No premalignant lesion is identifiable on clinical examination, thus no comparative histological features to denote the stages of carcinogenesis for HPV driven HNSCC are identifiable. This is in contrast to HPV-driven cervical carcinoma, making screening a challenge for the head and neck region. However, HPV proffers a favorable prognosis in the head and neck region, with better overall survival rates in contrast to its HPV negative counterparts. This has resulted in extensive research into de-intensifying therapies aiming to minimize the morbidity induced by standard concurrent chemo-radiotherapy without compromising efficacy. Despite the favorable prognosis, cases of recurrence and/or metastasis of HPV positive HNSCC do occur, and are linked with poor outcomes. HPV 16 is the most frequent genotype identified in HNSCC, yet there is limited research to date studying the impact of other HPV genotype with respect to overall survival. A similar situation pertains to genetic aberrations associated in those with HPV positive HNSCC who recur, with only four published studies to date. Somatic mutations in TSC2, BRIP1, NBN, TACC3, NFE2l2, STK11, HRAS, PIK3R1, TP63, and FAT1 have been identified in recurrent HPV positive OPSCC. Finding alternative therapeutic strategies for this young cohort may depend on upfront identification of HPV genotypes and mutations which are linked with worse outcomes, thus ensuring appropriate stratification of treatment regimens.The Human Papilloma Virus (HPV) is an oncogenic virus which is associated with the development of head and neck squamous cell carcinoma (HNSCC), predominantly within the oropharynx. Approximately 25% of oropharyngeal squamous cell carcinoma (OPSCC) cases worldwide are attributable to HPV infection, with an estimated 65% in the United States. Transmission is  Oncogenic Human Papilloma Virus (HPV) is associated with the development of squamous cell carcinoma of the anogenital  and the head and neck region . HPV posEarly studies on HPV associated cancer arose from work on cervical squamous cell carcinomas . HoweverWhen stratified into HPV positive and negative OPSCCs, it has been identified that HPV positive OPSCCs have a distinctive mutational landscape compared with HPV negative counterparts . Thus, aThe purpose of this article is to review HPV carcinogenesis within the head and neck region, focusing on pathological and molecular discrepancies in contrast to the cervical region. In view of the favorable prognosis for HPV positive OPSCC, studies have primarily focused on discerning a tailored therapeutic strategy for HPV positive OPSCC to minimize long-term side-effects derived from intensive chemotherapy, radiation therapy (RT) and/or surgical intervention . There ivia skin-to-skin contact, skin-to-mucosa contact, or mucosa-to-mucosa contact. HPV transmission is thus predominantly via sexual behaviors including oral sex, vaginal sex and oral-anal contact , HPV 33 (4%), HPV 35 (2%), HPV 39 (2%), HPV 45 (6%), HPV 53 (<1%), HPV 56 (<1%) . An inteAs noted earlier, research from the 1990s has provided instrumental knowledge regarding the carcinogenesis of HNSCC . The preIn 2011, two studies utilized whole genome sequencing to analyze the genetic framework of squamous cell carcinomas of the head and neck region . These sIn 2015 the Cancer Genome Atlas (TCGA) published its work underpinning the landscape of genomic alterations associated with HNSCC . In totaThe most frequently encountered mutated genes amongst HPV positive and negative OPSCC were TP53, CDKN2A, FAT1, PIK3CA, Lysine Methyltransferase 2D (KMT2D) and NOTCH1. These play a key role in the RTK/RAS/PI3K pathway. Other frequent mutations of OPSCC include let-7c and FGFR3, which are also linked to the RTK/RAS/PI3K pathway. Reviewing the Phosphoinositide 3 kinase pathway, genome analysis of OPSCC completed by next generation sequencing and copy number alterations displayed that regardless of HPV status and anatomic site, the somatic mutations are missense mutations on chromosome 3q26. 3q26 is the location of the PIK3CA gene . PhosphoThe PI3K/PTEN/AKT/mTOR pathway has been identified as a key component of carcinogenesis in the HPV positive cohort in the literature to date. Given its prevalence in OPSCC, targeted agent against components of the pathway, including mTOR inhibitors, are being trialed in patients with advanced/metastatic disease irrespective of HPV status .HPV positive OPSCC are associated with young white males; a male to female ratio of 3:1; classically non-smokers; and it has been deemed to be a distinct clinical entity from HPV negative counterparts. Although HPV is a risk factor for the development of OPSCC, the overall prognostics are favorable in contrast to those who are negative . From thSubsequent studies have demonstrated increased sensitivity to treatment, with higher response rates to RT, induction chemotherapy and chemo-radiation therapy (CRT) . The two1) Given the characteristic younger age at diagnosis, individuals may have less co-morbidities, with a good baseline performance status prior to starting treatment .via p53 activating pro-apoptotic genes such as BCL2 Associated X, Apoptosis Regulator (Bax) (2) Studies have shown that HPV OPSCC are more radio-sensitive. Mechanisms by which this has been illustrated include the role of TP53 tumor suppressor gene and high oxygenation levels within HPV positive tumors in contrast to HPV negative tumors . HPV negor (Bax) . This isor (Bax) .The following are some of the factors believed to contribute to the differences in outcome:via Immune regulation. Orthotopic models have shown immunocompetent mice infected with HPV positive cancer do better than those who are immunosuppressed (3) Another proposed mechanism is ppressed . HPV OPSppressed . Studiesppressed . The pro4) Finally, differences in the mutational status of HPV positive vs. HPV negative OPSCC are believed to benefit the HPV positive cohort which will be discussed in detail below.A further theory pertains to the knowledge that HPV positive OPSCC are less hypoxic than HPV negative OPSCC. Oxygen is required for free radical formation, and thus the effectiveness of RT. In summation, higher oxygen and oxygen free radicals levels derived from HPV may thus be linked to overall improved radio-sensitivity .3) AnothDespite outcomes varying, at present treatment guidelines for HPV positive disease are as per those for HPV negative . In keepCurative therapies, although effective in their aim to improve PFS and OS, are not without their risks and complications. The toxicity associated with chemotherapeutics and RT can be extremely challenging and debilitating for patients, thus the importance of providing an adequate dosage of a therapeutic agent without inducing localized and/or widespread complications. This is extremely important given the younger cohort of patients with HPV positive disease, who potentially will live longer enduring side effects including xerostomia, dysphagia and the requirement for parenteral feeding tubes . It is kIntensity modulated RT involves administration of a reduced treatment dose, with studies administering fractions ranging from 30 to 60 Gy . PrelimiAs highlighted above, mechanisms to reduce toxicity and subsequently improve quality of life relate to administration of an alternative agent to the mainstay systemic chemotherapeutic agents. Such agents utilized include Cisplatin, or a platinum therapy combined with 5-Fluorouracil. One of the proposed substitutes involves targeting receptors linked to pro-oncogenic activity in conjunction or as an alternative to systemic chemotherapy. Molecular analysis of OPSCC patients identified high levels of epidermal growth factor receptor (EGFR) expression, predominantly in the HPV negative cohort, as illustrated in EGFR targeted therapies for HNSCC have shown to improve OS benefit when administered with RT, with a landmark study showing such in 2000 by Bonner et al. Further de-intensification measures such as replacing CRT with RT alone have been trialed. A retrospective study from 2013 showed a comparable outcome for HPV positive OPSCC treated with RT alone in contrast to those treated with CRT . HoweverAdditional studies are reviewing various modalities to reduce intensity and thus toxicity of therapy without compromising cancer control. An example include a Phase II trial examining administration of induction Cisplatin chemotherapy, followed by Cetuximab with reduced dose RT for HPV-positive resectable stage III/IV OPSCC . The stuThe utilization of ‘organ preservation surgery’ to ensure minimal long term morbidity is an increasing are of interest acknowledged in current guidelines . ResultsDespite the definitive association of HPV positivity with overall better outcomes in OPSCC, there is limited research to date studying the impact of each HPV genotype with respect to OS. This may be as the HPV 16 genotype is the most prevalent genotype identified OPSCC . Lei et Regarding the head and neck region, a multicenter study in Korea published in 2015 revealed that tonsillar carcinomas which were positive for the HPV 18 genotype were associated with shorter disease-free intervals than that of the HPV‐negative and HPV 16 positive groups . Data frTo date there is limited data examining the genetic aberrations associated in those with HPV positive OPSCC who recur . Two key. FGFR3, B2M, TRAF3, SMAD2, ARID, MAPK1 and MAPK2K2 mutations were observed in those who did not recur  are involved in the PI3K/AKT/mTOR pathway. Tuberous Sclerosis Complex subunit 2 (TSC2) is a tumor suppressor gene which produces the protein tuberin. This protein interacts with the hamartin protein produced by Tuberous Sclerosis Complex subunit 1 (TSC1) . HamartiTP63 is a tumor suppressor gene from the same family of a TP53. The TP63 gene is expressed in numerous isoforms, most relevant thus far to tumorigenesis are TAp63 and ΔNp63. TP63 haThe next gene mutation linked with HPV positive OPSCC who recur is the BRIP1. This gene is associated with Fanconi’s anemia, a condition linked to HNSCC. NBN genvia a transforming acidic coiled-coil protein 3 (TACC3)/colonic, hepatic tumor overexpressed gene (ch-TOG)/clathrin complex (A further mutation discovered includes a mutation in transforming acidic coiled coil containing protein 3 (TACC3) gene. This gene produces a non-motor protein that functions to promote microtubule growth from the centrosome, thus providing stability to the mitotic spindle. This occurs  complex .Additional mutation of nuclear factor–erythroid-2–related factor-2 (NFE2L2), a gene that encodes the transcription factor NF-E2-Related Factor 2 (Nrf2). Nrf2 plays a role in the antioxidant response within the cell, with mutation leading to resistance of the cell to anti-oxidant damage and ensuring cell survival . FAT1 geHarvey rat sarcoma viral oncogene homolog is a member of the RAS family of oncogenes frequently mutated in a variety of cancers. The family is subdivided into HRAS, KRAS and NRAS. The RAS family of oncogenes are extremely important in tumorigenesis owing to their key role in signal transduction pathways. The RAS protein alternates between being bound to a GTP or GDP, with GDP being the inactive state. RAS signaling plays a key function for cellular aspects of cellular proliferation, survival, angiogenesis and differentiation .There have been studies conducted to elicit whether particular somatic mutations are identified in this cohort of patients, in an attempt to correlate which patients are associated with a worse overall outcome. NOTCH1 and PTEN mutations were linked with a reduced recurrence free survival in patients with HPV positive OPSCC , with PIThere a two principal parting points regarding genetic aberrations in OPSCC tumors associated with disease progression and/or recurrence. Firstly, although hypothesized that the recurrence group have genetic mutations in line with the HPV negative cohort, Harbison et al. concluded that the HPV related OPSCC that did or did not recur had more genomic features in common with each other than with HPV-unrelated tumors . SecondlIn patients with recurrent and/or metastatic disease, the mainstay of treatment is dependent on numerous factors including site of recurrence, resectability and prior treatment . TreatmeCancer development results in cancer cells being capable of evading immune response to ensure their survival. Non-cancer cells possess immune-checkpoint proteins, which act as a protective mechanism, preventing an autoimmune attack on the host. In cancer evolution, neoplastic cells possess such checkpoint proteins, and thus evade the host immune system . TargetiLyford-Park et al., in 2013 published a central paper demonstrating the role of PD1/PDL1 in providing an environment for HPV infection and persistence, with provision to eliminate the immune regulation and resultant tumorigenesis . StudiesNivolumab, an IgG4 anti–PD1 monoclonal antibody, has been trialed for recurrent HNSCC, irrespective of HPV status, and has shown improved OS in contrast to those on single agent Docetaxel, Methotrexate or Cetuximab . AlthougA further phase II trial, HAWK, examined the utilization of Durvalumab monotherapy vs. systemic chemotherapy in patients with recurrent or metastatic HNSCC that had high PDL1 expression in tumor cells. When stratified based on site and HPV status, 30% of patients with HPV-positive OPSCC had a response in contrast to 10.8% in the HPV negative cohort .via hematological confirmation by circulating free DNA (cfDNA) detection in plasma (One area that has been studied in recent times involves the prospect of detecting HPV DNA in the oral cavity by oral rinses or swabs, or n plasma . Such meThe utilization of viral DNA detection as a surrogate marker for treatment response within the head and neck region is not a new entity. The Epstein-Barr Virus is a known oncogenic virus linked to the development of nasopharyngeal carcinoma . In 1998A systematic review of eight studies found the sensitivity and specificity for HPV detection based on oral rinse or an oral swab ranged from 72 to 92% respectively . Conversvia the methods outlined is the potential to assess if viral clearance has been achieved post cancer treatment. A study by Fahkry et al. published in 2019 reviewed HPV status from oral rinses post treatment regimens for patients with oral and oropharyngeal cancers and highlighted the percentage of patients who never achieve full clearance of their HPV (via various therapeutic modalities, HPV clearance is not always achieved.An interesting perspective from HPV detection heir HPV . For indheir HPV . Thus, dTranslating such findings into the clinical domain, Fahkry et al. showed that patients with cfHPVDNA post treatment have a 2-years incidence of recurrence without detectable tumor HPV DNA of 45.3%, in contrast to 12.2%. This was linked to an increased risk of local and regional recurrence . Ahn et In relation to the particular genotype strains detected, certain studies reviewed only HPV 16 DNA post-treatment, whereas Ahn et al. reviewed numerous HR and LR HPV subtypes . As expeIn summary, HPV detection by routine procedures such as oral rinses or blood analysis in patients who have undergone treatment may in the future be utilized as a screening mechanism to stratify patients into those who are higher risk of recurrence based on their inability to clear the oncogenic virus.HPV driven cancers within the oropharynx have marked pathogenic differences in contrast to HPV driven cervical cancers. HPV positive status proffers a favorable prognosis for HPV positive OPSCC in contrast to its HPV negative counterparts. However, cases of HPV positive OPSCC recurrence do occur, and are linked with poor poorer overall outcomes. There is limited research to date examining the genotypic strains and the mutational landscape of HPV positive OPSCC patients who experience local and/or distant recurrences. With ongoing research into de-intensifying therapeutic regimens for HPV positive OPSCC, the identification of those individuals likely to progress or re-present with recurrence or metastasis is paramount. Concentrating on genotypic and/or mutational patterns in the recurrence group, as well as identification of individuals with residual virus within the oral cavity post cancer treatment, may result in improved stratification of treatment for patients with HPV positive OPSCC.


Koreans and Korean Americans (KAs) have limited HPV knowledge and awareness. KAs share a culture with Koreans, and this culture has affected their behavior around HPV. This systematic review aimed to synthesize the factors associated with HPV vaccination among Koreans and KAs. The literature search was done with four databases. The vaccination rate, awareness and knowledge of HPV, and factors associated with vaccination intention were identified. Eighteen articles were selected. Koreans and KAs had low levels of HPV knowledge and awareness. Perceived benefits and seriousness were associated with vaccination intention. Cervical cancer history, beliefs that their daughters need a pap smear test, sexual intercourse experiences, occupation, low education, and income were associated with vaccination intention. This systematic review discovered that HPV vaccination behavior is associated with HPV vaccine awareness, perceived benefits of the vaccine, and the perceived seriousness of HPV infection among Koreans and KAs. Based on the results, we suggest healthcare providers provide a HPV vaccine recommendation by emphasizing the benefits of the vaccination to Koreans and KAs. This study can be the basis for developing interventions to increase HPV vaccination by guiding the target population and variables, as well as the intervention content. Human papillomavirus (HPV) infection is the most prevalent sexually transmitted infection (STI) in the United States (US) . HPV infKorean Americans (KAs) are the fifth-largest Asian American subgroup , and 1,8In general, immigrant populations are affected by the culture of their original country, especially the culture of their mother . To incrA systematic review of the literature was performed, following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines . We inclThe search databases used were PubMed, CINAHL, PsycInfo, and Web of Science, and we used a combination of the keywords HPV or human papillomavirus AND Korean or Korean American or Asian American from the last five years . An author and a certified librarian searched the literature and screened the initial selection for validation of the inclusion criteria.From each study, we extracted the theoretical framework, study design, population and basic demographic information, acculturation, the purpose of the study, outcome, implication, and levels of evidence. Two authors reviewed the titles and abstracts for the screening. Full texts were reviewed by the authors independently for study selection.After the full-text review, 19 studies were found. Most studies surveyed only females. Six targeted females and males, and one targeted only males. The targeted age group was mostly adults, accounting for 16 of the studies. Among these 16 articles, the targeted populations included college students, any parents of adolescents, mothers of adolescent daughters, patients who had cervical conization, women who did not take mammograms and/or pap tests in the past two years, women without cervical cancer, school nursing teachers, and general adults. In the studies not targeting adults, one targeted high school and university students aged 15–27, and the other targeted elementary school students aged 11 and 12.Thirteen studies were based on behavioral theory. The theoretical frameworks used in these were the health belief model (HBM), the information–motivation–behavioral skills model, the behavioral model of health services use, and a self-construal. Three studies combined theories: one used both the HBM and the theory of reasoned action, and two combined the revised network episode model and the theory of planned behavior. Overall, the HBM was the most commonly used; seven studies solely used the HBM, and one used the HBM combined with the theory of reasoned action. The HBM describes health behaviors as being influenced by health belief factors, such as perceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action, and self-efficacy . The behSome studies reported the vaccination rate: roughly 2.4% or 12 out of 495 young men , about 6Most studies measured their knowledge levels. One  measuredQuestions concerning HPV knowledge were about whether HPV infection affects both males and females, the method of transmission of infection, how to prevent HPV, whether HPV can be cured, whether the vaccine is for both males and females, HPV’s relationship with cervical cancer, signs and symptoms of HPV, and ages for the vaccination. The reported knowledge was represented as a percentage of correct answers or the percentage of participants who answered correctly. The range of percentage of correct answers was from 2.2%  to 79.8%As with the knowledge levels, the awareness of HPV was varied. The percentage of HPV awareness ranged from 1.71% to 86.6%. Awareness of the HPV vaccine was higher than the awareness of HPV itself, ranging from 48% to 92.4%. Two studies asked about awareness of a cervical cancer vaccine rather than an HPV vaccine, and the results ranged from 66.3%  to 84.1%Six studies analyzed the factors associated with vaccination intention by using multiple regression . One stuIn terms of the people who were eligible to receive the HPV vaccine, the population was high school students and university students. Age was not a significant factor, but university students had higher vaccination intention than high school students . Sexual Parents, young age, being female, higher educational level, higher income, occupational status, cervical cancer history, belief that their daughter needs a pap smear test, perceived benefits, and perceived seriousness were significantly associated with HPV vaccination intention for their daughters ,32. AddiThis systematic review synthesized the factors associated with HPV vaccination intention in KAs and Koreans. Eighteen articles were utilized for the synthesis, and the findings were as follows.The results showed were variable for HPV awareness. All the studies reported more awareness of the HPV vaccine than awareness of HPV itself. Awareness of the HPV vaccine was especially increased when the researchers asked about awareness of a cervical cancer vaccine rather than HPV or an HPV vaccine. This result is consistent with the results of an earlier study; 96.5% of women know about a cervical cancer vaccine, while only 52% of women had heard about HPV . A possiIn terms of HPV-related knowledge, all the studies reported limited HPV knowledge. The highest correct answer rate was 66.2% from school nursing teachers. It was not possible to compare with other studies that targeted other ethnicities, because each study used different measurements. However, the literature about other Asian Americans has also reported low levels of HPV-related awareness and knowledge in this population ,38 and tThe significant factors associated with HPV vaccination intention were young age, being female, higher education, higher income, occupational status, sexual intercourse experience, cervical cancer experiences, beliefs that their daughters need a pap smear, awareness of the HPV vaccine, HPV knowledge, recommendations from parents, perceived benefits, and perceived seriousness. The significance of young age, being female, higher education, higher income, and occupational status were the same as previous studies of Americans and immigrants in America . It is aAmong the HPV-related factors, the synthesis of the results in terms of HPV knowledge was inconsistent. Only one study found a significant relationship between HPV knowledge and HPV vaccination behavior, while two studies found a nonsignificant relationship. HPV knowledge was measured using different measurement scales in each study. The inconsistent results might be a result of using different measurement scales. Using different measurement scales could lead studies to be noncomparable. There are some studies that have developed measurement scales of HPV knowledge using a standardized development process ,40. SincHPV vaccine awareness was a significant factor for HPV vaccination intention, while the awareness of HPV itself was not a significant factor for HPV vaccination intention. A possible explanation may be that the HPV vaccine was advertised as a cervical cancer vaccine at the time it was first licensed . AwareneAmong the behavior-related factors, perceived benefits and perceived seriousness were significantly associated with HPV vaccination intention. In particular, all the studies that measured perceived benefits reported the significance of this variable’s relationship with HPV vaccine intention. Three studies targeted Koreans, analyzing high school or university students, school teachers, or mothers. This might imply Koreans will receive the HPV vaccine when they receive information about the benefits of the HPV vaccine. Additionally, the HPV vaccine was not yet free at the time the surveys were conducted, so the results of this study could emphasize the importance of providing information on the vaccine’s benefits. This could have an even greater relationship with vaccination now that the HPV vaccine is freely available to adolescent Korean women. One study conducted after the free national HPV vaccination program  reportedWhile reviewing the results of the included studies, we found that some studies reported a discrepancy in results between the manuscript and tables. Where there was a discrepancy between the article itself and its tables, this study followed the results from the manuscript. Moreover, one article included incorrect information about their measurements. Future studies have to consider the quality of manuscripts by writing and reviewing articles more carefully.In addition, studies about KAs did not clearly state their definition of KAs. Most studies indicated their inclusion criteria as being a self-identified KA or including both first- and second-generation KAs. There was no limitation for the years of living in the US or their age when they arrived in the US as inclusion criteria. Immigrant generation can be classified in various methodologies such as age at arrival in the US, nativity, or parents’ ethnicity . MoreoveHPV vaccination is the primary preventive measure against HPV infection and its development into cancers. However, racial disparities among HPV vaccination have been reported . While tThe major limitation of this study is the analysis of KAs and Koreans as a whole. The reason we did not exclude either group is that it is likely first-generation immigrants are affected by their original culture and have the unique health-related beliefs of that culture . FurtherAnother limitation in the studies about Koreans was that all the studies except one were conducted before the HPV vaccine became free to female adolescents as one of the national vaccination programs in 2016. The vaccine for male adolescents in Korea is not complementary. Therefore, future studies are recommended to examine the attitudes regarding the HPV vaccine and HPV knowledge and see how these have been changed in comparison to the previous studies, as well as how they may differ by sex.This systematic review identified the behavioral aspects for HPV vaccination and what factors are associated with HPV vaccination intention in KAs and Koreans. In general, KAs and Koreans have low levels of knowledge and awareness about HPV. While HPV knowledge and awareness had mixed significance across the studies, perceived benefits had consistent results that were significant to HPV vaccination intention. As well as perceived benefits, some demographic factors such as cervical cancer experience or sexual intercourse experience were significantly associated with HPV vaccination intention. The awareness of HPV itself was not a significant factor; on the other hand, awareness of the HPV vaccination was a significant factor.


People with Fanconi anemia (FA) are genetically susceptible to gynecological cancers and cancers of the head and neck. There are known associations between oral infection with human papillomavirus (HPV) and development of head and neck cancers. This study sought to measure how common oral HPV positivity is in a large sample of people with FA followed over 8 years, while also evaluating serum titers to ascertain natural exposure to HPV, and how well people with FA who were vaccinated responded to HPV vaccination. We found that oral HPV positivity is significantly higher in individuals with FA compared to family and unrelated controls, but that response to HPV vaccination between FA and controls is similar. Common risk factors associated with HPV in the general population did not predict oral DNA positivity in FA, unlike unrelated controls. Future mechanistic and vaccinations studies are needed to understand this phenomenon.p = 0.07), siblings , and unrelated controls . A FA diagnosis increased HPV positivity 4.84-fold (95% CI: 1.96–11.93) in adjusted models compared to unrelated controls. Common risk factors associated with HPV in the general population did not predict oral positivity in FA, unlike unrelated controls. Seropositivity and anti-HPV titers did not significantly differ in FA versus unrelated controls regardless of HPV vaccination status. We conclude that individuals with FA are uniquely susceptible to oral HPV independent of conventional risk factors.High-risk human papillomavirus (HPV) is prevalent and known to cause 5% of all cancers worldwide. The rare, cancer prone Fanconi anemia (FA) population is characterized by a predisposition to both head and neck squamous cell carcinomas and gynecological cancers, but the role of HPV in these cancers remains unclear. Prompted by a patient-family advocacy organization, oral HPV and HPV serological studies were simultaneously undertaken. Oral DNA samples from 201 individuals with FA, 303 unaffected family members, and 107 unrelated controls were tested for 37 HPV types. Serum samples from 115 individuals with FA and 55 unrelated controls were tested for antibodies against 9 HPV types. Oral HPV prevalence was higher for individuals with FA (20%) versus their parents (13%;  High-risk human papillomavirus (HPV) is prevalent and is known to cause 5% of all cancers worldwide ,2. AccorSome populations are at greater risk of HPV-associated cancer than others . NotablyWe have previously reported that loss of function of the FA-related DNA repair pathway in high-risk HPV positive epidermis stimulates production of the E7 oncogene, increases viral replicative capacity, and expands the basal stem and progenitor cell compartment ,21. Thesp = 0.003). Interestingly, even those with FA who had not been sexually active tested oral HPV DNA positive more often than siblings, although the difference was not statistically significant (8.7% vs. 2.9% p = 0.44) [Our earlier epidemiologic work in this area was first prompted by the Fanconi Anemia Research Fund (FARF), a patient-family advocacy organization with a governing board of patients, family members, clinicians and scientists. In response to their call, we sought to understand the nature of HPV susceptibility in FA by characterizing the epidemiology of oral HPV infections and host immunological responses in individuals with FA and family controls, considering that all parents of those with FA, and a portion of siblings are heterozygotes with mutations in one of the 22 Fanconi genes. We found that participants with FA are particularly susceptible to oral HPV infection with 11.1% of FA individuals testing oral HPV DNA positive to at least one of 37 HPV types by Roche Linear Array, significantly more than first degree family members  . IncreasOver a period of eight years, we consented one of the largest cohorts of families to date, to test oral HPV DNA prevalence in individuals with FA compared to siblings, parents and unrelated controls, as well as take an early look at the incidence and persistence of oral HPV positivity. This allowed for close examination of risk factors typically associated with HPV infection in the general population including early life exposures, hygiene practices, use and exposure to tobacco products, alcohol consumption and sexual practices. Further, antibody responses to HPV are generally type-specific making evaluation of antibodies in blood serum one of the best approaches to simultaneously monitor response to natural infection and gauge the protective effect of HPV vaccines . ConsequAt the invitation of the Fanconi Anemia Research Fund (FARF), we invited attendees of the FARF’s Adult and Family Meetings to participate. Additionally, patients visiting the Cincinnati Children’s FA Comprehensive Care Center were also invited. Participants were from 15 countries and 34 states across the USA. Individuals of all ages were eligible to participate if they reported a diagnosis of FA or were a parent or sibling of an individual with FA and were willing to complete study-related surveys and provide oral rinse samples. Healthy controls were recruited either at Cincinnati Children’s Hospital Medical Center or at a FARF sponsored event and were eligible if they did not report a diagnosis of FA, did not have a family member with FA, and had no other known bone marrow or immune abnormalities. A total of 212 subjects with FA were enrolled in the study, as well as 331 family members , and 111 unrelated controls. Of these, 201 individuals with FA, 88 siblings, 215 parents and 107 unrelated controls have at least one sample for HPV DNA testing, while 128 participants with FA and 151 family members have two or more samples tested over an 8-year period. In addition, 115 individuals with FA and 55 unrelated controls had at least one blood sample for antibody testing. The majority of control samples were collected at a single time point at various times throughout the life of the study. The Institutional Review Board at Cincinnati Children’s Hospital Medical Center approved this study.The surveys were typically self-administered either via paper (years 1 and 2 only) or online (Research electronic data capture (REDCap) software) . ParticiOral rinse and gargle samples were collected in 15 mL normal sterile saline and processed as previously described . BrieflyBlood samples were collected using appropriate precautions by trained personnel. Fifty percent of the samples from participants with FA were collected at Cincinnati Children’s Hospital, with the remainder collected at FARF events as previously described . Eighty-Statistical analyses were performed using Statistical Analysis Software (SAS), version 9.4 . Prior to analyses, data qualities were examined. Demographic variables and potential risk factors of HPV were summarized as frequencies (proportions) and compared between FA, siblings, parents and unrelated controls using chi-square or Fisher’s exact tests. These factors were then compared between oral HPV negative and positive individuals with FA, and in unrelated controls. The effects of HPV risk factors on HPV positivity were also tested using multi-variable logistic regression.p-value ≤ 0.05.For our analysis of oral HPV prevalence, any sample testing positive with any HPV type was considered oral HPV positive. For our analysis of risk, high-risk types 16, 18, 35, 45, 51, 52, 56, 58, 59, 66, 68, were included in our high-risk group; all others were considered low risk. For the analysis, if a sample tested positive for a high-risk type (regardless of having low risk types), they were in the high-risk category; the low-risk category only included participants with low risk types. A similar analysis was performed focusing only on those types targeted in the 4-valent Gardasil vaccine. Multiple HPV positive results over events includes participants testing positive for multiple HPV types in a single sample. Cumulative incidence was defined as the proportion of new HPV positive cases during the entire study over the total subjects at risk (subjects that tested negative when entering the study). Participants with no oral sample (37), those with samples inadequate for evaluation  and those positive for HPV in the first/baseline test (48) were removed from our analysis of incidence. Of the remaining 558 subjects, 114 individuals with FA, 105 parents, 35 siblings, and 6 unrelated controls had ≥2 oral test results. Those with more than one sample had up to 6 follow-up time points with median time between sampling events being 1.13 years. Because only 6 unrelated controls had testing at more than one-time point, they were not included in our analysis of incidence. Finally, consecutive oral HPV positivity was defined as participants testing positive for any HPV type in subsequent samples (having the same HPV type across samples). Serotiters were analyzed using Wilcoxon rank sum tests. Titers under the detection limits were imputed as half of the detection limits. Geometric mean titers (GMT) were calculated and compared between groups using a student t-test assuming log normal distribution. Statistical significance was indicated by a p < 0.001) (p < 0.001). Overall, the parents of those with FA were highly educated; only 16% and 23% of fathers and mothers, respectively, having less than a college education  . Among iducation . More thp < 0.001); it was also higher than their first-degree family members, parents  and siblings  respectively (Fisher’s exact test) (p = 0.90) or within the FA group (p = 0.77). Indeed, among 18 oral DNA positive individuals with FA who had not been vaccinated for HPV at the time of sampling, 14 (78%) were oral HPV positive for a type targeted in the 4-valent vaccine. Among the 23 (56%) oral positive vaccinated individuals with FA, 19 (83%) tested oral HPV DNA positive for a type targeted in the 4-valent vaccine. There were only three vaccinated siblings and four unvaccinated siblings testing positive to a 4-valent type. No parents or unrelated controls who tested oral positive had been vaccinated at the time of sampling.Oral HPV positivity was evaluated among all study participants with oral samples regardless of their HPV vaccination status. Among individuals with FA, 41 (20%) tested oral positive for at least one type of HPV at one or more time points. The oral HPV prevalence in FA was significantly higher than in unrelated controls (6%), (ct test) . Considect test) . The preThe most common HPV type detected across groups was HPV16, consistent with published studies ,29. Indep = 0.025), parents , and unrelated controls . In participants less than 12 years old, oral HPV positivity did not significantly differ in those with FA (15%) compared to siblings  or unrelated controls . Notably, in oral HPV positive individuals with FA, 13 (32%) had multiple HPV types in the same sample, including two individuals younger than 12 years old. None of the oral HPV positive siblings, 3 parents (10%), and only a single unrelated control (17%) had multiple oral HPV types detected  than siblings , but did not reach statistical significance when compared with siblings (p = 0.20)  . Given tLooking over time, only five individuals with FA tested oral HPV positive in consecutive samplings compared to four parents . Type-spp < 0.001) (p = 0.03 and 10% p < 0.001 respectively) and at lower birth weight (35% vs. 4% and 10% p < 0.001 respectively) (We next assessed exposure and behavior-related factors that could influence positivity between case and control groups . Deliver< 0.001) . In compctively) .p < 0.001). This difference was not influenced by a history of HSCT.With regard to oral hygiene, the siblings’ brushing and flossing habits were more similar to the FA group; but significant differences were observed between FA and parents and unrelated controls . Not surp < 0.001, respectively). However, exposure to second-hand smoke did not significantly differ between groups   .p < 0.003 and p = 0.05). Indeed, 31% of individuals with FA reported ever having had a STD vs. 12% of siblings, 14% of parents and 13% of controls  , we next examined differences between groups in reported STDs . Those w= 0.012) . Sexual Among unrelated controls, only 6 tested positive for oral HPV; 2 males and 4 females. None of the risk factors examined were significantly associated with oral HPV positivity. However, with the exception of sex , the associated trends in our unrelated controls were similar to those previously reported , with alp = 0.08), but not head and neck and oral cancers (p = 0.36). At least 46 participants (21.7%) with FA have died  since the cohort was first initiated.Among subjects with FA, only sex and race were significantly associated with oral HPV positivity . Males hConsidering only those with FA and unrelated controls, multivariable models including FA status, gender and race indicated that having FA was the greatest risk factor  for being oral HPV positive.p = 0.05) . Among unvaccinated individuals, differences in seropositivity between individuals with FA and unrelated controls were not statistically significant . Among t = 0.05) . Because = 0.05) . Althoug = 0.05) .p = 0.27). Non-transplanted participants with FA had higher GMTs than the transplanted FA group regardless of vaccination status consistent with our previous findings [p = 0.029, 0.05 and 0.015). Notably, HPV31 and 58 are not represented in the original 4-valent Gardasil vaccine, but have been added to the 9-valent vaccine.We next compared anti-HPV titers in non-transplanted individuals with FA to those transplanted, stratified by vaccination history. Only post-transplant vaccinations were included in the analysis. There were no statistically significant differences between the median time of sampling for this study and time post HPV vaccination for non-transplanted  and transplanted groups   who all are obligate carriers of a single mutation in one of the FA genes  that may play a role in viral susceptibility ,34. In tRegardless of vaccination status, we also found that individuals with FA had a higher oral HPV prevalence. Indeed, among those testing positive, 83% were still oral DNA positive for a type targeted in the 4-plex vaccine post vaccination, suggesting that either vaccination does not completely protect individuals from oral HPV types included in the vaccine or exposure to and/or infection with HPV occurs before vaccination. As post-vaccination HPV seropositivity for the four HPV types targeted in the 4-valent Gardasil vaccine was not significantly different between FA and unrelated controls , exposurTo gain a better understanding of natural exposure to HPV, we examined the serology of unvaccinated children ≤13 years old with FA. Of the 46 children, 65% were seropositive to at least one of the nine HPV types targeted in the 9-valent Gardasil vaccine. Of the 19 total unvaccinated, unrelated controls ≤13 years old (58%) were also seropositive . These dConsidering this possibility, we observed that individuals with FA were also more likely to report being born prematurely and at lower birth weights . Of thosTo further complicate matters, individuals with FA are likely to require a hematopoietic stem cell transplant in childhood (average age 11 years old) . StudiesWhile immune factors are likely to be a critical factor, they alone do not explain the observed increase in HPV prevalence in FA. Indeed, tissue-specific mechanisms might also contribute. We have previously shown that increased accumulation of the HPV E7 protein, and/or viral replication in HPV16 and HPV31 positive 3D epidermal organoid models without intact FA DNA repair machinery results in greater HPV proliferation and/or viral load . Other sp = 0.08). Future longitudinal studies are needed to further assess the risk of cancer in FA compared to family and/or unrelated controls.As with any observational study, there were a number of limitations. First, the age ranges of parent and sibling groups differed from those with FA. Not surprising, parents were much older and more likely to have had sex, and siblings were on average younger and less likely to have had sex. For this reason, we added an unrelated control group. Further, persistence of oral HPV did not significantly differ between individuals with FA and either the family or unrelated control groups . This isTaken together, while the underlying molecular and cellular pathways have yet to be fully elucidated, we conclude that individuals with FA represent a population uniquely susceptible to HPV and perhaps other DNA tumor viruses, and that relevant mechanisms are independent of conventional risk factors. As individuals with FA are at considerably greater risk of HNSCC, identifying factors that augment risk of oral HPV DNA positivity could ultimately lead to viable preventive measures or interventions for those with FA and others with genome instability syndromes. Further mechanistic, observational and vaccination studies are needed to explore these possibilities.


Background and Objectives: Cervical cancer (CC) is the eighth most common cancer among Saudi women of all ages. With limited national data, we aimed to evaluate the public awareness of cervical cancer, CC risk factors, HPV infection, and HPV vaccines in different regions of Saudi Arabia. Materials and Methods: This was a survey-based cross-sectional study that encompassed 564 Saudi women over a period of a month. A self-administrated questionnaire was distributed through different social media platforms. Results: The collected data included sociodemographic variables and questions assessing awareness of CC, and the attitudes toward CC screening and human papillomavirus (HPV) vaccination. Most respondents were aware of CC (84.0%), although their primary source of information was the internet. However, only 45 females (8.0%) had a history of cervical screening. Furthermore, most females did not know that HPV was transmitted sexually (78.9%), or that it caused genital warts (81.7%) and CC (81.9%). Regarding the HPV vaccine, 100 females (17.7%) had heard about it, but only 11 (2.0%) took the vaccine, although more than half of the respondents (54.1%) were willing to take the vaccine after being informed about it. Conclusions: We noticed a remarkable lack of awareness among the respondents regarding HPV’s clinical implications; and the HPV vaccine, and its importance and availability. The main source of information for most of the Saudi women in this study was the internet, which may be an unreliable source, or provide misleading information that may delay screening or discourage vaccination. Thus, organized campaigns by the Ministry of Health or other health-advocating agencies, in addition to screening and vaccination programs, are strongly encouraged. Cervical cancer (CC) is one of the most common causes of gynecological cancer-related morbidity and mortality . It is tWomen in developing countries, especially those with a low socioeconomic status or educational level, are at higher risk of certain health issues, including acquired immunodeficiency syndrome, maternal mortality, and cervical cancer . AlthougHPV is considered the most important etiological and prognostic factor of CC; therefore, the implementation of early CC screening and HPV vaccination are highly efficacious in decreasing the prevalence and incidence of CC . RegrettThis is an observational, cross-sectional, survey-based study that was conducted in Saudi Arabia using an online self-administered questionnaire that was electronically distributed through social media from June to July 2020. All study procedures were performed in accordance with the ethical standards of the Declaration of Helsinki and the universal International Conference on Harmonization-Good Clinical Practice Guidelines. The Institutional Review Board, College of Medicine, King Saud University Hospital approved the study protocol (no. E-20-5069 on 8th September 2020). Written informed consent was obtained from all the participants.2 pq / e 2), in which: Z is the level of confidence ; p is the estimated proportion of the population ; q is 1 − p (1 − 0.5 = 0.5); and e is the margin of error, which is 5% (=0.05).The study included all Saudi women (from different regions of Saudi Arabia) who agreed to participate. Males and non-Saudi women were not included in this study. The sample size was calculated to assess the awareness level of cervical cancer using the Cochran’s Formula n0 = (Z The study’s sample size was expanded to help decrease the risk of sampling errors. The current study included a simple random sample of 564 out of 1692 (33.3%) individuals who fulfilled the study requirements and completed the questionnaire. A pilot study was carried out on seven women to demonstrate the feasibility of the questionnaire, and changes were made according to the results. The final questionnaire contained three main sections with close-ended items. The first part included personal data: age, place of residence, educational level, smoking status, marital status, occupation, monthly income, number of children, and type of contraception used. The second part assessed the participants’ awareness level by asking them whether they had heard the following information before: the presence of cervical cancer, that cervical cancer is treatable if caught early, that cervical cancer is caused by HPV, that HPV can be transmitted sexually, that HPV can cause genital warts, and the existence of an HPV vaccine. In the third part, participants were asked about their source of information. In addition, they were asked whether they had ever had the following: cervical cancer, a cervical cancer screening test, and the HPV vaccine. Finally, the respondents were asked about their willingness to take the HPV vaccine if offered, and to advise their friends and family to take the HPV vaccine. The data were extracted and analyzed using SPSS statics for windows software version 25 . Categorical variables were expressed as percentages. In order to assess the level of awareness, a score out of 6 was given to each participant based on their answers to the second part of the questionnaire (high scores represent a high level of awareness); after that, multivariable ordinal logistic regression was used for the final score to assess the factors associated with the level of awareness regarding cervical cancer. This study was approved by the King Saud University’s Institutional Review Board. In addition, the participants filled out the online surveys anonymously and voluntarily for the purpose of this study. Furthermore, each participant had a unique IP address to prevent duplications, and the participants were not offered rewards or incentives for participation.Among a total of 718 surveys, 564 were completed and unduplicated. The sociodemographic data of the participants are shown in The data collected on the level of awareness are shown in Most of the participants stated that their primary source of information about cervical cancer was the internet (31.4%). Despite these results, only 45 females (8.0%) had a history of cervical screening. Among the 564 participants, only 2 (0.4%) were diagnosed with cervical cancer. By admission, only 11 (2.0%) had taken the vaccine. However, more than half of the respondents (54.1%) were willing to take the vaccine if offered, and most of them (72.9%) were inclined to advise friends and family to take it, as shown in p-value < 0.007, 95% CI = 0.071−0.441); high educational level ; and marital status, in which widows were more aware .The associations between the sociodemographic variables and the level of awareness are shown in Despite the fact that most of the participants were educated (95% with a high school education or higher) and had heard about CC 84%), most of them had never had a cervical screening test. These results are in accordance with similar studies conducted in different cities, including Qassim, Jeddah, Riyadh, Hail, and Tabuk ,17,18,194%, most Our data showed that 8.6% of the respondents had had relations outside of their current marriage, and we know that a portion of these results refer to previous marriages (6%) or polygamy (6.2%). Unfortunately, most females did not know that HPV is transmitted sexually (78.9%), or that it causes genital warts (81.7%) and CC (81.9%). These results are similar to those published in previous regional studies, which showed that only 14.4% of the general population were aware of HPV as a cause of CC [The HPV vaccine has been available since 2006, yet 83.2% of women have never heard about it, and among those who knew about the vaccine, only 11 have taken it . As per previously published data ,18, moreAlthough the literature revealed few studies, conducted in individual hospitals or limited to a certain area of the Kingdom, we found that our cross-sectional data, which included females from all geographical regions, are compatible with the previously published regional studies, and highlight shared aspects ,19,20. TData from other Arab countries are not far from what we found according to the systematic review done by Rihab Gamaoun. She found that the level of awareness of HPV was low to moderate in all Arab countries. The main obstacles against implementing national programs were mainly due to cultural and religious sensitivities, in addition to the fear of the cost of such vaccinations. Interestingly, acceptability and willingness to get screened or to receive the vaccine was high if the service was free of cost. It was shown in this review that, up to date, the only Arab country which succeeded in implementing a national vaccination program was the United Arab Emirates: their experience showed an absorption rate of 77% in the 1st year, which declined later to 59%. It was blamed on the insufficient media support, and the controversy regarding the side effects of the vaccine .We believe that the current cultural changes in Saudi Arabia which happened over the last twenty years, given that the majority (around 65%) are relatively young and educated people, have helped women to become more confident in discussing such important health issues, and this should lead to more realistic results and a better understanding of HPV status. It is known to gynecologic oncologists that cervical cancer patients are relatively young, and present with advanced stages in most cases . It is rA very positive report was just published from the United Kingdom showing important and encouraging results of 87% eradication of cervical cancer due to successful implementation of an HPV vaccination program. Such a report should encourage the Saudi government to talk with confidence about its initiative .Saudi women, like all Arab women, are showing very low awareness about CC prevention, screening, its relation to HPV infection, and the value of anti-HPV vaccination. The government has recently developed a very positive initiative. Scaling-up of this national strategy by the Ministry of Health or other health advocacy agencies is becoming indispensable to overtake the Saudi structural and societal gaps. Countries with no CC screening programs nor HPV vaccination programs should not delay that further, as the value is unquestionable. Our recommendations should be applicable to all Arab countries or any country which is similar in conservative culture and religious beliefs.


Among community-dwelling adults ages 65 and older, approximately 11% have experienced elder mistreatment (EM), including physical, emotional or sexual abuse, neglect, or financial exploitation. EM research typically focuses on this age group; however, Veterans receiving Veterans Health Administration (VHA) care have increased earlier morbidity, which may accelerate the impacts of EM. Using a cohort of all VHA Veterans 50 years and older with VHA use in 2018-2020, we examined correlates of EM. ICD-10 codes from clinical encounters identified Veterans with indications of EM . A 10% sample of Veterans without indications of EM was selected for comparison . Logistic regression compared EM+ Veterans to the comparison sample and assessed overall demographic and clinical differences as well as differences by age, i.e. 50-64 versus 65 and older. Overall, female gender , non-white race/ethnicity , dementia , PTSD , anxiety , military service connected disability status , and higher Elixhauser medical morbidity scores  were associated with EM. Prior year ER visits , inpatient stays , and mental health visits  also predicted EM+ status. Forty-six percent of VHA Veterans with indicators of EM were aged 50-64. For these Veterans, female gender, PTSD, service connection, and mental health visits were associated with increased risk of EM compared to Veterans 65+. Findings highlight clinical correlates of EMs among Veterans in VHA care. Increased awareness of EM risk factors is warranted and may inform VHA efforts for EM prevention, detection and intervention.


Head and neck cancers can be divided in two major groups according to their risk factors, being high-risk human papillomavirus related (HPV-positive) and alcohol and tobacco related (HPV-negative) head and neck cancers. The majority of the locally advanced patients are treated with radiotherapy. However, up to 50% of these patients show local recurrences. The majority of these recurrences are linked to resistance to radiotherapy treatment. It is known that the response to DNA damage, also a process called the DNA damage response, is an important factor that determines the effectivity of radiotherapy. Here, we assessed the role of the DNA damage response in the resistance process to radiotherapy of head and neck cancers, by generating head and neck cancer cells resistant to radiotherapy. We show that the DNA damage response is differentially involved in the resistance process of HPV-positive and HPV-negative head and neck cancer cells. More specifically, HPV-positive radiotherapy-resistant cells showed increased ability to repair the DNA damage induced by radiotherapy. HPV-negative radiotherapy-resistant cells showed increased capacity to replicate after radiotherapy treatment. Despite this difference, inhibition of the DNA damage response enhanced the effect of radiotherapy in both groups. Radioresistance is a major cause of recurrences and radiotherapy (RT) failure in head and neck squamous cell carcinoma (HNSCC). DNA damage response (DDR) is known to be important for RT response, but its role in radioresistance is not fully understood. Here, we assessed the role of DDR in the radioresistance process of HNSCC by generating radioresistant clones from both HPV-positive SCC154 and HPV-negative SCC61 cells. We show that fractionated RT decreased RT response of HPV-positive and HPV-negative radioresistant clones in vitro and in vivo. Moreover, HPV-positive and HPV-negative radioresistant clones were characterized by differential DDR response. HPV-positive radioresistant clones showed less residual double-strand break damage and increased G2/M arrest recovery after RT, indicating an acquisition of increased DDR kinetics. In contrast, HPV-negative radioresistant clones showed less micronucleated cells after RT and increased survival upon checkpoint inhibition, indicating an increased replicative capacity. Inhibiting key factors of DDR in combination with RT rescued the radioresistant phenotype of both HPV-positive and HPV-negative radioresistant clones. Altogether, our results not only highlight the importance of DDR response in the radioresistance process of HPV-positive and HPV-negative HNSCC, but also provide possibilities for new therapies for HNSCC patients in recurrent settings. With approximately 600,000 new cases yearly, head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide . The twoUp to 26% and 10% of patients with locally advanced HPV-negative and HPV-positive HNSCC, respectively, are affected with locoregional recurrences after treatment ,5. RadioHere, we investigate the importance of DDR in a radioresistant setting for both HPV-positive and HPV-negative HNSCC. Radioresistant cells were generated through fractionated irradiation to mimic patient relapse in vitro, since matched primary and recurrent tumor samples are unavailable to study the mechanisms underlying radioresistance. Next, we elucidated the role of DDR by targeted inhibition in both HPV-positive and HPV-negative radioresistant cells. All used cell lines were authenticated and cultured as previously described in . HPV-posInhibitors ABT-888, AZD6738, AZD7762, LY2603618 and NU7441 were purchased from Selleck Chemicals . Inhibitors were dissolved in dimethylsulfoxide .The in vitro irradiation experiments were performed with Baltograph  and Small Animal Radiation Research Platform . The in vivo irradiation experiments were performed with Small Animal Radiation Research Platform .(−αD−βD²)).Established HNSCC cell lines were treated with a fractionated RT treatment schedule of 2 Gy per fraction given two times a week, up to a total dose of 60 Gy. Between fractions and after completion of the treatment schedule, recovery periods of 2–3 days and 10–12 days were implemented, respectively. In addition, cells were subcultured independent of fractionated irradiation, preventing the risk of radiosensitive cells overgrowing radioresistant cells as described in . Clonal Cells were seeded and after attachment overnight, treated with DMSO (0 µM) or indicated concentration of drug 2 h before exposure to increasing RT doses of 0, 2, 4 and 6 Gy. Drug concentrations were chosen based on previous studies and with no/limited baseline toxicities . Medium The CRISPR-Cas9 screen was performed as previously reported in . More spDT = doubling time, T = the incubation time in any unit, Xe = the cell number at the end of the incubation time, Xb = the cell number at the beginning of the incubation time). Cells were seeded in 96-well plates and fixed from day 1 until day 7 via SRB assay. The population doubling time was determined in the exponential growth phase with the following formula  containing 100 µg/mL RNase A . For Bromodeoxyuridine (BrdU) analysis, cells were incubated with 75 µM 5’-Bromo-2’-Deoxyuridine  for 2 h and further processed via BD Biosciences protocol (Anti-BrdU B44). Cell cycle distribution was assessed with BD FACSVerse .Proteins were extracted with RIPA buffer  containing protease and phosphatase inhibitors . Protein concentrations were determined using Bradford assay  with albumin bovine serum . Ten-15 µg of protein was loaded onto SDS-PAGE . The following antibodies were used: pATM S1981 , ATM , pATR S428 , ATR , pCHK2 T68 , CHK2 , pCHK1 S345 , pCHK1 S296 , CHK1 , pH3 S10 , pDNApk S2056 , DNApk total , PAR , Vinculin  and beta-actin . Protein bands were detected using enhanced chemiluminescence  and visualized with AI680 Western blot imager . Densitometry was performed via Image J. All protein values were corrected to their loading control and phosphorylated protein values were corrected to their total protein values. Relative values were calculated by dividing each value by the control (untreated) value.Cells were seeded in µClear 96-well plates  and fixed with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100  in 0.5% BSA-PBS, cells were stained with primary antibody against pH2AX Ser139  followed by staining with 488 Alexa fluor secondary antibody . Nuclei were counterstained with DAPI . For micronuclei analysis, cells were fixed as described above and stained with DAPI . Immunofluorescence images were acquired with In Cell analyzer 2000 . 3. Tumor volumes were determined with caliper measurements. Body weight and health status of the mice were monitored daily.HPV-positive and HPV-negative parental and radioresistant cells were subcutaneously injected in both flanks of female nu/nu NMRI  mice. Experiments were performed according to the ethical committee of KU Leuven (P163/2017). Mice were divided in a control group and RT group. Mice from the latter group were treated with fractionated RT with 2 Gy per fraction for 3 or 5 consecutive days when tumors reached a volume of approximately 100 mmp-values < 0.05 were considered statistically significant.Statistical significance was tested using one-way ANOVA, two-way ANOVA and extra-sum of squares F-test using Graphpad prism 6.01 software . To assess the underlying mechanisms of radioresistance, we generated isogenic radioresistant clones R61-1 and R61-4 from HPV-negative cell line SCC61, and R154-1 and R154-4 from HPV-positive cell line SCC154, by fractionated RT with 2 Gy per fraction up to total dose of 60 Gy. Compared to parental cells SCC154 and SCC61, fractionated RT significantly increased the relative clonogenic survival of R154-1, R154-4, R61-1 and R61-4 clones, with 1.6-, 1.2-, 1.6- and 1.4-fold increase at 2 Gy, respectively A. AlthouNext, we assessed whether we could detect the decrease in radiosensitivity also in vivo conditions by investigating the delay in tumor regrowth after RT treatment in HPV-positive and HPV-negative radioresistant xenografts compared to their parental xenografts B,C. TumoIn line with the in vitro data, HPV-positive radioresistant models showed higher variation in RT response compared to HPV-negative radioresistant models. Fifty percent of the mice reached a TVD at day 5 and day 15 for R154-1 and R154-4 respectively, while 50% of SCC154 in vivo xenografts reached TVD at day 19, indicating a tumor growth delay of at least 4 days compared to the radioresistant clones. In addition, HPV-negative radioresistant clones showed an increase in radioresistance in vivo, with 50% of the R61-1 and R61-4 xenografts reaching TVD at day 21 and day 22 respectively. In contrast, 50% of SCC61 xenografts reached TVD at day 45, indicating a tumor growth delay of 23 days. We also determined the TVD in non-irradiated conditions for all the in vivo models B,C. AlthSeveral studies demonstrated that fractionated RT can result in alterations in DDR ,21,22. TIn line with previous studies ,24,25,26HPV-negative SCC61 cells and radioresistant clones showed a similar decrease in γH2AX foci levels at 16 h and 24 h after RT, indicating the absence of residual DSB damage and suggesting the absence of differential DDR after RT . No diffThis was verified by similar cell cycle distribution after RT, with similar G2/M arrest induction at 12 h and similar recovery of G2/M arrest after RT in both HPV-negative radioresistant clones and parental cells .Since increased ability of cells to divide and survive with RT-induced DNA damage is a characteristic of radioresistant cells , we asseIn contrast, HPV-negative radioresistant clones showed significantly less micronucleated cells compared to SCC61 parental cells at 48 h and 72 h after RT . Hence, ATR is an important regulator of S-phase progression and G2/M cell cycle checkpoint activation after replication-based DNA damage and genomic instability . MoreoveOur previous study showed that inhibition of DDR resulted in radiosensitization of both HPV-positive and HPV-negative HNSCC models . Therefo2Gy of 3.0 for both HPV-positive and HPV-negative radioresistant cells. Inhibition of cell cycle checkpoints CHK1/2 resulted in average DEF2Gy of 1.5 for all radioresistant cells, whereas inhibition of BER showed the least radiosensitizing potential with average DEF2Gy of 1.3. In addition, Western blot analysis showed targeted inhibition of all drugs  radioresistant cells and tumor biopsies of esophageal cancer patients, highlighting the importance of DDR in the radioresistance process. Moreover, the overlap of dysregulated pathways and genes between ESCC cells and tumor samples from recurrent ESCC patients show the potential of using in vitro radioresistant models for determining underlying processes of radioresistance . In lineThe ATR pathway is known to prevent replication stress and mitotic catastrophe by functioning on downstream cell cycle checkpoints . DiffereInterestingly, radioresistant cells generated from the SCC61 cell line by Bansal et al.  showed dSeveral studies have shown that DDR inhibition resulted in radiosensitization of HPV-positive and HPV-negative HNSCC using established cell lines ,15,16,17Although we were able to generate radioresistant cell lines from both HPV-positive and HPV-negative groups of HNSCC, we only used a single cell line for each group to investigate radioresistance. Expanding the number of isogenic radioresistant cells derived from additional HPV-positive and HPV-negative HNSCC cell lines and validating these models with patient-derived tumor xenografts will increase the clinical relevance. Another limitation of this study is that we only investigated the role of DDR in the radioresistance process. Although we have shown that DDR was differentially involved in HPV-positive and HPV-negative radioresistant clones, these differences were small and the driving forces of radioresistance were not unraveled. Further studies investigating the changes at the genomic, transcriptomic and epigenetic level could further broaden the understanding of the processes driving radioresistance and the involvement of DDR therein.In conclusion, we have shown that DDR is differentially involved in the radioresistant process of HPV-positive and HPV-negative HNSCC cells. HPV-positive radioresistant cells showed an increased DNA repair capacity, whereas HPV-negative radioresistant cells showed an increased replicative capacity. Despite these differences, combining DDR inhibitors together with RT renders both HPV-positive and HPV-negative radioresistant cells more sensitive to RT, offering possibilities for new therapies for patients with recurrent HNSCC, a population with limited therapeutic options.


Neuronal development is a tightly controlled process involving multi-layered regulatory mechanisms. While transcriptional pathways regulating neurodevelopment are well characterized, post-transcriptional programs are still poorly understood. TIA1 is an RNA-binding protein that can regulate splicing, stability, or translation of target mRNAs, and has been shown to play critical roles in stress response and neurodevelopment. However, the identity of mRNAs regulated by TIA1 during neurodevelopment under unstressed conditions is still unknown.To identify the mRNAs targeted by TIA1 during the first stages of human neurodevelopment, we performed RNA immunoprecipitation-sequencing (RIP-seq) on human embryonic stem cells (hESCs) and derived neural progenitor cells (NPCs), and cortical neurons under unstressed conditions. While there was no change in TIA1 protein levels, the number of TIA1 targeted mRNAs decreased from pluripotent cells to neurons. We identified 2400, 845, and 330 TIA1 mRNA targets in hESCs, NPC, and neurons, respectively. The vast majority of mRNA targets in hESC were genes associated with neurodevelopment and included autism spectrum disorder-risk genes that were not bound in neurons. Additionally, we found that most TIA1 mRNA targets have reduced ribosomal engagement levels.Our results reveal TIA1 mRNA targets in hESCs and during human neurodevelopment, indicate that translation repression is a key process targeted by TIA1 binding and implicate TIA1 function in neuronal differentiation.The online version contains supplementary material available at 10.1007/s11033-021-06634-0. Post-transcriptional regulation is the collection of mechanisms regulating gene expression that start operating co-transcriptionally through the processing of the pre-mRNAs, and play critical roles in cellular homeostasis and development . Post-trPost-transcriptional regulation mechanisms play pivotal roles during neurodevelopment and in neuronal function –9. ImporTIA1 is an RBP that recruits RNAs into stress granules as part of the adaptive response to stress , 12. HowImmunoprecipitation of TIA1 followed by transcriptome-wide analyses of co-eluted RNAs have been carried out on colorectal carcinoma (RKO) and HEK293 cells for unbiased identification of TIA1 target RNAs in non-stressed conditions , 20. In Here we performed RNA immunoprecipitation (RIP) of TIA1 protein followed by next-generation sequencing of co-eluted RNAs (RIP-seq) using an in vitro model of neurodevelopment involving differentiation from human embryonic stem cells (hESCs), to neural progenitor cells (NPCs) and then to cortical neurons under unstressed conditions. To precisely map and quantify mRNAs bound by TIA1 at the 3′UTR isoform level, we performed 3′UTR RNA-seq on transcripts precipitated by a TIA1 specific antibody and normalized these to RNAs eluted with an isogenic IgG control. We found thousands of high confidence target mRNAs in hESCs. Interestingly, the number of targets and their binding efficiencies decrease significantly in NPCs and neurons, indicating that the TIA1 regulatory network of target mRNAs decreases as hESCs differentiate into neurons. Most TIA1 mRNA targets have reduced ribosomal engagement levels, suggesting that translational repression throughout the first stages of neurodevelopment is a key process associated with TIA1 binding.MECP2 and UBE3A mRNAs, known TIA1 mRNA targets with validated significance for neurodevelopment and neurodevelopmental disorders [MECP2 and UBE3A mRNAs were enriched in TIA1 IP but not in IgG IP samples as quantified by qRT-PCR. 18S RNA was used as negative RNA control.To identify TIA1 mRNA targets transcriptome-wide in an in vitro model of neurodevelopment, we conducted RIP-seq on samples collected from each stage of neuronal differentiation as reported previously  isorders , 27, expOverall quality of total RNA used as input samples from all aliquots and cell types was determined by bioanalyzer analysis, which showed that all input samples had RNA integrity numbers (RIN) values greater than 7 . Quality of RNA samples from TIA1 or IgG IPs could not be determined by bioanalyzer since these samples lack the ribosomal RNA molecules used to calculate RIN values. Nonetheless, analyses of RNA length showed that the fraction of RNAs larger than 200 nucleotides (nt) was above 90% in all TIA1 IP samples, indicating that the majority of RNA molecules remained intact after immunoprecipitation and RNA purification steps . In all three independent biological replicates for each cell type the total RNA recovered from IgG IP samples was significantly lower than the TIA1 IP samples. Two of the IgG samples, one replicate each of NPCs and neurons, did not have sufficient RNA material for library prep despite the high number of cells used and our efforts to collect all possible co-eluted RNA . This resulted in the use of two replicates of IgG IP samples for NPC and neuron cell types for downstream analyses. The low recovery of RNAs from IgG IP samples underscores that our IP method resulted in low background of non-specific RNAs bound to the magnetic beads.After sequencing, we confirmed the cellular identities using cell-type gene-specific markers from the input samples Fig. A. High rTP53 [MECP2 [UBE3A [To identify TIA1 mRNA targets we first calculated TIA1 binding efficiency (BE) by determining the ratios of all detected mRNAs in IP over the input samples , 28 Fig” sectionTP53 , MECP2 [3 [MECP2 , and UBE2 [UBE3A  were suc2 [UBE3A , 13, 20 To orthogonally validate the identification of TIA1 target mRNAs from our study, we compared our dataset with a previous CLIP-seq experiment that identified TIA1 mRNA targets in HEK293 cells . ApproxiAFF2 and CHD8), genes implicated in neuronal syndromes (USP9X), and genes found to be translationally dysregulated in Rett syndrome cortical neurons (NEDD4L) . Interestingly, in hESCs we identified an enrichment for terms associated with development and cellular differentiation Fig. A. Examinrons NEDDL  as TIA1 mRNA targets that are consistently expressed across all cell types . Interestingly, of these, 80.4% are bound by TIA1 in either hESCs, or in both hESCs and NPCs but not in neurons (Supplementary Table 3). Only 17 ASD-risk genes were bound by TIA1 in neurons, accounting for 11.9% of all TIA1 ASD-risk gene targets, and 14 of which are consistently bound by TIA1 in all three cell types. Together, our data suggests that TIA1 regulates neurodevelopment and ASD-relevant transcripts in the early stages of development, and this regulation is reduced as cells progress through neurodevelopment.TMEM196 as a neuron-specific target gene (Supplementary Table 4). Altogether, these results indicate that while not changing its protein abundances, TIA1 plays a major role in the first stages of human neurodevelopment where most of its targeted mRNAs in hESCs continue to be expressed in NPCs and neurons but are no longer targeted by TIA1.Finally, we investigated the potential cell-type specific functions of TIA1 mRNA binding. We looked at the genes expressed in all cell types that were at the bottom 20 percentile in the other two cell types. Using this threshold, we found that out of the 2400 hESC TIA1 targets, only 47 are hESC specific (i.e. not expressed in NPCs or neurons) Fig. E. No moltranslating ribosome affinity purification-seq) and RNA-seq that quantified changes in ribosomal engagement, hence translation regulation, transcriptome-wide in similar conditions of in vitro neurodevelopment [Since TIA1 has been shown to be a translational regulator in hESC , we testelopment . Intereselopment .Here we investigated the roles of the RBP TIA1 during typical neurodevelopment by determining its network of targeted mRNAs in a hESC-derived neuronal developmental model. We performed TIA1 RIP-seq on multiple replicates of hESC, -derived NPCs and cortical neurons under unstressed conditions and calculated its BE to RNAs transcriptome-wide. We also deployed an isogenic IgG antibody as a negative control and to set the minimal BE threshold for target-mRNA calling. siRNA-mediated KD of TIA1 was used to validate the specificity of the TIA1 antibody using western blots on independent cell samples, but KD was not used as a negative control in the RIP-Seq due to concerns about stress induction.With this experimental set-up, we identified TIA1 target mRNAs with high confidence while minimizing false-positive calls, yielding 2400 TIA1 targets in hESCs, 845 in NPCs, and 330 in neurons. Interestingly, these numbers of target mRNAs represent 18% of hESCs transcriptome, but only 7% and 3% of the transcriptome in NPCs and neurons, respectively, even though the vast majority of the target mRNAs were expressed in all three cell types and TIA1 protein levels did not change significantly. We found only 47 TIA1 targets in hESCs that are not expressed in either NPCs or neurons. Additionally, the remaining TIA1 target mRNAs in NPCs and neurons had significantly decreased BE relative to hESCs. The decrease in the number of TIA1 target mRNAs and reduction of BE upon differentiation indicate that TIA1 dynamically regulates mRNA metabolism in the early stages of human neurodevelopment.The underlying causes of the observed decrease in TIA1 binding during neurodevelopment are still unclear. One possibility is that the changes in the TIA1 mRNA network may be due to post-translational modifications to the protein changing its binding dynamics, which would not be without precedent. For example, CPEB3 is an RBP that regulates mRNA translation in neurons and, like TIA1, CPEB3 is a prion-like protein that can self-aggregate via a prion-like domain . Post-trThe decrease in TIA1 BE in neurons could also be due to structural changes in the mRNA targets themselves, or the expression of other RBPs or miRNAs impacting TIA1-binding. Post-transcriptional regulation typically involves multiple regulators and is determined by the local concentrations of these various regulators in the cellular environment. Changes in the 3′UTR of TIA1 targets during differentiation could alter the balance between regulators and affect TIA1 binding . CLIP-seELAVL2, ELAVL3, and ELAVL4, respectively) are expressed exclusively in a neuronal context [UBE3A [Another possible explanation for the decrease in TIA1 binding in neurons is an increase in the abundance of other regulatory molecules that share a binding site with TIA1. At higher abundances, these factors can outcompete TIA1 for binding to transcripts changing their regulatory profile as has been previously shown , 39. The context , 39, 40.t [UBE3A .Together, our results provide important insights into the network of TIA1 target mRNAs during neurodevelopment and its roles in neuronal differentiation. TIA1 appears to regulate neurodevelopmentally relevant transcripts in the early stages of development, and the network of regulated mRNAs decreases as cells differentiate to form neurons, despite the continued presence of TIA1 in the cells. This decrease in TIA1 binding may play an important role in regulating the transition from stem cells and progenitor cells to functional mature neurons.H9 hESCs obtained from the National Stem Cell Bank (WiCell) were cultured with mTeSR1 culture medium (STEMCELL Technologies) on Becton Dickinson hESC-qualified matrigel. Pluripotent stem cell work was approved by the Canadian Institutes of Health Research Stem Cell Oversight Committee and The Hospital for Sick Children Research Ethics Board.l-ornithine + laminin coated dishes and grown in N2 media + laminin (1 μl/ml). After 7 days, neural rosettes were manually picked and transferred to poly-L-ornithine + laminin coated wells. After 7 days, neural rosettes were picked a second time, digested with Accutase and plated on poly-l-ornithine + laminin coated wells. Resulting neural precursor cells were grown as a monolayer and split every 5–7 days in NPC media , 2 μg/ml Heparin, 1 μg/ml laminin). To generate neurons, NPCs were plated on poly-l-ornithine + laminin-coated plates at a density of 106 cells per 10 cm dish and cultured for 4 weeks in neural differentiation medium .An embryoid body (EB)-based method was used for neuronal induction of NPCs as described previously . hESCs whESCs were transfected with Silencer select siRNAs (ThermoFisher) to knockdown TIA1 (# s14133 and s14132) following manufacturer’s instructions. Cells were subjected to two consecutive transfections of siRNAs at two-day intervals in the week leading up to protein harvest.Cells were washed in ice-cold PBS and total protein extracted in radioimmune precipitation assay (RIPA) buffer . Equivalent protein mass was loaded on SDS-PAGE and transferred to Hybond ECL  nitrocellulose membrane. Membranes were incubated with TIA1 and Actin B antibodies  in 5% milk in PBS + Tween 20 (0.05%). Near-Infra Red-conjugated secondary antibodies (LI-COR) were used and membranes scanned using LI-COR Odyssey CLx scanner according to manufacturer’s instructions. Acquired images were analyzed using ImageStudio v5.2.5.7 cells per plate, on six 10 cm plates, and grown overnight prior to lysate collection. To collect neuronal samples, NPCs were seeded on 10 cm plates and maintained in neuronal differentiation media for 4-weeks prior to lysate collection. Lysate collection and RNA immunoprecipitations were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore #17-700) following manufacturer’s instructions. Briefly, cells were washed twice in ice-cold PBS and lysed in RIP Lysis Buffer complemented with Protease and RNase Inhibitor Cocktails. Prior to immunoprecipitation, magnetic protein A/G beads were prepared with 5 μg of rabbit anti-TIA1 (Abcam-#ab40693) or normal rabbit IgG antibodies (from Magna RIP kit). The RIP lysates were centrifuged at 12.000 g for 10 min at 4 °C. The lysate was then split into three fractions: 10 μl was set aside as an input sample and frozen at − 80 °C until the RNA purification step, 100 μl of RIP lysate was added to the tube containing the TIA1 bound beads, and the IgG bound beads. The RIP lysates were incubated with the antibody-bead complexes overnight at 4 °C. Total RNA was isolated and purified from the input lysates and the immunoprecipitation samples using phenol/chloroform and resuspended in 10 μl of RNase-free water. Quality control of the RIP samples was assessed by qRT-PCR (see below). RNA samples were sent to The Centre for Applied Genomics (TCAG) for bioanalyzer analysis to assess the quality of the RNA.For each RIP lysate hESCs or NPCs were seeded at a density of 1 × 10−(ΔΔCt) method, and 18 s rRNA as a normalizing control. Primers used were: MECP2 forward: GCUCUGCUGGGAAGUAUGAUG, reverse: TTTGGGCTTCTTAGGTGGTTT; UBE3A forward: GTTCTGATTAGGGAGTTCTGGG, reverse: TCCTTTGGCATACGTGATGG; and 18S forward: GATGGGCGGCGGAAAATAG, reverse: GCGTGGATTCTGCATAATGGT. Technical duplicates and biological replicates were combined to calculate an average fold change. For comparison of mRNA levels between input and IP samples, results are displayed as a percentage of input.RNA was reversed transcribed to cDNA using SuperScript III reverse transcriptase, and random hexamer primers. qRT-PCR was performed using SYBR Select PCR master mix on a ViiA7 Real-Time PCR System (both from ThermoFisher). Fold changes were calculated by the 2Sequencing libraries were prepared using the Agilent Bravo NGS workstation  using a previously established protocol:https://www.agilent.com/cs/library/applications/5991-8601EN_Auto_NGS_Application.pdf). Library preparation was done using the QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina (Lexogen #015.24), following manufacturer’s instructions. To minimize variability in the library preparation, 17 ng of RNA was used in the library preparations of all input and TIA1 IP samples. The RNA was spiked with SIRV Set 3 Iso Mix E0/ERCC controls (Lexogen 025.03) prior to the start of library prep. Due to low RNA levels in the IgG samples the maximum amount of 5 μL of RNA was used for each IgG sample. Each sample was dual indexed during the final library amplification using the Lexogen’s i5 6 nt Dual Indexing Add-on Kit (#047) to allow for differentiation of the samples after sequencing.(The sequencing was then conducted by The Centre for Applied Genomics (TCAG) using the Illumina HiSeq 2500 and two lanes of the Rapid Run Mode flow cell. The samples were sequenced as single-ended reads, with a read length of 100 base pairs, at a sequencing depth of approximately 20 million reads for each sample.The unique molecular identifiers were used to remove PCR duplicates. Reads were then aligned to the human genome hg38 using the STAR aligner , with devst function in DESeq2 package). Then, PCA of transformed counts was used to validate clustering of samples from same assay and cell type (plotPCA in DESeq2 package).Raw gene counts were normalized to corresponding medians of each replicate and cell-type. Then, log10 of normalized counts were plotted in a heatmap showing abundance of cell-type specific markers. Second, we used variance stabilizing transformation on raw gene counts  analysis was done using g:profiler and default parameters . Genes tAll datasets and statistical tests were done using R software. For comparison of TIA1 binding efficiency of different transcripts within the same samples statistical analysis was performed using two-tailed student’s t-test. For comparison of ribosomal engagement of the same transcripts in different cell types statistical analysis was done using a paired t-test. For RIP-seq data analysis, the p-values were adjusted using the Benjamini–Hochberg method. In all cases, fold-change differences with a p-value ≤ 0.05 were considered significant.A RNA Integrity Numbers (RIN) from all RNA samples show that all input samples have RIN values above 7. B percentage of RNA molecules larger than 200 nucleotides for each sample. C RNA concentration of all samples show that IgG samples had low yields in RNA recovery. Supplementary file1 (JPG 494 kb)Supplementary Figure 1. Quality control of RNA samples used for sequencing. Supplementary file2 (XLSX 177 kb)Supplementary file3 (XLSX 188 kb)Supplementary file4 (XLSX 14 kb)Supplementary file5 (XLSX 10 kb)Below is the link to the electronic supplementary material.