505). All previous analyses were based on the remaining 49,655 women from whom a sample for HPV assay was collected at a routine smear test that gave an adequate cytological result [Samples were excluded if they were taken before July 1988 due to storage procedure (n = 24) that tested HC2-positive but were insufficient for further typing were assumed to be HR-HPV-positive. The cohort has been followed via national registration for cancer incidence and mortality  until May 2020 resulting in 24,496 women contributing a total of 409,094 person-years . All LBC samples taken (as part of the NHSCSP) from women in the cohort were tested for HPV until 2009. Women were randomised in a 3:1 ratio to have their HPV result revealed. Women with abnormal cytology were managed according to current guidelines and hence the only difference between the arms of the trial was among women with normal cytology, where women with HPV were offered repeat HPV testing and if persistent, colposcopy. Hybrid Capture 2 (HC2) was used as the primary HPV assay with HC2-positive and a sample of HC2-negative samples further tested with PCR-based assays  [Women from the random sample in the Manchester cohort (MC) and all women from the ARTISTIC cohort (AC) were classified hierarchically into three mutually exclusive groups: HPV16 or HPV18, any other HR-HPV  without HPV16 or HPV18, and HPV-negative. Those testing positive for types other than these 13 HR types were defined as HR-HPV-negative. Cumulative cancer and CIN3 risks were estimated by Kaplan–Meier methods using the date of cancer registration as the outcome with censoring at the date of emigration, date of death, age 85 years, and on March 31, 2019 to allow for late registration of CIN3 and cancer. For the CIN3 analysis, women were censored at age 70, rather than age 85, as they are rarely screened after this age.Total person-years were calculated and follow-up time was stratified by time period (in 5-year periods from 1987 for the MC and 2002 for the AC) and age group (in 5-year age groups from age 15 to age 69 for CIN3 and age 84 for cancer). English national cancer incidence rates and mortality rates for England and Wales were used to calculate standard incidence ratios (SIRs) and standard mortality ratios (SMRs), respectively, for selected gynaecological and other common cancers . Cervical cancer incidence rates are around 50% higher in the NW (northwest) than in the London (southeast) region of England and about 20% higher than the average for England. There is a similar north/south divide regarding lung cancer incidence, which is around 35% higher in the NW than in England as a whole. Rates in the NW are similar to national rates for breast, vagina, vulva, anus, ovarian and colon cancers . For theWithin 30 years of follow-up invasive cervical cancer (ICC) was diagnosed in 144 of the 47,625 women in the MC, 35 of whom died with cervical cancer as the stated cause. Among the 24,496 women in the AC, 32 were diagnosed with ICC over 17 years of follow-up. Table Lung cancer incidence and mortality ratios were elevated in the MC  but not in the AC  and CIN3 . There were insufficient numbers of vaginal cancers to estimate rate ratios but elevated risks were seen for cancers of the vulva  and anus  in those testing HPV-positive at baseline. There was some evidence of elevated lung cancer incidence in HPV-positive women in the MC , but not in the AC (RR = 1.0) . Referral criteria based on cytology alone were similar in both cohorts except for HPV-infected women in the AC with normal cytology, but the modern sampling and cytology methods employed by the ARTISTIC trial . Due to the referral procedures, the majority of CIN3s were diagnosed following abnormal cytology in both cohorts, but only 16% (8/49) of CIN3s in the MC had high-grade cytology at baseline, compared to 52% (175/336) in the AC. The pattern of cumulative risk of CIN3  in 21,692 HPV-negative women in the AC. The higher risk for women who tested HPV-negative in the MC is likely to be due to false negative results due to a combination of poor sample quality and lower test sensitivity. Sample collection in the MC was sub-optimal by today’s standards: a wooden spatula was used, the tip of which was broken off and stored in the sample medium before being removed sometime later [The woman diagnosed with ICC within 5 years of a negative HPV test in the MC , but not in the AC .Despite the rarity of other HPV-related cancers, we showed strong elevated risks of vulva and anal cancers associated with cervical HPV infection. There were insufficient numbers of vaginal and oropharyngeal cancers to explore the relationship with HPV. We observed an overall increased risk of lung cancer in the MC both compared with national rates  and following HPV positivity  for the MC, indicating some confounding by deprivation.The overall excess presumably reflects the high local rates, but HPV has been suggested as a risk factor for lung cancer. HPV DNA has been identified in lung cancer biopsies , includiThe main strength of these analyses is the almost complete follow-up over 30 years for the MC and 17 years for the AC. The MC samples were collected before HPV testing techniques had been developed and the samples were processed and stored in sub-optimal conditions, but HPV-positive results are likely to be reliable despite the potentially lower sensitivity for detecting HPV infection. Only an eighth of the entire MC were originally tested for HPV infection, so a nested-case control study is currently underway in which stored samples from women who later developed ICC and other HPV-related cancers are being tested by modern sensitive PCR. Samples stored from the AC are much better quality, but were originally tested with HC2 which may not be comparable to modern HPV assays. HR-HPV was not detected in 27% of the HC2-positive samples after genotyping .To maximise the numbers included in the analysis, we included 65 women aged under 20 with HR-HPV and 262 who were HR-HPV-negative in the MC analyses. They appear separately in Table The women participating in the AC consented to be randomised into a trial, and were probably more selected than those participating in the MC, who consented for their samples to be used for research. Any social class bias might account for the lower lung and cervical cancer rates seen in the AC. More regular attendance for women in the AC would also partly explain their higher CIN3 rates.There is a degree of under-registration for carcinoma-in-situ. In order to compare the cohorts, we restricted the analysis to CIN3 diagnoses with national registration. Including the additional 96 CIN3s with no cancer registration recorded by the local pathology laboratory during the first 8 years of the AC [HPV infections, particularly HPV types 16 and 18, continue to confer a higher risk of invasive cervical cancer 15–30 years after infection. Women testing HPV-positive should therefore be followed until they clear their infection, and it is unsafe to discharge women from screening programmes if they remain HPV-positive even if they have negative cytology and colposcopy. By comparing these two cohorts, we have shown how the sensitivity of cytology influences the rate at which CIN3 is detected and that more sensitive HPV testing predicts future risk of CIN3 and ICC more accurately. The aim of the screening programme is to stratify women into groups reflective of their future risk of CIN3 and ICC. Those with minimal risk can be screened less often, while those at the highest risk can be screened more frequently to prevent ICC or diagnose it at an earlier stage. Sensitive HPV testing should be considered in primary screening, provided that appropriate triage testing is used to distinguish transient and persistent HPV infections.Supplementary Material


TTN) mutations, whether the TTN gene mutation may be a predictor, and its impact on mortality and survival in these patients. A total of 585 samples from patients with ovarian serous cystadenocarcinoma were collected from The Cancer Genome Atlas and PanCancer Atlas through the cBioPortal for analysis of social, genetic, and histopathological factors. Logistic regression was used to investigate whether TTN mutation could be a predictor, and the Kaplan–Meier method was applied to analyze survival time. TTN mutation frequency did not differ between age at diagnosis, tumor stage, and race, and was related to increased Buffa hypoxia score (p = 0.004), mutation count (p < 0.0001), Winter hypoxia Score (p = 0.030), nonsynonymous tumor mutation burden (TMB) (p < 0.0001), and reduced microsatellite instability sensor score (p = 0.010). The number of mutations (p < 0.0001) and winter hypoxia score (p = 0.008) were positively associated with TTN mutations, and nonsynonymous TMB (p < 0.0001) proved to be a predictor. Mutated TTN affects the score of genetic variables involved in cancer cell metabolism in ovarian cystadenocarcinoma.Several factors may increase the risk of development of ovarian cancer. In this study, we investigated the relationship between social, genetic, and histopathologic factors in women with ovarian serous cystadenocarcinoma and titin ( Ovarian cancer is the sixth most common and deadliest malignancy in women . The incRegarding mortality, a study based on the Global Cancer Observatory (GLOBOCAN) database showed that mortality rates vary among different countries due to differences in the Human Development Index (HDI) . AccordiHistologically, ovarian carcinoma is divided into five types: serous, endometrioid, mucinous, clear cells, and squamous cells, which are further divided in subtypes ,5. AmongBRCA1/2), tumor protein p53 (TP53), epidermal growth factor receptor (EGFR), and AKT serine/threonine kinase 2 (AKT-2) [Regarding genetic factors, although certain gene mutations, such as breast cancer gene 1 and 2 ( (AKT-2) , are inv (AKT-2) .TTN) represents a tumor mutation burden (TMB) associated with various cancers, including ovarian cancer [TTN gene acts in chromosome condensation and segregation (https://genome-euro.ucsc.edu/ accessed on 23 December 2022). Therefore, the presence of a mutation can compromise gene function at this stage of the cell cycle and favor the emergence of neoplasms, such as ovarian cancer. Analysis of the frequency of this mutation in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database (http://www.sanger.ac.uk/cosmic accessed on 23 December 2022) [TTN mutation and OSC may enable the discovery of a new biomarker for the screening and management of the disease in clinical practice.A recent study showed that a spontaneous mutation of the titin gene (n cancer . The TTNer 2022)  revealedTTN-related hypoxia scores can provide information regarding important characteristics of the tumor and establish relationships with tumor severity and progression in affected individuals [TTN mutation may provide evidence for a new prognostic and predictive biomarker for the risk and aggressiveness of ovarian cancer in women with the mutated gene.In addition, analysis of mutation types, the presence of microsatellite instability (MSI), and ividuals ,12. TherTTN gene mutation with social, genetic, and histopathological factors in women diagnosed with OSC, to verify whether this mutation can be a predictor and if it impacts the mortality and survival of these individuals.In this study, an analysis of OSC samples from The Cancer Genome Atlas (TCGA) was performed to identify the relationship of TTN mutations. Furthermore, it is hypothesized that TTN mutation may be related to a higher risk of OSC, increased mortality, and reduced survival in women diagnosed with OSC.We hypothesized that social, genetic, and histopathological variables are related to the presence of Thus, the results of this study may contribute to early disease screening and the development of personalized treatments for patients with OSC and provide a better understanding of the molecular mechanisms of the disease.The open-access database used in this research is maintained by the Memorial Sloan Kettering Cancer Center (MSK). These individuals were not identified and contained anonymous data; therefore, according to the National Health Council (CNS) Resolution No. 510, 7 April 2016, evaluation by the research ethics committee was not required .Observational study with ecological design having descriptive and inferential bioinformatics analysis.The study included 585 women diagnosed with OSC and the relevant data were collected in a public database from TCGA, PanCancer Atlas through the cBioPortal . SecondaAs inclusion criteria, we selected genomic samples with OSC, ICD-10: C56.9 , C48.2 , and C48.1 , using the filter to select samples with “mutation” Molecular Profile for TTN gene mutation types (ENST00000591111.5 transcript). As exclusion criteria, samples with incomplete information were not considered for the analysis.https://www.cbioportal.org accessed on 23 December 2022), maintained by the Center for Molecular Oncology at MSK [Data were collected using the database publicly available on the cBioPortal platform, an open source and open access resource for the interactive exploration of multidimensional cancer genomics datasets , which it defined as free for steps data OSC  [The data access policy regarding the availability of data for publication was consulted on the MSK Data Catalog platform (r Atlas) .TTN gene, a prior consultation was performed in the OncoKB portal. OncoKB is a knowledge base platform in precision oncology, which was developed at the MSK and is composed of biological and clinical information regarding genomic alterations in cancer (https://www.oncokb.org/cancerGenes accessed on 23 December 2022) [TTN has not been included in the list of cancer genes, highlighting the importance of studies assessing the potential of TTN as a cancer gene.For the er 2022) . The conData on 1838 clinical samples of serous ovarian cancer were available on the platform. For this study, only data referring to OSC derived from TCGA were selected via the cBioPortal.n = 475) and Genomic Profile for TTN mutation (n = 110) were used.We used a dataset containing clinical and summarized data from a sample of 585 serous ovarian cystadenocarcinomas of 585 patients from the PanCancer Atlas initiative, which comprehensively addresses cancer issues. To obtain these samples, the filters Mutation , time of overall survival (months), and disease-free survival (months).For analysis, we considered the following social variables: diagnosis age and race/skin color; genetic factors such as http://www.mutationaligner.org accessed on 23 December 2022) was used [The frequency by mutation type for the TTN gene, specifically for the ENST00000591111 transcript in OSC samples, was checked using cBioPortal. To explore the hotspots of somatic mutations in the protein domains, the MutationAligner platform (was used .2) or Mann–Whitney test was used.Descriptive analysis was expressed as frequency (absolute and relative), central tendency (median), and dispersion (interquartile range). To verify normality, the data were submitted to Kolmogorov–Smirnov test. To determine whether there were statistically significant differences between the frequency and median, the chi-square (χTTN) gene mutations could be a predictor. Initially, we performed univariate analysis considering a p-value < 0.20. To verify multicollinearity, the variance inflation factor (VIF) was calculated, and variables that presented a VIF value > 10 were removed from the final model. Statistical significance was set at p < 0.05. Odds ratios (OR) with 95% confidence intervals (95% CI) were used to quantify the degree of association.Binary logistic regression analysis was used to determine whether the Titin , Breslow (intermediary), and Tarone–Ware  tests were used to identify the occurrence of a statistically significant difference in different periods.For statistical analyses, we used the Statistical Package for the Social Sciences (SPSS) Version 26.0 .TTN gene and 110 with the mutation) were analyzed to assess the impact of TTN mutation on social variables, grid histopathology, and genetic factors.In this study, a sample of 585 individuals (475 without the mutation for the TTN gene and 110 with the mutation) were analyzed to assess the impact of TTN mutation on social variables, grid histopathology, and genetic factors.A sample of 585 individuals (475 without the mutation for the TTN gene mutation. The Buffa hypoxia score (p = 0.004), mutation count (p < 0.0001), Winter hypoxia score (p = 0.030), and nonsynonymous TMB (p < 0.0001) were higher in samples with mutation. Additionally, the MSI sensor score (p = 0.010) was lower in individuals with mutation.TTN gene showed that there was no significant statistical difference in relation to age at diagnosis (p = 0.419) and for race/skin color (p = 0.152), grade histologic neoplasm (p = 0.420), death (p = 0.937), and survival time (p = 0.082). However, one-tail analysis showed a lower survival time in the group with mutation (p = 0.041) (No TTN gene mutation: 34 (39) days; Mutation in TTN gene: 31 (35) days).As shown in p = 0.003), MSI sensor score , mutation count , and Winter hypoxia score  were associated positively with TTN mutation. The other variables were not different in relation to TTN mutations.In the univariate analysis presented in p < 0.0001) was found to be a predictor.In this sequence, multicollinearity of the variables was analyzed. The mutation count was removed for VIF values > 10. In the multivariate analysis, nonsynonymous TMB , followed by in-frame mutations (1 sample), truncations (8 samples), and multiple mutations (12 samples)  A. In tersamples) B.For missense mutations, the observed hotspots were fibronectin-type III (fn3) domain and the I-set immunoglobulin domain. Data from the InterPro description on the MultationAligner platform showed that fn3 domain has several important functions, including cell differentiation and migration, tumor metastasis, and cell adhesion activity. I-set domains are found in cell adhesion molecules, such as vascular, intercellular, and angiogenic molecules.OSC is the most common malignant ovarian tumor, accounting for approximately 50% of all cases. It occurs bilaterally in 30–50% of patients, affects women close to 60 years of age, is generally diagnosed late, and results in high lethality. In the context of late diagnosis and high lethality , studiesTTN mutations. Social factors, such as age at diagnosis and race did not affect the frequency of TTN gene mutations. Previous studies have found that mutation in this gene is observed in other types of tumors, but not in the variables of social factors in this case, which represent trends between populations. [TTN mutation and social, clinical, and genetic factors in ovarian cancer remains unexplored. Therefore, in this study, considering the database used and the Tissue Source Site, it is probable that the individuals are present in a certain geographical area that does not have large heterogeneity in social and demographic aspects [In the present study, social factors were not found to be associated with lations. ,20. To d aspects .TTN in ovarian cystadenocarcinoma. In this type of mutation, there is a variation in the DNA nucleotide in the coding region of the protein, which results in an alteration of the amino acid, and consequently of the codon, thereby influencing the structure, function, and level of the protein [TTN mutation in OSC may help characterize this histological subtype using a genetic marker.The frequency of TTN_ENST00000591111 based on the type of mutation, primarily missense mutation, is relevant in serous carcinoma, indicating that it may be a promising marker for detecting the type of missense mutation related to  protein . Therefo protein , as well protein . The preTTN mutations. This is likely due to the fact that the TTN ENST00000591111.5 transcript is a key component of striated muscles, with low levels of reads per kilobase of transcription per million reads mapped in ovarian tissue (https://genome-euro.ucsc.edu/ accessed on 23 December 2022). This may explain the lack of a histological relationship with TTN.Histopathological factors were not associated with TTN mutations to cancer cell activity and prognosis of oncogenic mutations in ovarian cancer. An analysis of the genomic sequencing profile of primary epithelial ovarian cancer samples showed that somatic mutations in multiple genes including TTN can be a biomarker for early diagnosis [Additionally, few studies have linked iagnosis .BRCA1/2 are two of the most well-known genes associated with the disease, and mutations in these genes increase the risk of developing both ovarian and breast cancer [TP53 is another commonly mutated gene in ovarian cancer, and its mutations are often associated with poor prognosis and resistance to chemotherapy [AKT-2 gene as a potential contributor to ovarian cancer development and progression [BRCA1 and BRCA2 genes have been successfully targeted with poly (ADP-ribose) polymerase (PARP) inhibitors, leading to significant improvements in response rates and overall survival in patients with BRCA-mutated ovarian cancers [BRCA1/2, TP53, and AKT-2 are among the most relevant gene factors associated with ovarian cancer, and their study is critical for improving our understanding and management of this disease.Ovarian cancer is a complex and heterogeneous disease with multiple genetic and environmental factors contributing to its development. Several genes have been identified as important players in the onset and progression of ovarian cancer. t cancer . TP53 isotherapy . Additiogression . These g cancers . However cancers . DespiteTTN is associated with high TMB [TTN mutation in OSC can also be used as a marker that should be further investigated in preclinical studies to test the in vitro activity of immunotherapy in a selected population with likely high TMB. However, the OncoKB portal showed no results regarding the TTN gene [However, a recent study indicated that high TMB . Tumors high TMB . In clinhigh TMB ,33. TherTTN gene , emphasiTTN mutation in OSC can also be used as a marker that should be further investigated in preclinical studies to test the in vitro activity of immunotherapy in a population with likely high TMB selected as a biomarker.Therefore, considering previous findings and the results of the present study, it is possible that the presence of a TTN gene mutation and OSC. The mutation count was higher in women with ovarian cystadenocarcinoma and was positively associated with TTN gene mutation. The TTN gene is susceptible to cancer protein kinase, encodes the major polypeptide expressed in oncogenesis, and is found in many functional cell types [TTN ENST00000591111.5 transcript plays an important role in chromosomal condensation and segregation during mitosis [TTN gene may affect cell cycle and contribute to tumor occurrence and progression. In addition, we found that missense point mutations in the TTN transcript for the fn3 and I-set domains can affect cellular processes such as angiogenesis.In this study, we found that genetic factors are associated with ll types . In part mitosis . TherefoTTN gene mutation. When a tumor develops, the homeostasis of tissue oxygenation is altered because of the increased demand for oxygen by the cancer tissue, leading to the rapid and erratic formation of new blood vessels and increased demand with regard to the available supply of oxygen [TTN mutation and hypoxia score in ovarian cancer has not yet been researched.When we examined the relationship between Winter and Buffa hypoxia scores, we found an increase in hypoxia with f oxygen . This isf oxygen . AlthougTTN-related hypoxia score indicated that dysregulation of these parameters can offer valuable information about tumor characteristics and its relationship with disease progression and severity in affected individuals [Molecular analysis of mutation types, MSI status, and ividuals . Tumor bividuals , therebyTTN gene as the score increased. It is anticipated that MSI  will be increasingly employed in cancer diagnostics because it is crucial for understanding cancer progression, extracting information about familial risk, and developing treatment methods [The MSI sensor score was lower in individuals with mutations but was positively correlated with a mutant  methods .TTN gene mutation, and the levels of nonsynonymous TMB were greater and more positively correlated. TMB is a biomarker for immuno-oncology that has a high rate of immunotherapy response and is used to quantify the burden of neoantigen tumors by examining both the quantity and quality of mutations [TTN mutation serves as a risk factor for ovarian cancer [When women with ovarian cystadenocarcinoma had the utations ,40. Althutations ,42,43. On cancer .Sociodemographic variables, such as family history, demography, and low/high income, were not analyzed because of database limitations. In addition, given the different natural histories and clinical backgrounds of the five ovarian cancer histotypes, the results of this study are only applicable to the analyzed subtypes. Nevertheless, our study limitation concerns the ecological design, where association between clusters does not necessarily indicate that it occurs at the individual level, when investigating samples from secondary databases . HoweverTTN mutations. However, we identified genetic factors associated with TTN mutations in women diagnosed with OSC. The Buffa hypoxia score, mutation count, Winter hypoxia score, and nonsynonymous TMB were higher and positively associated with TTN gene mutation in women with ovarian cystadenocarcinoma. MSI sensor scores were lower in those with mutation but were positively associated with mutated TTN gene. The survival time was lower in women with TTN mutation. Nonsynonymous TMB proved to be a predictor of interaction with other variables. In addition, missense point mutations in the TTN transcript for the fn3 and I-set domains may affect cellular processes such as angiogenesis. In summary, these findings indicate that TTN mutations and TMB may act as target genetic markers to assist in the detection of OSC and in the clinical management of the disease.Social and histopathological factors were not associated with


APP) and Down syndrome cell adhesion molecule (DSCAM) regulate neuronal morphology and contribute to neuronal aberrations in DS. Here, we report the functional interaction between the Drosophila homologs of these two genes, Amyloid precursor protein-like (Appl) and Dscam (Dscam1). We show that Appl requires Dscam to promote axon terminal growth in sensory neurons. Moreover, Appl increases Dscam protein expression post-transcriptionally. We further demonstrate that regulation of Dscam by Appl does not require the Appl intracellular domain or second extracellular domain. This study presents an example of functional interactions between HSA21 genes, providing insights into the pathogenesis of neuronal aberrations in DS.Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21). Although several HSA21 genes have been found to be responsible for aspects of DS, whether and how HSA21 genes interact with each other is poorly understood. DS patients and animal models present with a number of neurological changes, including aberrant connectivity and neuronal morphology. Previous studies have indicated that amyloid precursor protein ( Summary: Down syndrome is caused by an extra copy of human chromosome 21 (HSA21). This study reveals that the interaction between two HSA21 genes is important for axon development. Down syndrome (DS) occurs at a prevalence of 1 in approximately every 700 live births in the United States . DS is cDrosophila homolog of the HSA21 gene Down syndrome cell adhesion molecule (DSCAM), termed Dscam1 (referred to as Dscam hereon), promotes axon terminal growth in a dose-dependent manner , the Drosophila homolog of human amyloid precursor protein (APP)  appears to act as a dominant negative, decreasing the phosphorylation of Abelson tyrosine kinase (Abl) below wild-type levels . The domin (APP) , has bee systems . APP is  neurons , and thi neurons . We thusAppl and Dscam interact to regulate the growth of axon terminals during development. Co-overexpression of Appl and Dscam increased axon terminal length more than expressing either transgene independently. We found that Appl promotes axon terminal growth by upregulating the expression of Dscam. Appl increases the level of Dscam protein, but not that of the transcript, through an intracellular-domain-independent mechanism. This study reveals a previously unknown interaction between two HSA21 genes that are important for axon development.In this study, we show that Drosophila larvae (Drosophila homologs Appl and Dscam.Class IV dendritic arborization (C4da) neurons provide an excellent model for studying neuronal morphology in a larvae . The cela larvae A. This aa larvae . These aa larvae , collecta larvae   promoter  to visualize single C4da neurons homozygous for mutations or overexpressing transgenes in an otherwise heterozygous or wild-type background (UAS-mCD8::GFP) was expressed to label the morphology of single C4da neurons. Axon terminal length was measured from the axon branch point in the CNS to the axon endings , (2) overexpressing only a V5-tagged Appl transgene (UAS-Appl::V5), (3) overexpressing only a GFP-tagged Dscam transgene (UAS-Dscam::GFP), and (4) overexpressing both Appl::V5 and Dscam::GFP. In order to control for UAS-site competition for binding the GAL4 transcription factor, the negative control group, Appl::V5-only group and Dscam::GFP-only group also expressed an unrelated transgene – a LacZ::GFP fusion protein with a nuclear localization signal (UAS-LacZ::GFP.nls). This additional UAS-transgene in the Dscam::GFP-only group titrated Dscam expression to a level that did not drive significant changes in axon terminal length. Expressing Dscam::GFP at a non-phenotypic level allowed us to test whether Dscam co-expression with Appl::V5 could drive more axon growth than expression of either construct independently.Because overexpression of ckground . In addi endings . Using tAppl and Dscam increased axon terminal length more than expressing either transgene alone . Thus, Dscam can drive axon terminal growth without Appl, suggesting that Appl is not downstream of Dscam.As shown previously , Dscam ol length A,C. ImpoDscam is downstream of Appl by determining whether Appl overexpression increases axon terminal length in the absence of Dscam. As previously demonstrated by Dscam (18Dscam) caused a decrease in axon terminal length  promoter (nSyb-GAL4) to overexpress a V5-tagged Appl (Appl::V5), and then assessed proteins extracted from larval CNS via western blotting. Chemiluminescence of the protein bands was normalized to that of either α-Tubulin or Elav  (Appl::V5 overexpression increased the level of endogenous Dscam compared to that of the no-UAS control (Appl (dAppl) decreased the amount of endogenous Dscam to one-third the amount observed in the wild-type control  . Appl::V control A,B. Conv control C,D. By c control , Fragile control  or Cyste control  . Two separate primer pairs (Dscam#1 and Dscam#2) that catch all known isoforms   A (FossgrP695ΔCT)  and compP695ΔCT) B,C. ThisAppl-driven axon terminal growth requires the Appl extracellular domains. APP and Appl have two highly conserved extracellular domains – extracellular domain 1 (E1) and extracellular domain 2 (E2)  and lacked the required binding sites for all known cleavage (UAS-ApplΔE2) still caused twice the number of average C4da axon tracts, compared to that in the control (Appl construct lacking E1 (UAS-ApplΔE1) did not cause detectable overgrowth. This suggests that Appl requires the E1, but not the E2, domain to drive C4da axon terminal growth.Because the cytoplasmic domain of APP is not required to promote axon growth, we directly tested whether the n 2 (E2) . To detecleavage D  experiments suggest that Dscam and Appl do not form a protein complex, as Appl was not pulled down together with Dscam (Drosophila Schneider 2 (S2) cells in the presence of the mRNA translation inhibitor cycloheximide (CHX). Dscam::GFP was co-expressed with either Appl or an exogenous membrane protein (CD8::GFP) as a negative control. CHX was then added to the cells to prevent further protein synthesis , Appl overexpression, Dscam overexpression and Appl–Dscam co-overexpression conditions. Similar to the experiments for evaluating axon terminal growth, the negative control group, Appl overexpression group and Dscam overexpression group also expressed UAS-LacZ::GFP.nls so that the number of UAS-transgenes in them was the same as that in the Appl-Dscam co-overexpression group. The additional UAS-LacZ::GFP.nls in the Dscam overexpression group titrated Dscam expression to a level that did not cause significant changes in axon terminal length . The resulting pUASTattB-Appl::V5 transgene was then injected into embryos carrying the attP40 docking site for making transgenic lines. We generated constructs for S2 cell transfection by inserting the Appl, CD8 and Dscam sequences into the pAc5.1-V5/His or the pAc5.1-GFP plasmid backbone.The G13FRT and 19AFRT lines were heat shocked for 15 min, and no horseradish peroxidase (HRP) staining was performed. Axon terminals were measured using Neurolucida software (MBF Bioscience), and branches under 5 µm were excluded. G13ppk-Gal4, hsFLP, UAS-mCD8::GFP; tub-Gal80, FRT flies were mated with (1) G13, UAS-LacZ::GFP.nls, FRTG13UAS-Appl::V5, FRT; (2) G13UAS-Appl::V5, UAS-LacZ::GFP.nls, FRT; (3) G13UAS-DscamTM2::GFP, UAS-LacZ::GFP.nls, FRT; (4) G13UAS-Appl::V5, UAS-DscamTM2::GFP, FRT; (5) 18, FRTG13Dscam; (6) 18Dscam, G13UAS-Appl::V5, FRT; or (7) G13FRT flies  d, FRT19AAppl; (2) 19AFRT; (3) 19AUAS-DscamTM2::GFP, FRT; (4) d, FRT19AAppl; or (5) d,UAS-DscamTM2::GFP, FRT19AAppl flies  of late-third-instar larvae or S2 cells as described previously . BrieflyPrimary antibodies used were rabbit anti-GFP  , mouse aDrosophila Genomics Resource Center (S2-DRSC). The vendor authenticated the cell line. S2 cells were cultured in Schneider's Drosophila medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 28°C. Five-hundred microliters of cells were plated at a density of 0.8×106 24 h before transfection with home-made polyethylenimine (PEI). DNA plasmids were mixed with Opti-Mem solution (Thermo Fisher Scientific), and PEI was used at a ratio to DNA of 1:5. Forty-eight hours after transfection, S2 cells were collected in ice-cold 1× PBS, centrifuged at 200 g for 2.5 min to remove medium and suspended in 50 µl 2× SDS sample buffer containing β-mercaptoethanol. Cells were mechanically disrupted and sonicated prior to western blotting.S2 cells were purchased from For the protein degradation assay, CHX was dissolved in a minimal amount of dimethyl sulfoxide and added to each well at time zero (T0) at a concentration of 0.5 ng/µl. Cells in each well were collected individually at 4-h time increments, as shown in 2, 50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and EDTA-free protease inhibitor cocktail ]. The supernatant (300 µl) was incubated with anti-GFP antibody-conjugated magnetic beads  that were prepared according to the manufacturer's instructions. After incubating the cell lysates with the magnetic beads for 3 h at 4°C, the beads were washed three times in the IP buffer (5 min each) and three times in 1× PBS containing PMSF. The proteins bound to the beads were then eluted in 2× sample buffer without β-mercaptoethanol. The eluates were transferred to a new tube, before β-mercaptoethanol was added, and boiled for 5 min before loading to SDS-PAGE gels and analyzed with western blotting.Forty-eight hours after transfection, S2 cells were solubilized in the IP buffer [150 mM NaCl, 1 mM CaClChmp1 was used as the reference gene. Two sets of primers were used to catch all known Dscam isoforms. Analysis was performed using a QuantStudio 5 Real-Time PCR Systems with Design and Analysis software 2.5 (Thermo Fisher Scientific). The primer sequences were as follows . The plate was covered with 750 µl water and pre-heated to 33°C. Behavioral responses were video recorded for 60 s. Videos were analyzed by LabGym software .t-test, and multiple group comparisons were made using one-way ANOVA with Tukey multiple comparisons post hoc analysis. For data with non-normal distribution, two-group comparisons were made using two-tailed Mann–Whitney U-test, and multiple group comparisons were made using Kruskal–Wallis with Dunn's multiple comparisons post hoc analysis. For normal data compared to a theoretical value, such as ratios where the null hypothesis would be a ratio of 1, one-sample t-tests were applied. For behavioral data, two-group comparisons were made using two-sided Chi-square tests. P<0.05 was considered significant.All statistical analysis was performed using GraphPad Prism. Researchers were unaware of genotype/group when performing analyses. Normality was assessed for all groups. For data with normal distribution, two-group comparisons were made using an unpaired two-tailed 10.1242/dmm.049725_sup1Supplementary informationClick here for additional data file.


Referral rates for colposcopy and biopsy and repeat testing (HPV + cytology) after triage were significantly higher among DNA-tested (24.9% and 27.9%) compared to mRNA-tested women (18.3% and 5.1%), as were cervical intraepithelial neoplasia grade 3 or worse (CIN3+) detection rates . Ten cancer cases were diagnosed during follow-up; eight were in DNA-tested women. Conclusion: We observed significantly higher referral rates and CIN3+ detection rates in young women with ASC-US/LSIL when the HPV DNA test was used at triage. The mRNA test was as functional in cancer prevention, with considerably less healthcare utilization.Background: A specific, cost-effective triage test for minor cytological abnormalities is essential for cervical cancer screening among younger women to reduce overmanagement and unnecessary healthcare utilization. We compared the triage performance of one 13-type human papillomavirus (HPV) DNA test and one 5-type HPV mRNA test. Methods: We included 4115 women aged 25–33 years with a screening result of atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesions (LSIL) recorded in the Norwegian Cancer Registry during 2005–2010. According to Norwegian guidelines, these women went to triage . Women were followed through December 2013. Results: HPV positivity rates at triage were 52.8% and 23.3% among DNA- and mRNA-tested women ( Cytology-based screening has reduced the incidence and mortality of cervical cancer in countries with organized screening programs . HoweverIndeed, HPV test-based screening for women aged 30 years or younger is not cost-effective, given the high prevalence of HPV infection  and low In a previous study, the reported 5-year risks of CIN3+ for women with screening results of ASC-US and LSIL were 2.6% and 5.2%, respectively . MoreoveIt has been shown that a 5-type HPV mRNA test has a higher clinical specificity and positive predictive value than a 14-type HPV DNA test in the triage of women with minor cytological abnormalities at screening . In the Data were obtained from the Norwegian Cervical Cancer Screening Programme (NCCSP), a division of the Cancer Registry of Norway (CRN). All cytology and pathology departments in Norway must report their results to the CRN, where results are classified according to the Bethesda System  and the In the present analysis, we aimed to assess the performance of HPV DNA testing (using Hybrid Capture II) and HPV mRNA testing (using PreTect HPV-Proofer) in a specific subpopulation of women in Norway. Our study sample consisted of women aged 25–33 years who underwent cervical cancer screening between 2005–2010 and had a recorded screening result of ASC-US or LSIL in the NCCSP. Inclusion in the study sample required the absence of a previous diagnosis of CIN1+ or HSIL. Pragmatically, we identified 4115 women who met these criteria, thus forming our final study sample. Subsequently, we followed their medical records until 31 December 2013. This population was of interest as, unlike women aged 34–69 years and older who have been screened using HPV testing since 2015, women aged 25–33 years underwent cytological screening, and in cases with minor cytological abnormalities, HPV testing was used for triage. At the end of the post-screening follow-up period, we defined women with indication for colposcopy and biopsy or repeat testing (HPV and cytology) without it being performed as women with incomplete follow-up.The screening algorithm that was in effect in Norway during the study period recommended that women with screening results of ASC-US/LSIL attend triage within 6–12 months thereafter; triage consisted of both HPV testing and repeat cytology. Depending on the triage results, the screening algorithm recommended either further follow-up or a return to the 3-year screening interval without further follow-up. To reflect best practices, we categorized the post-triage study sample into three screening algorithm-recommended (SAR) groups: SAR referral for colposcopy and biopsy, SAR referral for repeat testing (HPV and cytology), or SAR return to the 3-year screening interval . Total nucleic acids were isolated from 1 mL of the leftover LBC material preserved in ThinPrep using PreTect X  and subsequently analyzed for mRNA expression according to the manufacturer’s instructions. The HPV DNA status of the specimens was detected by the hc2 HPV kit following the manufacturer’s protocols [Data on the type of HPV test used for triage in different laboratories were taken from the NCCSP database. The type of HPV test was not randomized; laboratories in each geographical region made their own decisions about which commercial HPV test to use. When women with minor cytological abnormalities were triaged using conventional or liquid-based cytology (LBC), extra specimens were collected and placed in preservation and transport media for DNA (digene Specimen Transport Medium ) and mRNrotocols . The HC2The primary study outcomes were HPV positivity rates at triage, SAR referral rates for colposcopy and biopsy or repeat testing after triage, and CIN3+ detection rates from triage to the end of follow-up (31 December 2013). We also explored CIN3+ detection rates at 42  and 78 months  post-screening. We compared these outcomes between women who received HPV DNA and HPV mRNA testing at triage.p-value < 0.05 as the significance level. In order to compare the age distributions among HPV DNA-tested and HPV mRNA-tested women, we divided women into age groups of 25–29 and 30–33 years old.All analyses were conducted in SPSS 29.0. Chi-square tests were performed for categorical variables, with a Of the 4115 included women, 62.1% (2556/4115) received HPV DNA testing and 37.9% (1559/4115) received HPV mRNA testing at triage. The distributions for age, screening result (ASC-US or LSIL), and the most recent cytology result before screening are reported for DNA-tested and mRNA-tested women in p < 0.001) (p < 0.01), a pattern that was also observed for the SAR referral rate for repeat testing after triage . Consequently, according to the screening algorithm, more mRNA-tested than DNA-tested women should have returned to routine screening   . Moreove < 0.01) .p = 0.72) (p = 0.73), as there were few cases of CIN3+ diagnosed at 42 months  . This si2 months .Eight cases of cervical cancer (5 squamous cell carcinomas and 3 adenocarcinomas) had been diagnosed at 78 months post-screening among DNA-tested women, while only two squamous cell carcinomas had been diagnosed among mRNA-tested women. Two of the DNA-tested women and one mRNA-tested woman most likely had false-negative HPV results at triage (data not shown).p < 0.001). The corresponding values at 78 months post-screening were 14.6% (372/2556) and 9.4% (147/1559) (p < 0.001). The proportion of both DNA-tested and mRNA-tested women with incomplete follow-up decreased from 42 to 78 months post-screening; accordingly, the proportion of women that returned to routine screening increased among all SAR groups after triage  and those who underwent HPV mRNA testing (using PreTect HPV-Proofer). We assessed factors that could impact the outcomes of the tests, including the age distribution, the distribution of ASC-US and LSIL results in the screening process, and the most recent cytology results before screening occurred. Our analysis indicated a high level of homogeneity between the two groups, thus reducing the risk of selection bias. The HPV DNA test detects 13 HPV types, whereas the HPV mRNA test only detects 5 types, leading to a higher HPV positivity rate among the DNA-tested women. In accordance with the Norwegian screening algorithm, this higher positivity rate resulted in a higher referral rate for colposcopy/biopsy and repeat testing for the DNA-tested women, leading to a higher overall rate of CIN3+ detection. However, this came at a cost of significantly more health resources, as measured by the number of follow-up visits and colposcopy/biopsy procedures. Our findings reveal that despite the higher resource utilization and CIN3+ detection rate in the DNA-tested women, the HPV DNA test was not more effective in preventing cervical cancer, which was the primary goal of the screening.In our low-risk population of women aged 25–33 years with ASC-US/LSIL, the HPV positivity rate at triage among DNA-tested women was lower (52.8%) than the global rate observed among women of all ages with ASC-US/LSIL who received this triage test (59.4%) . The HPVAccording to Norwegian guidelines, significantly more DNA-tested women in our study sample were to be referred for colposcopy and biopsy and repeat testing. In the Danish study, which included women under 30 years of age with ASC-US/LSIL, biopsy rates in DNA- (67%), 14-type mRNA- (77%), and 5-type mRNA-tested women (58%)  were genAs a consequence of the higher SAR referral rates for colposcopy and biopsy and repeat testing, DNA-tested women had a significantly higher overall CIN3+ rate compared to mRNA-tested women at 42 months post-screening. Our results support the general conclusions that, in the triage of women with minor cytological abnormalities, an HPV mRNA test has lower referral rates than an HPV DNA test ,34,35. HThe number of HPV types included in the test may influence positivity, referral, and CIN3+ detection rates. A previous report showed that only 5–6 HPV types  were present in 85% of invasive cervical cancer cases, while the other eight HPV types included in 13–14 type HPV DNA tests were detected just in 1.5% of invasive cervical cancer cases . Thus, t5 in 2010 to 14.5/105 in 2019) before decreasing slightly to 12.6/105 in 2021 [The Norwegian health care system has allocated limited resources for colposcopy and biopsy examinations ; therefo in 2021 .The higher overall CIN3+ detection rate among our DNA-tested women compared to mRNA-tested women was attributable to significantly higher healthcare utilization in the former group, as measured by the number of SAR referrals for colposcopy and biopsy or repeat testing. Overall, 345 DNA-tested women (13.1%) had been diagnosed with CIN3+ at 42 months post-screening, with 17 more diagnosed at 78 months post-screening. Among mRNA-tested women, 129 (8.3%) had been diagnosed with CIN3+ at 42 months, and 33 more at 78 months, post-screening. This revealed the burden of overmanagement among DNA-tested women, as 216 (345 − 129 = 216) more DNA-tested women with CIN3+ received treatment to avoid 16 (33 − 17 = 16) more women having CIN3+ results within 36 months, i.e., before the next screening round.The number of treatments using the loop electrical excision procedure (LEEP) in Norway has increased from 3743 in 2010 to 7354 in 2021 without any reduction in the number of women with cervical cancer . A low SNot all CIN3 cases, and only a minority of CIN2 cases, will progress to cancer ,43,44. RThe importance of this study lies in the long-term comparison of the predictive and performative value of HPV DNA and HPV mRNA tests in the triage of a large number of 25–33-year-old women with minor cytological abnormalities at screening. We used the NCCSP database, a nationwide, register-based platform embedded within the CRN, which allowed us to identify women with minor cytological abnormalities in Norway. Moreover, we used the 11-digit personal identification number assigned to all persons at the time of birth or immigration in Norway to merge data on cytology results, histological results, cancer cases, etc.Although ours is not a randomized study, we consider the similarity in the characteristics of our DNA- and mRNA-tested women  as an adOur analysis assumed that women, general practitioners, and gynecologists consistently followed the screening algorithm perfectly; however, in real clinical practice, there are probably more referrals for cytology/biopsy and repeat testing. Therefore, we also investigated the CIN3+ detection rate among women who were returned to screening , to reflIn this study, we evaluated the performance of HPV DNA testing (using Hybrid Capture II) and HPV mRNA testing (using PreTect HPV-Proofer) in a population of young women with ASC-US/LSIL. We found that when the HPV DNA test was used for triage, there was a significantly higher rate of referral for colposcopy and biopsy, repeat testing, and CIN3+ detection. However, this did not result in improved cancer prevention compared to the mRNA test. The mRNA test demonstrated similar efficacy in cancer prevention while requiring significantly less healthcare utilization. Based on these findings, healthcare authorities considering a triage test for minor cytological abnormalities among younger women may prefer the mRNA test to minimize overmanagement and reduce unnecessary healthcare utilization.