Squamous cell papilloma (SCP) in the upper aero-digestive tract is a rare disease entity with bimodal age presentation both at childhood and in adults. It originates from stratified squamous and/or respiratory epithelium. Traditionally, SCPs have been linked to chemical or mechanical irritation but, since the 1980s, they have also been associated with human papillomavirus (HPV) infection. Approximately 30% of the head and neck SCPs are associated with HPV infection, with this association being highest for laryngeal papillomas (76–94%), followed by oral (27–48%), sinonasal (25–40%), and oropharyngeal papillomas (6–7%). There is, however, a wide variation in HPV prevalence, the highest being in esophageal SCPs (11–57%). HPV6 and HPV11 are the two main HPV genotypes present, but these are also high-risk HPVs as they are infrequently detected. Some 20% of the oral and oropharyngeal papillomas also contain cutaneous HPV genotypes. Despite their benign morphology, some SCPs tend to recur and even undergo malignant transformation. The highest malignant potential is associated with sinonasal inverted papillomas (7–11%). This review discusses the evidence regarding HPV etiology of benign SCPs in the upper aero-digestive tract and their HPV-related malignant transformation. In addition, studies on HPV exposure at an early age are discussed, as are the animal models shedding light on HPV transmission, viral latency, and its reactivation. The upper aero-digestive tract is composed of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, hypopharynx, larynx, trachea, and esophagus. Squamous cell papillomas (SCPs) are histologically benign growths encountered practically at all body sites where squamous epithelium exists  . SCPs arIn the aero-digestive tract, the most common sites of SCPs are the oral cavity and the larynx, while sinonasal and esophageal areas are less commonly involved. Only few SCPs have been described in the naso- and oropharynx  ,5,6,7,8.The etiology of SCP is not universally confirmed but mechanical and chemical irritation and/or infection with human papillomaviruses (HPV) have been prime culprits . The mosThe two most frequent HPV genotypes associated with SCPs are HPV6 and HPV11, but high-risk HPV16 and 18, and other mucosal alpha-HPVs, have been detected at a lower frequency ,9,10,11.This review summarizes the key features of SCPs in the upper aero-digestive tract , includiSinonasal papilloma (SNPs), identified in the 1850s has frequently been called the Schneiderian papilloma. This is because these lesions develop from the Schneiderian membrane, i.e., an ectodermally derived respiratory mucosa covering the nasal cavity and paranasal sinuses, in contrast to the other mucosa of the upper respiratory tract, which is derived from an embryological endoderm. The latest 2017) WHO classification omitted the eponymous “Schneiderian” designation in favor of the more simple sinonasal papilloma (SNP)  WHO clas,20,21.The symptoms of SNP include unilateral nasal obstruction, epistaxis, bloody nasal discharge, headache, facial pain, anosmia, dysosmia, and epiphora. SNPs are characterized by three typical features: 1) a locally destructive growth pattern, (2) development of frequent recurrences, and (3) the potential for malignant progression  a locall,23,24. BThere are three histological subtypes of SNP: 1) inverted SNP, (2) exophytic SNP (previously known as fungiform papilloma), and (3) oncocytic SNP   inverted,25.Sinonasal inverted papilloma (SNIP) is the most commonly diagnosed subtype, accounting for almost 65–75% of all SNPs ,19,25,26Sinonasal exophytic papillomas (SNEP) account for 20–25% of all SNPs ,22,25, aSinonasal oncocytic papilloma (SNOP) is the most uncommon type, representing only 3–6% of all SNPs ,22,25. IThe etiology of SNPs is not yet universally accepted. Several risk factors have been identified, including cigarette smoking, alcohol consumption, allergic rhinitis, essential hypertension, anticoagulant medication, as well as exposure to environmental and occupational noxious agents such as organic solvents, welding fumes, and nickel compounds ,31,32. SSince pioneering original observations in the early 1980s, implicating HPV as the potential etiological agent of SNPs, the discussion is still ongoing regarding the etiological role of HPV in SNPs and their malignant transformation. This discussion has been updated in recently published systematic reviews and meta-analyses.n = 760) cases were HPV-positive, with HPV prevalence being highest (65.3%) in SNEPs, followed by SNIP (37.8%) and SNOP (22.5%) [In 2013, we published a systematic review and meta-analysis of the literature reporting on HPV detection in SNPs up until April 2012. Seventy-six studies were eligible, covering 1956 SNPs from different geographic regions. Altogether, 38.8% ( (22.5%) . The lik (22.5%) .n = 40/76) D-SNIPs, and 23.6% (29/123) M-SNIPs tested HPV-positive, thus disclosing the highest HPV prevalence (>50%) in SNIP with concomitant dysplasia [Gupta et al. (2020) summarized the results from 21 additional studies published since the appearance of our meta-analysis . Of the ysplasia .Recently, Pähler Vor der Holte et al. 2020) reported that 31 of 80 SNIPs were HPV- positive (38.8%). The most commonly detected HPV genotypes were HPV6  and HPV16 , followed by HPV11 , HPV58 , HPV42, and HPV83 . Most patients displayed an infection with just one HPV type. In total, 16.3% of the SNIPs had a co-infection with two HPV types, but one patient had co-infection with four different HPV types. In that series, only four SNEPs were available, all being HPV-positive: HPV6, HPV11, and HPV 91. Of the two SNOPs, the larger one, spanning three paranasal sinuses, presented co-infections with HPV6, HPV16, and HPV42 [020 reporThe role of cutaneous β- and γ-HPVs in SNPs is practically unexplored, although evidence is available that these viruses are present as asymptomatic infections both in the nasal cavity and in oral mucosa ,13. Due p = 0.039), the absence of an inflammatory polyp (p = 0.033), an increase in the ratio of neoplastic epithelium to stroma (p = 0.037), an increase in hyperkeratosis (p = 0.030), and a decrease in the number of eosinophils (p = 0.037) [The estimated overall rate of malignant transformation of SNPs is approximately 9% (95% CI 7–11%), based on the systematic review by Re et al. (2017), who included 29 studies with a total of 3177 patients . The risa p = 0.0, an incr= 0.037) .Lawson et al. (2008) reviewed the SNP literature published between January 1987 and December 2006 to address the following three important questions: (1) Why do HPV detection rates vary so greatly? (2) What is the relationship between LR- and HR-HPV types and HPV detection rates? (3) Is there a relationship between certain HPV genotypes and lesion recurrence or their malignant progression? . The firp < 0.02) in SNIP with high-grade dysplasia  and SCC  compared to SNIP with no dysplasia or mild dysplasia . Furthermore, the preponderance of LR-HPV in benign SNIP (ratio LR/HR = 4.8:1) shifted in dysplastic and malignant SNPs. The LR/HR ratio was 1.1:1 for SNIPs with high-grade dysplasia, but reverted in favor of HR-HPV (1:2.4) in SNIPs with malignant transformation [HPV detection rates significantly increased (ormation . Recurreormation .p = 0.343), HPV11 , and HPV 6/11  infections were not significantly associated with the risk of malignant SNIP. In contrast, the risk of malignant SNIP was significantly increased in patients infected with HPV16 , HPV11/16 , HPV18 , and HPV16/18  [Ding et al. (2020) authored a systematic review and meta-analysis on malignant transformation in SNIP stratified by HPV genotypes . Altoget< 0.001) .p = 0.905), indicating that there was not a similar increase in HPV-associated SCC in sinonasal tract such as that found in HPV-associated oropharyngeal carcinomas. Importantly, the positive association of HPV with malignant SNIP transformation was confirmed in all three geographic regions of the study: North America, Europe, and Asia [Another meta-analysis by Stepp and coworkers (2021) focused on the role of HPV in the malignant transformation of SNIP , which wand Asia .EGFR mutations and HR-HPV infection represent essential, alternative oncogenic mechanisms in SNIP and SNIP-associated SCC. Furthermore, alterations in TP53 and CDKN2A are likely to be early markers for malignant progression of SNIP. Thus, there is a need for molecular sub-classification of SNP and sinonasal carcinomas because it may contribute to prognostic predictions and the design of personalized molecular-targeted precision medicine.Molecular mechanisms underlying SNP, or their malignant progression, are still incompletely understood, and the number of cases studied is relatively small. This notwithstanding, the results are consistent and merit review in this paper ,43,44,45LINE-1 was determined, as its hypo-methylation is known to be significantly associated with squamous histology, tobacco smoking, and poor prognosis. All SNOP lesions were HR-HPV-negative but had KRAS mutations, while all SNIPs were also negative for HR-HPV but had no KRAS mutations. HR-HPV was observed in only 13% of SNIP-associated SCC and in 8% of de novo-SCC patients. EGFR mutations were more common in SNIP than in SNIP-associated SCC (72% vs. 30%) or de novo-SCCs (17%) [EGFR-mutated ISPs compared to 76% (13/17) of the patients with EGFR-wild-type ISP (p = 0.0044). LINE-1 hypomethylation significantly increased from papilloma to early stage SCC, and further to advanced stage SCC (p = 0.03), and it was associated with occupational exposure (p = 0.01) and worse prognosis (p = 0.09) [Sahnane et al. (2019) genotyped 10 genes involved in EGFR signaling in 25 SNIP, 5 SNOP, and 35 SCC samples from 54 patients . AdditioCs (17%) . At 5-ye = 0.09) .EGFR mutation, EGFR copy number gain (CNG), KRAS mutation, and HR-HPV infection in 146 SNIP-associated SCCs [EGFR mutations were present in 21 (14.7%) of 143 SNIP-SCCs, including 13/14 (92.9%) SNIP-SCCs and 8/129 (6.2%) non–SNIP-SCCs (p < 0.0001). The majority of EGFR mutations were exon 20 insertions, with the remainder composed of deletions and single-nucleotide substitutions in exons 19 and 20. KRAS mutation was not detected in any of the 142 SNIP-SCCs. EGFR CNG was detected in 41 (28.1%) of 146 SNIP-SCCs; all of them were HPV-negative [EGFR mutation, EGFR CNG, and HR-HPV were essentially mutually exclusive, and each subgroup had distinct clinic-pathological features. The HPV-negative/EGFR-mutant group, the HPV-negative/EGFR CNG-positive group, and the triple-negative group had significantly worse prognoses than the HPV-positive group . Thus, Hongo et al. (2020) confirmed the results by Sahnane et al. (2019), in that EGFR mutation may play a pathogenically important role in a subset of SNIP-SCCs [Hongo et al. (2020) determined the prevalence, mutual relationships, and clinic-pathological significance of ted SCCs . HR-HPV In yet another study, Brown et al. (2021) characterized mutations and copy number alterations in 29 SNP-associated SCCs utilizing targeted next-generation DNA sequencing (DNAseq) of frequently altered pan-cancer genes . They alCDKN2A alteration, and all except one (96.6%) harbored at least one TP53 or CDKN2A alteration. Importantly, none of these alterations were detected in the subset of matched SNP, indicating that TP53 and CDKN2A are likely to be early molecular events in the malignant progression to SNP-SCC. EGFR and KRAS mutations were significantly enriched in SNP-associated SCC relative to other aero-digestive tract SCC (p < 0.001); CDKN2A mutations, TERT copy number gains, and LR--HPV infection also occur more frequently in these tumors (p < 0.05) [According to these data, the vast majority of SNP-SCC (72.4%) showed evidence of at least one  < 0.05) . These rEGFR mutations (detected by Sanger sequencing) and HPV infections represent alternate oncogenic pathways [EGFR mutation or an HPV infection. Similarly, as reported by Sahnane et al. (2019) and Hongo et al. (2020), HPV and EGFR mutation was mutually exclusive in all cases of SNIP-associated SCC and all but one SNIP [EGFR genotypes. Malignant progression of SNIP was significantly associated with the presence of HPV infection and the absence of an EGFR mutation  [In a previous study, the same research group also showed that pathways . That coone SNIP ,43,44. H= 0.002) .At birth, nasopharynx is lined by typical respiratory epithelium, but this pseudostratified columnar ciliated epithelium is gradually replaced by stratified, non-ciliated epithelium and, with advancing age, by mature squamous epithelium. A transformation zone with distinct squamo-columnar junctions is typical of the nasopharynx; thus, one would expect to find this area to be a typical site for SCP. However, primary nasopharyngeal papilloma is an extreme rarity, as determined from the literature. Only 12 well defined “old” cases could be derived from the archives of The Armed Forces Institute of Pathology (AFIP) . These lThe existent of nasopharyngeal papilloma is also ignored in the latest WHO book, 2017 . Two of The oral cavity is unique in its anatomy and dual function, comprising the first portion of the digestive tract but also part of the upper airways . DependiOral squamous cell papilloma (OP) is still a controversial issue. The discussion is ongoing as to whether these lesions represent a reaction to injury rather than a true benign neoplasm . In animBased on clinical examinations, Knapp et al. (1971) reported that OP was the second most common lesion among 181,338 consecutive oral examinations made in the US Army . OPs occSome rare genetic syndromes are known to be associated with multiple OPs, including (1) focal dermal hypoplasia syndrome, (2) WHIM syndrome, (3) acrodermatitis enteropatica, (4) Cowden syndrome, (5) nevus unius lateralis, (6) Costello syndrome, and (7) Down syndrome . WHIM syFrithiof and Wersall (1967), as well as Gysland et al. (1976), were the first to identify intra-nuclear viral particles closely resembling HPV in the epithelial cells of an OP and oral condyloma, respectively ,53. We rn = 4/19) were HPV-positive and HPV6 or HPV11 were the only genotypes detected. Danna et al. (2017) analyzed 83 SCPs derived from different sites of the respiratory tract  [Trzcinska et al. (2020) studied the presence of HPV in a series of 131 SCPs of the head and neck region, including 19 OPs [ryngeal) . Twenty-ryngeal) .We recently published data on 83 oral epithelial lesions, of which 33 (39.8%) were diagnosed as OP, 40 (48.2%) as papillary hyperplasia, 6 (7.2%) as verruca vulgaris, and 4 (4.8%) as others . The oveAs discussed in relation to SNIP, it is important to elucidate the role of cutaneous HPV genotypes in both benign and premalignant and malignant oral lesions. It is currently known that asymptomatic HPV infections with a wide spectrum of β- and γ-HPV genera are found in oral and oropharyngeal mucosa ,55. BottOral mucosa contains a wide spectrum of HPV genotypes, predominantly of the β-HPV and γ-HPV genera. Future studies are clearly warranted on the whole spectra of HPV genotypes present in oral mucosa to gain further insights into the biology of HPV and its association with oral neoplasia.Unfortunately, the data are scant regarding oropharyngeal SCPs. Kim et al. (2019) authored a retrospective study on oropharyngeal papillomas diagnosed between 2013 and 2015 . In theiHPV presence in oropharyngeal papillomas has not been systematically studied, and the limited data discussed here are extracted from a few recent studies on SCPs with a focus on several sites of the respiratory tract. As already discussed, Dona et al. (2017) studied 83 papillomas, of which 43 were of oropharyngeal origin . Only 3 Laryngeal papilloma (LP) is a disease with a long history, described as “warts in the throat” in the seventeenth century, and it was first recognized as an entity separate from other laryngeal lesions by MacKenzie 1871) . Origina871 . OriThe estimated incidence of RRP varies from 0.6 to 4.3/100,000 among children and 1.8 to 2.3/100,000 in adults. ,58. AccoClinically, LP is a multi-nodular, cauliflower-like exophytic tumor composed of stratified squamous epithelium with vascular connective tissue core, and it is similar to the histology of SCPs at other mucosal sites. The course of these benign lesions is highly variable; some patients have spontaneous remission, whereas, in others, morbidity is high because these lesions are difficult to eradicate due to their high tendency for recurrence and spread throughout the aero-digestive tract. In adults, RRP lesions are more often solitary and less severe, and upper airway obstruction occurs less frequently than in children. Malignant transformation into laryngeal  SCCs does occur, but rarely in a proportion of disease prevalence .Ulmann (1923) suspected PV etiology of LPs long before HPV particles could be demonstrated by electron microscopy . He alson = 14) in these co-infections, followed by HPV45 (n = 3), HPV18 (n = 2), HPV16 (n = 1), HPV31 (n = 1), and HPV 35 (n = 1). Notably, 14 patients (6.3%) were negative for any tested HPVs, and 6 of them developed SCC. Using metagenomic sequencing, one patient also proved to have HPV8, which is a cutaneous HR-HPV. Co-infection with HR-HPV or being HPV-negative was significantly more common among the patients with AO-RRPs than in those with JO-RRPs. Interestingly, HPV11 was more prevalent in children than in adults [Since 1998, an increasing number of studies on HPV presence in RRP have been published, and the proportion of HPV positivity in RRPs has increased close to 90–95%, with HPV6 and HPV11 still being the two most predominant genotypes. As the diversity of HPV has expanded to cover >200 genotypes, the role of other HPV genotypes in RRP has become more relevant. Omland et al. (2014) screened 221 patients (174 AO-RRP and 50 JO-RRP) for HPV genotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68a, and 68b . In totan adults .Recently, Hoesli et al. (2020) studied 184 patients fulfilling the following strict inclusion criteria of RRP: (1) Visually obvious papillomas, (2) recurrence requiring multiple surgeries, and (3) histologically confirmed diagnosis of an LP . AltogetThese data implicate that a minority of RRPs are HPV-negative, which, however, can also progress to malignant lesions. Clearly, RRPs can be caused by HPV genotypes other than HPV6/11, and a minority of those can undergo malignant transformation.Kashima et al. (1992) concluded that JO-RRP patients were more often firstborn, delivered vaginally, and born to teenage mothers . On the Recent evidence supports the view that there is an immunologic tolerance to HPV6 and HPV11 in patients with JO-RRP ,67. AddiIn conclusion, research is accumulating to support the notion that the RRP microenvironment is immunocompromised; thus, regaining Th1 cell function may be a durable approach to prevent persistent infection.p = 0.03), whereas HPV11 was more prevalent in JO-RRP cases (p = 0.02), and even more prevalent in high-risk JO-RRP cases  (p = 0.04) [The outcomes of RRP are variable among individual patients, and several studies have focused on the risk factors associated with a more aggressive outcome. Most studies agree that a younger age at onset of JO-RRP is an important independent predictor of disease severity ,15,16,64 = 0.04) . Similar = 0.04) .Studies on the genetic variants or viral integration of HPV6 and HPV11 genotypes are lacking, and the results from scattered studies have been disappointing. Do Bonfim et al. (2015) reported that JO-RRP lesions harbored exclusively HPV-6vc-related variants of the five variants studied, whereas, among AO-RRP, HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference . IkegamiIn 2012, Ure and Forslund analyzed the methylation status of the HPV6 upstream regulatory region (URR) in SCPs from the upper aero-digestive tract of six adult patients. All CpGs in the URR were non-methylated, from both basal/intermediate and superficial cells, suggesting that methylation is not involved in the regulation of transcription from the HPV6 URR, regardless of epithelial differentiation . SimilarDistal spread of RPP occurs in 5% to 48% of cases. Various hypotheses have been proposed to explain this distal spread, including the extension of papillomas by contiguity, diffuse viral contamination, and iatrogenic factors, such as laryngoscopy, bronchoscopy, tracheostomy, and surgical manipulation. Risk factors for the spread of RRP toward the lower respiratory tract include HPV11 infection, age younger than 5 years, tracheostomy performed to avoid airway obstruction, and previous invasive procedures ,81. GéliMalignant transformation (to SCC) in AO-RRP occurs in 3% to 6% of the patients, while it is rarer in JO-RRP patients, <1%. As expected with JO-RPP, malignant transformation occurs in older children; the median time between the diagnosis of JO-RRP and the diagnosis of SCC is 19 years (range 4–45 years) . HPV11 sp = 0.0005). Of the 63 JO-RRP patients, there were no cases of dysplasia, but 3 (5%) patients developed an invasive SCC, all with pulmonary involvement. The JO-RRP patients with SCC had a younger age of disease onset  and a higher rate of tracheal involvement than those who did not develop SCC. Gender, smoking history, number of operations, or use of cidofovir showed no association with the development of dysplasia or SCC in either the AO-RRP or the JO-RRP cohort [Karatayli-Ozgursoy et al. (2016) performed a study on 159 RRP patients, comprising 96 AO-RRP and 63 JO-RRP patients . In theiP cohort .10p15.1, resulting in the loss of this gene’s expression. Alterations in AKR1C gene expression have previously been implicated in the pathogenesis of other HPV-related malignancies [Until recently, malignant transformation had been reported only for HPV11-associated RRP in 2–4% of all RRP-cases, but not for HPV6. However, Huebbers et al. described a novel molecular mechanism in the first case of HPV6-associated laryngeal SCC identified in JO-RRP. HPV6 was shown to be integrated in the aldo-keto reductase 1C3 gene (AKR1C3) on chromosome gnancies .p = 0.034). Among the 15 incident cases of JO-RPP , none of the mothers were vaccinated before pregnancy, 20% had maternal history of genital warts, 60% were first-born, and 13/15 were born vaginally. Of the examined cases, only HPV6 (n = 4) or HPV11 (n = 3) were detected. Of the two cases reported in 2017, one was a probable case of non-laryngeal JO-RRP and the other occurred in a child who was not born in Australia. In 2019, no cases of JO-RRP were reported to the APSU in any Australian state or territory for a second consecutive year. Thus, in the eight years of JO-RRP surveillance, seventeen confirmed cases were reported, with one to four cases identified per year until the end of 2017 [Reliable data on the efficacy of HPV vaccines in preventing SCP in the aero-digestive tract are available only for RRP. The off-label HPV vaccines used for treatment of these lesions fall outside the scope of this discourse. Australia is the pioneer and model country in implementation of national HPV vaccination programs. This is because the 4-valent vaccine introduced in 2007 for girls and 2013 for boys has had an excellent uptake, with over 80% among girls and 75% among boys, with at least two doses. Since October 2011, surveillance of JO-RRP has been conducted by the the Australian Pediatric Surveillance Unit (APSU) . Thus fa of 2017 .Taken together, the widespread routine HPV vaccination  of both girls and boys has interrupted community circulation of the causal HPV types of JO-RRP and has consequently dramatically reduced the risk of perinatal exposure and subsequent disease in Australia. It remains to be seen whether the effect is equally dramatic for the other HPV-associated pediatric SCPs.Tracheal papilloma (TP) is a distinct rarity, mostly manifested as the tracheal involvement of RRP. TPs have both a juvenile and an adult onset, presenting with either a JO- or AO-papillomatosis, mostly due to the distal spread of RRP from the larynx ,81,82. TThe presence of TPs has also been associated with primary biliary cirrhosis, Cowden disease, and tuberculosis ,92,93. TInterestingly, TPs associated with laryngeal lesions have low incidence of malignant transformation, in contrast to patients whose lesions are limited to trachea and bronchi, usually manifested in adulthood, and showing a higher incidence of malignant transformation ,94,95. AEsophageal squamous cell papillomas (ESP) are rare tumors that were first described in 1928 by Patterson et al.  and histESP is detected mostly incidentally by esophagogastroduodenoscopies in 0.01–0.45% of cases. However, although ESP is rare, its prevalence has been increasing; Pantham et al. (2017) reported an increase in ESP prevalence from 0.13% in 2000 to 0.57% in 2013 . ESP is The exact etiology of ESP is still uncertain, but several etiologic factors have been proposed such as chemical factors, mechanical factors, and infections with HPV and/or EBV ,100. TheThe role of HPV infection in ESP is still a controversial issue despite the fact that 40 years have elapsed since our original observations in the early 1980s. We performed a systematic review and meta-analysis of the literature reporting on HPV detection ESP, covering the literature through to May 2012. In total, 39 studies were eligible, covering 427 ESPs from different geographic regions . AltogetHelicobacter Pylori (H. pylori). Altogether, 19% (7/38) of the ESP analyzed were positive for HPV DNA; three for HPV6 and one each for HPV16, 18, 31, and 81. There was no correlation between the presence of HPV and patient’s age, reflux esophagitis, H. pylori, smoking habits, or location of ESP [Tiftikçi et al. (2017) analyzed HPV in ESPs diagnosed in 21 women and 17 men with a mean age of 41 years (range 17–67 years). Most of the ESPs were located at mid-esophagus (68%) . Eight on of ESP .n = 19 studies, 95% CI: 2–29%) and 26%  in BE samples. HPV prevalence was higher in EAC tissue than in esophageal tissue from healthy controls . The prevalence of EBV in EAC was 6%  [An association between HPV and esophageal adenocarcinoma (EAC) and Barret’s esophagus (BE) has been reported, as was summarized in a recent systemic review by Kunzmann et al. 2017, which examined a total of 30 studies . The poo: 0–27%) .ESPs have also been detected in children, as found for all SCPs in different sites of the aero-digestive tract. Recently, Tou and Al-Nimr (2021) completed a retrospective search through all endoscopies (EGD) performed (with various indications) in children under 18 years of age from 2000 to 2014 in their pediatric hospital . Of the It is currently well established that HPV infection is not exclusively a sexually transmitted infection (STI). HPV infection can be acquired at an early age or even during birth, as suggested by the presence of HPV in the placenta, amniotic fluid, and cord blood A feature common to SCPs in the aero-digestive tract is that they all present a bimodal incidence and prevalence pattern, being present in children and adults irrespective of their anatomic site. Thus far, however, only JO-RRP has been closely associated with maternal HPV transmission, though HPV can also infect both oral and pharyngeal mucosa vertically. This raises important questions challenging the future HPV studies: (1) can HPV spread from oral and pharyngeal sites distally to the larynx and esophagus? (2) Can HPV acquired at an early age become a latent HPV infection which becomes activated later in life in these sites? (3) Is HPV transmission via blood possible, as recently suggested by animal studies? These important issues are discussed here, and because animal studies have also shed light on this topic, they will be addressed in A meta-analysis of 3128 mother–child pairs confirmed that children born to HPV-positive mothers are 33% more likely to be HPV-positive than children born to HPV-negative mothers are. This risk was even higher (45%) when only HR-HPV infections were considered . A recenp < 0.001). Currently, the natural history of β-HPVs in children is practically unknown. However, though not widely studied yet, HPV-genotypes belonging to either β-, or γ-genera have been detected in sinonasal-, oral- and pharyngeal SCPs. [The transmission of cutaneous HPV genotypes from a mother to her newborn is thought to occur through skin-to-skin contact and during breastfeeding. Recently, Dassi et al. (2020) collected nasopharyngeal specimens from 0–12-month-old infants born by vaginal delivery (and breastfed at the time of sample collection) to investigate the perinatal transmission of α- and β-HPVs . In totaal SCPs. . More stn = 331 children; n = 329 mothers; and n = 131 fathers). The prevalence of oral HPV in these children varied from 8.7% to 22.8% over time, being highest at birth and lowest at the 3-year follow-up visit. Altogether, 18 different HPV genotypes were identified in the oral mucosa, of which HPV16 was the most prevalent, followed by HPV18, 6, 33, and 31. The prevalence of multiple-type infections varied from 0.3% to 3.7%. Only 41.4% (135/329) of the children remained HPV-negative for all oral samples , collected during the entire 6-year follow-up period [The next pertinent question is, what is the outcome of vertically transmitted α-HPV infections? We recently published data on oral HPV infections during the first 6 years of children born to 329 mothers included in the Prospective Finnish Family HPV Study (ongoing since 1989: p period .n = 4) acquired only HPV16 or HPV18 genotypes during the follow-up. When analyzed by gender, HPV prevalence was higher at 1-, 2-, 12-, and 36-month visits in boys, but not at the 6-year visit. Importantly, HPV positivity at birth or later was unrelated to the mode of delivery. The results showed that, during the 6-year follow-up, 63% (26/41) of these children carried the same genotype that was already detectable at birth, including 4/11 children positive with HPV6 at birth who still had this genotype at their 6-year follow-up visit [None of the HPV genotypes present at birth seemed to promote the acquisition of another specific HPV genotype, not even an HPV from the same clade. However, newborns with oral HPV6 or HPV11  ,123 examHPVs cannot be directly studied in animal models because PVs are strictly host species-specific. However, several animal models are useful for studying PV biology and immunity, as reviewed by Doorbar in 2016 . notablyNatural history studies on HPV infections of SCPs in the head and neck are lacking. However, the outcome of oral HPV infections most probably mimics that described in the early experimental study by Parsons and Kidd in 1943 . Thus, iExperimental transmission of OPs was successful in 81.5% (66/81) of the rabbits using crude suspensions of Bekerfeld filtrates of ground papillomas. New papillomas developed after 6–36 average 14) days, increased in size for about 1 month, and some papillomas persisted for as long as 400 days. The isolated virus, ROPV, was stable and a high temperature (75 °C to 80 °C) for 30 min was needed to inhibit viral infectivity. Rabbits with OPs already regressed or undergoing regression were resistant to ROPV re-infection. In some of the rabbits, the virus remained latent after clearance of the papilloma but could be activated simply by injuring or irritating the area. Viruses could be washed out of the mouths in the rabbits carrying the growths and from the mouths of rabbits with normal mucosa but carrying a latent infection. Transmission of the virus from a mother to her offspring was shown, and there were “papilloma families”, in which transmission of the virus from a mother to her offspring occurred via saliva during licking [ days, inA similar situation is seen in the multimammate mouse , in which latent MnPV  acquired early in the animal’s life can subsequently be activated to produce papillomas, keratoacanthomas, and other skin tumors .Activation of latent HPV infection could be the most likely source of primary and recurrent papilloma in the aero-digestive tract, as it is in the genital tract. Several animal studies have confirmed the ability of CRPV to persist at low DNA levels in clinically normal epithelium without visible disease. However, mild mechanical irritation or exposure to ultraviolet light could activate the virus, leading to the emergence of clinical lesions ,125,126,The evidence for HPV in blood mononuclear cells of women with genital HPV lesions was presented in 1991 , but theHPV transmission by blood represents a conceptually novel idea and, if accepted more generally, this would change the current thinking about the modes of HPV spread within the host.SCP in the upper aero-digestive tract is a rare disease entity, as summarized in HPV6 and HPV11 are the two major HPV genotypes present in all papillomas, irrespective of their anatomic location. However, HPV detection rate is highest in RRP, followed by exophytic SNP, OP, and SNIP. Importantly, although HPV association is highest with RRP, there is always a minor proportion of RRPs that tests HPV-negative (4–6%), and these HPV-negative papillomas can also progress to malignancy. Recent evidence has also shown that not only mucosal α-HPV genotypes but also cutaneous HPVs from the β- and γ-genera are detectable in sinonasal-, nasopharyngeal-, oropharyngeal-, and oral papillomas; however, until now, only one RRP case has been reported as testing HPV8-positive.HPV diversity in these lesions is much wider than previously considered, as summarized in


Among hr-HPV-infected participants, 82.8% (312/377) were infected with at least one of 9 valent vaccine-covered hr-HPV genotypes. From regression analysis, detection of abnormal cytology correlated positively with HIV infection (OR 2.17 [95% CI 1.51–3.13]), number of hr-HPV genotypes infected (OR 1.83 [1.59–2.10]), history of STI (OR 1.58 [1.14–2.22]) and No. of lifetime sexual partners (OR 1.56 [1.10–2.21]), albeit multivariate analysis identified the number of detected hr-HPV genotypes (adjusted OR 1.78 [1.54–2.06]) as the independent risk factor for abnormal cytology. High rates of anal hr-HPV infection, especially 9-valent HPV vaccine-preventable hr-HPV were detected among our MSM participants in Japan. HPV vaccination should also be encouraged for MSM in Japan.Anal high-risk human papillomavirus (hr-HPV) infection is widely considered a cause of anal cancer. However, epidemiological data are quite limited in Japan. This study investigated anal HPV infections and cytological abnormalities among MSM with or without HIV infection. Anal swabs were obtained, and cytological results were examined. Hybrid capture-based methodology was used for hr-HPV genotyping. The exclusion criterion was a history of vaccination against HPV. 644 subjects participated, and the overall prevalence of hr-HPV was 59.7% (95% CI 54.7–62.3), HIV-infected had higher prevalence than HIV-uninfected (68.9% vs 40.6%)  Anal cancer screening using anal cytology has recently been performed in a few developed countries6, and in such countries, HPV vaccination is recommended for all male and female adolescents (from 9 to 26 years). Moreover, the Advisory Committee on Immunization Practices (ACIPs) in 2019 recommended shared clinical decision-making regarding HPV vaccination for some adults aged 27 through 45 years who are not adequately vaccinated8, even though preventive effects of vaccination against anal cancer or new acquisition of hr-HPV in sexually active adult populations have not been clearly demonstrated.Anal chronic infection by high-risk types of human papillomavirus (hr-HPV) and HPV-related malignancies are a growing concern of public health among men who have sex with men (MSM). Indeed, the prevalence of hr-HPV is extremely high not only among MSM with anal cancer but also among asymptomatic MSM9.In Japan, the HPV vaccine for male individuals was approved only very recently, whereas genotyping of anal hr-HPV and anal-canal cancer screening are not currently approved. Furthermore, epidemiological data for MSM, including prevalent hr-HPV genotypes and related histopathological abnormalities, are quite limited, despite preliminary data suggesting high prevalenceIn the present study, we assessed the epidemiology of anal colonization by hr-HPV and HPV-related abnormal cytology among HIV-infected and uninfected MSM and sought to discuss future strategies to prevent HPV-related anal cancer in Japan.p < 0.001).We enrolled a total of 644 MSM, including 437 HIV-infected and 207 HIV-uninfected eligible consenting participants. The demographic characteristics according to HIV-infected and uninfected subjects are presented in Table 9/L (IQR 512–821).Compared to HIV-uninfected subjects, infected subjects had a significantly larger number of sexual partners in their lifetime but had fewer sexual partners in the past 6 months. There was no significant difference in the percentage use of condoms during anal sex in either group, but a history of STIs was significantly higher in HIV-infected than -uninfected subjects. All HIV-infected individuals were being treated with anti-retroviral therapy (ART), and plasma HIV RNA was undetectable (< 20 copies/mL) in 97.7% (427/437). For viremic patients, viral load was in the range of 21–119 copies/mL. CD4 counts were high, with a median of 666 × 10p < 0.001); HPV16, -52 and -58 were the most common HPV genotypes. The number of hr-HPV genotypes found in each participant stratified by HIV serostatus is illustrated in Fig. p < 0.001)), subjects with multiple infections were more prevalent among the latter than the former (182/425 (42.8%) vs 29/207 (14.0%) (p < 0.001).Among the participants, sufficient anal samples for hr-HPV genotyping were available for 632/644 98.1%) (425 HIV infected and 207 HIV uninfected). Figure % (425 HIp = 0.249), carried at least one of 9 valent vaccine-covered hr-HPV genotypes  (see Supplementary Table p = 0.0081 and p = 0.0058 respectively), .Additionally, we observed that 312 of 377 h-HPV-infected subjects (82.8%), including 246 HIV-infected (84.0%) and 66 HIV-uninfected (78.6%) participants (p < 0.001). The prevalence of all hr-HPV types was 45.0% (182/404) for normal anal cytology, 79.2% (152/192) for ASC-US, 94.4% (17/18) for LSIL, 94.4% (17/18) for ASC-H, and 75.0% (9/12) for HSIL. Anal abnormal cytology results were observed in 37.3% (240/644) of all participants, with more cases among HIV-infected individuals  than HIV-uninfected individuals  (p < 0.001). Next, we assessed the impact of hr-HPV genotype on abnormal anal cytology between HIV-infected and -uninfected participants. Figure p < 0.001), but with a significant difference only detected for HPV31, -51, -56 and -68. Correlation between the frequency of anal abnormal cytology and the number of hr-HPV genotypes detected in a subject is shown in Fig. p < 0.001)  subjects. Findings among participants with NILM , termed as normal cytology, or ASCUS + , termed as abnormal cytology, are shown in Table p < 0.001).Finally, univariate and multivariate regression analyses were performed to identify factors affecting development of abnormal cytology. In univariate analysis, HIV infection , number of detected hr-HPV genotypes , past treatment history of STI  and No. of lifetime sexual partners  were significantly associated with abnormal cytology results, as indicated in Table 9. These 3 genotypes might be the predominant hr-HPV genotypes among Japanese MSM during the last decade. In this study, the prevalence of anal hr-HPV among HIV-infected subjects was 68.9% [95% CI 64.4–73.2], with a lower rate of 40.6% for HIV-uninfected subjects [95% CI 34.1–47.4]. The high prevalence of hr-HPV in HIV-seropositive MSM is similar to reports for other Asian countries, but the distribution of hr-HPV genotypes differs somewhat11. For example, the prevalence of hr-HPV infection is reportedly 85.2% in Taiwan, 82.7% in China and 85% in Thailand, and the most frequent hr-HPV genotypes are HPV-16, HPV-51 and HPV-52 in Taiwan and Thailand and HPV-16 and HPV-18 in China11.Anal HPV infection and its contribution to the risk of anal cancer is a growing concern, especially among MSM. However, epidemiological data remain limited for MSM in Japan, regardless of their HIV serostatus. Our study was performed at one of the largest healthcare centers for the HIV-infected population in Tokyo, and 90% of HIV seropositive individuals in Japan are MSM. The center also has the largest sexual health clinic for HIV-seronegative MSM in Japan as of now, where regular check-ups for HIV and STI testing, as well as HIV pre- and post-exposure prophylaxis, are carried out. Therefore, this study should generally represent a large proportion of MSM in eastern Japan. The results of the study showed that the prevalence of anal hr-HPV infection among all participants was 59.7% [95% CI 55.8–63.4] and that the most frequently detected hr-HPV genotypes were HPV-52 (18.8%), HPV-16 (17.2%), and HPV-58 (16.0%). Our previous study conducted 10 years ago in HIV-infected MSM identified a prevalence of hr-HPV infection as high as 75.9%, and the most frequent hr-HPV genotypes were HPV-58 (30.2%), HPV-16 (28.8%) and HPV-52 (22.2%)10. In addition, HIV-infected individuals were found to carry more hr-HPV genotypes than HIV-uninfected individuals and had an increased risk of abnormal anal cytology. Moreover, this study clearly documents for the first time in Japan that the prevalence of abnormal cytology correlates positively with the number of hr-HPV genotypes detected . Nevertheless, it is not clear from the present cross-sectional study whether HIV infection preceding hr-HPV infection causes impaired clearance of hr-HPV infection or whether HIV-infected individuals re-acquire hr-HPV more readily.In terms of anal cytology, the prevalence of hr-HPV was 45% for normal anal cytology, 79.2% for ASC-US, 94.4% for both LSIL and ASC-H, and 75.0% for HSIL. These findings are consistent with previous reportsted Fig. b, as was13.There are some limitations to be considered in the present study. First, this was a single-site, clinic-based, cross-sectional study in Tokyo that does not represent the entire MSM population in Japan. Second, the study did not assess whether a case of hr-HPV infection was a transient infection, a persistent infection, or a reinfection. Third, this study only analyzed hr-HPV genotypes; therefore, not all hr-HPV genotypes were assessed. Finally, cytology results for HIV-uninfected subjects were reported only through a Pap smear and then translated to the Bethesda system using a previously reported relationship for the two systemsIn conclusion, we found that hr-HPV, especially 9-valent HPV vaccine-preventable hr-HPV genotypes, is highly prevalent among MSM in Japan. Our study showed that an increased number of hr-HPV genotypes infected, is strongly associated with development of anal abnormal cytology; thus, HPV vaccination for Japanese MSM, those already infected or not, should be considered to prevent further acquisition of more hr-HPV genotypes, that would lead to HPV related anal malignancy. Further studies are needed to assess the health impact and cost-effectiveness of nine-valent HPV vaccination.Written informed consent for HPV and anal cytology testing was obtained from all participants. This study was approved by the ethics committee of the National Center for Global Health and Medicine . This study was implemented in accordance with the provisions of the Declaration of Helsinki.There is an existing group of HIV-infected MSM being treated at AIDS Clinical Center (ACC), National Center for Global Health and Medicine (NCGM), Tokyo, as well as a non-HIV-infected MSM group at the Sexual Health Clinic (SHC) outpatient in the same center. We conducted a cross-sectional study in these two groups between January 1, 2019, and March 31, 2019, for HIV-uninfected individuals and June 1 to August 31, 2019, for HIV-infected individuals. Eligibility criteria included male sex, age ≥ 16 years from SHC and age ≥ 20 years from ACC, self-reported history of having anal receptive sex with men, willingness to undergo anal swab sampling to test for hr-HPV infection, and anal cytology. Exclusion criteria was a history of HPV vaccination.Each participant completed a standardized questionnaire. The information gathered included sociodemographic data (age), sexual behavior data , and history of sexually transmitted infection .digene® specimen collection swabs, with a 5 cm insertion into the anal canal and a slow 360-degree rotating extraction. The swab was then shaken in BD SurePath liquid-based cytology fluid, and all samples were processed at the external laboratory LSI Medience Corporation Tokyo, Japan. HPV DNA was detected by hybrid capture-based methodology, as described previously14, which included only oncogenic or hr-HPV genotypes 15. Anal cytology slides were interpreted by two cytopathologists blinded to the HIV or HPV infection status. Liquid-based cytology was prepared for routine Pap staining. The cytology results were reported first using Papanicolaou Classification (Class I–V)16 and also reported using Bethesda system classification categories, as follows: NILM, ASC-US, LSIL , ASC-H  and HSIL 16. Slides with discordant interpretations were reviewed again under a multihead microscope to reach an agreement. Cytology results for HIV-uninfected subjects were reported only using Pap smears and then translated to the Bethesda system using the previously reported relationship of the two systems13; results for HIV-infected subjects were reported using both Pap smears and the Bethesda system.Samples were collected at the enrollment visit by well-trained health-care professionals using p < 0.05) in univariate analysis.All statistical tests were evaluated at a 0.05 level of significance. Statistical analyses were performed with GraphPad Prism 7.0 and regression analysis with Statistical Package for Social Sciences (SPSS) software, version 23.0. We report categorical data as numbers (n) and percentages (%) and continuous variables as median and interquartile range (IQR). We compared differences in sociodemographics and sexual behaviors between HIV-infected and uninfected subjects and performed descriptive analysis. Mann–Whitney U tests were employed for continuous variables, and Chi-squared/Fisher's exact tests were used for categorical variables. The odds ratios (ORs) of significant factors associated with hr-HPV infection and the detection of anal abnormal cytological lesions were calculated using logistic regression analysis. Multivariate logistic regression models were then applied to the significant factors (Supplementary Information 1.Supplementary Information 2.


Using RNA sequencing analysis, we quantified viral transcription efficiency and found higher levels of E6 and the alternative transcript E6*I in cervical tumors with HPV genotypes other than HPV 16. These findings were validated using whole transcriptome data from The Cancer Genome Atlas (n = 304). For the first time to our knowledge, transcript expression level of HPV E6*I was identified as a predictive biomarker of CRT outcome in our complete institutional data set (n = 88) and within the HPV 16+ subset (n = 36). In vitro characterization of HPV E6*I and E6 overexpression revealed that both induce CRT resistance through distinct mechanisms dependent upon p53–p21. Our findings suggest that high expression of E6*I and E6 may represent novel biomarkers of CRT efficacy, and these patients may benefit from alternative treatment strategies.Persistent HPV infection is causative for the majority of cervical cancer cases; however, current guidelines do not require HPV testing for newly diagnosed cervical cancer. Using an institutional cohort of 88 patients with cervical cancer treated uniformly with standard-of-care chemoradiation treatment (CRT) with prospectively collected clinical outcome data, we observed that patients with cervical tumors containing HPV genotypes other than HPV 16 have worse survival outcomes after CRT compared with patients with HPV 16 Cervical cancer is the fourth leading cause of cancer incidence and mortality in women worldwide . In 2021+ tumors  tumors (Other) conferring worse prognosis is not well understood. Additionally, even within previously reported studies, the HPV 16 patient subsets have PFS and OS below 75%, indicating that — within this clinically benefiting group — there may be some patients who are likely to fail their standard-of-care treatment and would benefit from a more personalized treatment plan . A secondary goal was to determine how HPV transcript expression, including the expression of alternatively spliced forms of HPV E6, affects cellular response to standard-of-care CRT.Previous studies have demonstrated that HPV status and HPV genotype are both prognostic indicators in cervical cancer patients –18. Cerv+ tumors , 14. How) tumors –18. Thisent plan , 17. TheOther genotypes or multiple HPV genotypes detected were grouped together as HPVOther. Forty-eight patients had HPV16+ tumors, and 29 were HPVOther: 9 HPV-18, 4 HPV-33, 3 HPV-45, 3 HPV-52, 3 HPV-59, 1 HPV-31, 1 HPV-56, 1 HPV-58, 1-HPV 68a, 1 HPV-82, and 2 patients with multiple HPV genotypes detected (16+ tumors had better PFS (P = 0.049) and OS (P = 0.004) compared with HPVOther patients (n = 57) had better PFS (P = 0.012) and OS (P = 0.084) compared with those with α7 clade (n =16) (https://doi.org/10.1172/jci.insight.138734DS1). Viral integration state was assessed using RNA sequencing (RNAseq) analysis, and integration events occurred in 70.3% of HPV16+ and 85.7% of HPVOther tumors (P = 0.067) and OS (P = 0.057) compared with patients with episomal HPV, although these results were not statistically significant  and increased mortality (P = 0.018) after CRT  and HPVOther (n = 17) patient groups. The HPV E6* and E7 transcripts were more highly expressed than HPV E6 in both HPV16+ and HPVOther groups (Other tumors had an average of 2.9-fold increase in HPV E6 (P = 0.0085) and 2.0-fold increase in E6* (P = 0.025) transcript expression compared with HPV16+ tumors. There was an average 1.5-fold increase in HPV E7 transcript expression of HPVOther compared with HPV16+ tumors; however, this was not statistically significant  and episomal HPV (n = 14) patient groups . Similar to our cohort, 56% of tumor samples were HPV16+ and 43% were HPVOther in the cervix TCGA cohort (Other patient group had an average fold increase of 3.0 for HPV E6 (P = 8.1 × 10–12), 2.2 for E6* (P = 2.4 × 10–9), and 1.4 for E7 (P = 0.0012)  .P = 0.047) and OS (P = 0.033) after CRT . HPV16+ patients with high HPV E6* expression again exhibited worse PFS (P = 0.38) and OS (P = 0.33)  and low (RPKM < upper quartile). High HPV E6* expression was a poor prognostic indicator for PFS (fter CRT . Interesfter CRT . To dete = 0.33) , althougP = 0.032) and SW756  cell lines with low transcript levels . CaSki (HPV 16) had higher levels of E6, E6*, and E7 transcript expression compared with SiHa (HPV 16) and SW756 (HPV 18) cells . Next, wt levels . Taken tP = 0.0107). E6* overexpression in SiHa cells increased p53 protein expression compared with WT SiHa   , consistP = 0.017), whereas WT cells and cells expressing E6* had residual γH2AX foci suggestive of unrepaired DNA breaks in the latter 2 lines  and NOXA (P = 0.0089) transcript expression, and the SiHa E6 overexpressing cell line had lower PUMA (P = 0.045) transcript expression compared with the WT SiHa cell line  (+ cells compared with WT SiHa (P = 0.029) and SiHa E6 cell lines (P = 0.057) (P = 0.2). Increase in p21 is known to induce G1 cell cycle arrest   . The fun 0.0028) . High p5 0.0028) –21. To t= 0.057) . There we arrest ; howevere arrest . Additioe arrest . To test = 0.01) . Both th = 0.01) , 25.Lastly, we tested whether knockdown of p21 is sufficient to rescue treatment sensitivity in the p21 high E6* overexpressing cell line . TransieOther genotypes had higher viral transcript expression of HPV E6 and E6*. High HPV E6* expression was significantly associated with patient risk of developing disease recurrence and was a predictive biomarker of poor disease response following CRT. Interestingly, the prognostic value of high HPV E6* expression was also observed when restricting analysis to the HPV 16 patient subset, indicating that it may be a valuable genotype-independent predictive biomarker. These results suggest that more detailed analysis of HPV genotype and HPV-related gene expression could be used to identify cervical cancer patients at risk for treatment failure from standard-of-care chemoradiation.Treatment for cervical cancer is currently guided by clinical staging, which includes the results of physical examination and whole-body imaging. In general, early-stage cervical cancers are managed by surgical resection, while locally advanced cases are treated with pelvic irradiation and the concurrent administration of cisplatin chemotherapy. While this treatment strategy is effective, 30%–50% of patients with locally advanced cervical cancer will fail CRT, necessitating the need to develop predictive biomarkers to assist in treatment planning. Current clinical guidelines do not require HPV testing of newly diagnosed cervical cancer. In this study, we demonstrate that expression of an HPV genotype other than HPV 16 is a predictive biomarker for a poor disease response to the standard-of-care CRT using a population of patients who were uniformly treated with standard-of-care CRT with prospectively collected clinical outcomes. Furthermore, using RNAseq, we demonstrated that cervical tumors with HPV+ head and neck squamous cell carcinoma (HNSCC) where HPV 18 genotype has been identified as an independent poor prognostic factor of OS  are HPV related, and patients with HPV-related OPSCC have favorable outcomes after chemoradiation , 27. Moror of OS . In addik cancer . Based u16+ patients had viral integration, whereas 83% of patients with HPVOther tumors had viral integration  and OS (P = 0.057) after CRT; however, perhaps due to the small cohort size, these survival differences did not meet the threshold for significance  and can reprogram cells of the microenvironment to a tumor-favorable phenotype . SenesceThe enhanced DNA damage repair we observed in high E6 expressing cells should be further investigated to determine the mechanism of repair being affected by HPV E6 expression. In + tumors indeed have lower transcriptional efficiency of HPV E6, E6*, and E7. Using cervical cancer cell lines engineered to overexpress E6 and E6* from HPV 16, we demonstrate that transcript variants of HPV E6 are associated with treatment resistance to standard-of-care CRT. While overexpression of full-length HPV E6 is associated with enhanced DNA repair, overexpression of HPV E6* is associated with tumor cell senescence. These results support more detailed examination of HPV genotype and viral transcript expression in patients with locally advanced cervical cancer and suggest that this information may be used in the future to identify patients at risk of CRT failure. In addition, these results provide important insights into treatment resistance mechanisms for HPV+ tumors.In conclusion, using a population of patients uniformly treated with standard-of-care CRT with prospectively collected clinical outcome data, this study confirms HPV genotype and identifies, for the first time to our knowledge, HPV transcriptional efficiency as predictive biomarkers of poor patient outcomes after standard-of-care chemoradiation in cervical cancer. We validated the association between HPV genotype and viral transcript expression efficiency using the cervix TCGA cohort, which also demonstrated that HPV 16 Patient tumor biopsies and blood were obtained prior to the initiation of therapy and stored at the Tissue Procurement Facility at Washington University in St. Louis School of Medicine. RT consisted of external beam irradiation and intracavitary brachytherapy, per institutional guidelines (Washington University in St. Louis School of Medicine), and concurrent cisplatin or carboplatin chemotherapy was also administered. Median follow-up time for patients alive at the time of last follow-up was 62 months . Inclusion in downstream data analysis required detectable HPV in patient tumors. In the reporting of this data, we have adhered to REMARK guidelines .18F-Flouro-deoxy-glucose positron emission tomography (FDG-PET) scans at 3 months after therapy to assess treatment response.Patients were followed at the following time points after the completion of CRT: 6 weeks, 3 months, 6 months, 12 months, 18 months, 2 years, 3 years and 5 years. Physical examination including a pelvic exam was performed at each visit. Patients received posttreatment 16+, HPVOther, or no HPV detected in the sample (HPV undetected). Patients with undetectable HPV were excluded from study analysis.DNA isolated from 88 pretreatment tumors were sequenced using next-generation sequencing, and probes for the following HPV genotypes were used to identify the HPV genotypes present within the tumor: high-risk HPV-16, -18, -31, -33-, 35, -39, -45, -51, -52 -56, -58, and -59 genotypes and low-risk HPV-6b, -11, -26, -30, -53, -54, -57, -61, -66, -68a, -70, -72, -73, -82, -98, -99, -100, -104, -105, and -113. SAMtools idxstats  was usedE6,JN) and E6*  were counted, as well as the number of reads aligned to E6 between the splice sites . The remaining aN reads align to the ranges over which E6 and E6* are identical. Total counts of E6 (E6N) and E6* (E6*N) reads were, therefore, obtained by assigning these reads to WT or E6* transcripts based on the proportions of each type of junction spanning read normalized by the size of the nt ranges from which such a read could arise, as in the following formulas:RNA was extracted, with 68 patients yielding at least the 0.5 μg of RNA with RIN > 7 sufficient for library preparation. RNAseq was performed for these patient samples using the Illumina HiSeq 3000. Raw sequencing reads were aligned to reference human and HPV viral genomes in GRCh38.d1.vd1 using STAR 2.7.0f, with chimSegmentMin set to 18 . STAR-geCounts of reads aligned to HPV E6, E6*, and E7 were then normalized by RPKM.Viral gene expression and integration status was obtained for 304 TCGA-CESC primary tumor samples using a similar method to that employed for our institutional data. RNAseq reads were extracted and aligned to reference human and HPV viral genomes in GRCh38.d1.vd1 using STAR 2.7.0f with chimSegmentMin set to 18. Reads that were chimeric between human and HPV references were extracted, and samples with greater than 1 such chimera per million total reads were designated as positive for HPV viral integration. Normalized HPV E6, E6*, and E7 expression levels were also obtained as above.2. Mycoplasma testing was performed periodically to verify no infection. Last date of mycoplasma testing for cell lines used in this study was July 17, 2019, for SiHa and January 22, 2020, for CaSki and SW756. Experiments were performed on cell lines under passage 30. CaSki, SiHa, and SW756 were used to evaluate endogenous expression of HPV E6, E6*, and E7 transcripts and determine cell sensitivity to radiation treatment by alamarBlue (Thermo Fisher Scientific). SiHa had the lowest transcript expression for all HPV transcripts evaluated and was selected to engineer cell lines for the overexpression of HPV 16 E6* and E6. The mammalian expression vector pCMV-6 was used as the vector for exogenous HPV transcript expression. Origene VC101902 HPV 16 E6 open reading frame (ORF) was used as the codon optimized sequence for HPV 16 E6 minigene (Integrated DNA Technologies) and customized for HPV 16 E6* with an early stop codon inserted at cDNA position 151 (G > T) (Origene); sequences are provided in Supplementary File 1. HPV 16 E6 and E6* ORFs were restriction enzyme digested with 10 U/μL SgfI and 10 U/μL MluI, ligated into pCMV-6 vectors using T4 DNA ligase, and transformed into 5-α competent E. coli (New England Biosciences [NEB]) overnight and then selected using 25 μg/mL kanamycin LB agar plates. Midiprep of plasmids was performed using ZymoPure II Plasmid Midiprep Kit (ZymoPure) according to manufacturer’s protocol. Purified vectors were transfected into SiHa cells using Lipofectamine 3000 (Thermo Fisher Scientific). Transformed cells were G418 selected, and resulting cell pools were validated using qPCR for vector E6* and E6 sequence expression.Cervical cancer cell lines were obtained from the American Type Culture collection (ATCC) and maintained in IMDM media (Invitrogen) with 10% heat-inactivated FBS and incubated at 37°C in 5% COCells were lysed with Cell Lysis Buffer , proteinase/phosphatase inhibitors and PMSF on ice for 30 minutes and then sonicated using the Bioruptor UCD-200 (Diagenode) on high for 5 minutes. NuPAGE LDS Sample Buffer (4×) (Invitrogen) and SDS buffer (2×) were added, samples were boiled at 95°C for 10 minutes and gel electrophoresed on 4-20% gradient gels , transferred to PVDF blot, and blocked with 5% TBST milk. Blots were incubated with primary antibodies at 4°C overnight; anti-p21 . HRP-conjugated secondary antibodies were incubated 1 hour at room temperature; anti-rabbit , P53-HRP , and actin-HRP (1:5000 sc-47778) (Santa Cruz Biotechnology Inc.) were used with ECL chemiluminescent reagent . Blots were visualized and quantified using the Bio-Rad ChemiDoc MP imaging system and Image Lab software (Bio-Rad).CDKN1A, NOXA, PUMA, BAX, GADD45A, and MDM2 (Thermo Fisher Scientific). Probes for the DREAM target genes — BIRC5, CDC25c, KIF23, MYBL2, and PLK4 — were obtained from Thermo Fisher Scientific. GAPDH and ACTB were used as reference genes. The qPCR was run at 50°C for 2 minutes, 95°C for 10 minutes, and 60°C for 1 minute using the 7900HT Fast Real-Time PCR System (Applied Biosystems). The specificity of the reaction was verified by melt curve analysis. Data processing was performed using SDS 2.4 software (Applied Biosystems), and the relative quantitation of each mRNA was performed using the comparative Ct method as described earlier . Altogether, 1 μg of total RNA was used to synthesize cDNA using High Capacity RNA-to-cDNA  kit per manufacturer’s protocol. Primers for HPV 16 and -18 native and vector sequences were synthesized by Integrated DNA Technologies, from reference sequences  or as de earlier .+ cells were quantified as number of cells per image frame with bright-field microscopy, in nonoverlapping regions.Cells were plated and grown in culture for 7 days, harvested, and stained using the Senescence β-Galactosidase Staining Kit  per manufacturers protocol. SA-β-gal6 cells per well and counted 3, 7, 9, and 12 days after plating on Vi-CELL cell counter (Beckman Coulter). For both cell viability and clonogenic survival, cells were treated with vehicle (0.01% DMSO) or 0.5 μM carboplatin 1 hour prior to radiation treatment (2 and 4 Gy). Cell viability was assessed 5 days after treatment using alamarBlue (DAL1025) (Thermo Fisher Scientific) per manufacturers protocol; viability was normalized to untreated samples. For colony formation assay, cells were treated as described, and 48 hours after treatment, cells were detached with 0.05% Trypsin and replated at 500 cells/well in a 6-well dish. Cells grew for 10–12 days, until untreated control wells reached colonies of > 50 cells. Plating efficiency (PE) was calculated as (number of colonies in control)/(500 cells seeded), and surviving fraction (SF) was calculated as (number of colonies in treated wells)/PE × (500 cells seeded). Cell lines were normalized to their own untreated control wells. For p21 knockdown colony formation assay, subconfluent (60%–70%) cells were transfected with 100 nM of p21 siRNA or control siRNA (Santa Cruz Biotechnology) using Lipofectamine RNAiMAX reagent (Invitrogen) in opti-MEM per manufacturer’s protocol. After 48 hours of siRNA transfection, transfected cells were irradiated and then processed as described.To assess cellular proliferation, cells were plated at a density of 0.4 × 10SiHa, E6, and E6* cells were seeded on chamber slides (Nunc Lab-Tek) and treated with single-fraction 2 Gy RT. Slides were fixed with 4% paraformaldehyde at 10 minutes, 2 hours, 6 hours, 12 hours, and 24 hours after irradiation. Cells were permeabilized and processed for immunofluorescence according to standard protocol. Slides were incubated with primary antibody 1:300 anti–phospho-Histone H2AX  at 4°C overnight. Secondary antibody 1:500 Alexa Fluor 488 goat anti–rabbit IgG  incubated 2 hours at room temperature. Slides were mounted using ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). Slides were imaged with the Axio (Carl Zeiss) microscope.Cell cycle and apoptosis were assayed using flow cytometry. For the following assays, cells were plated; the next day, they were treated with 0.5 μM carboplatin plus 4 Gy radiation. Cells were harvested at 24 and 48 hours following treatment. For cell cycle analysis, cells were washed and then stained with propidium iodide. For apoptosis, cells were washed then stained with annexin V and propidium iodide. Flow cytometry analysis was performed on MacQuant Analyzer and quantified on FlowJo. For each sample, 5000 events were collected.2 and Fisher exact tests were used for all other patient categorical variables. The Wilcoxon’s signed-rank test and 2-tailed Student’s t tests were used for in vitro experiments. One-way ANOVA was used when comparing more than 2 groups. P less than 0.05 was set as the threshold for significance for all study outcomes.Tumor recurrence and OS were the primary endpoints of the study. Survival outcomes were measured from the completion of treatment. Recurrence was defined as new or progressive disease both locally within the pelvis and distantly outside the RT field. The Kaplan-Meier method and log rank test were used to determine differences in PFS and OS. R version 3.5.2 and the packages survminer and survival were used for the analysis. Wilcoxon’s signed-rank test was used to compare patient age between HPV cohorts. The χThe study population included 88 patients prospectively enrolled into a tumor-banking protocol (January 2008 through July 2011). This study was approved by Washington University in St. Louis IRB, and all patients provided written informed consent for sequencing . RNAseq data can be accessed at Gene Expression Omnibus (GEO) with accession no. GSE151666.SM, PG, and JKS contributed by writing the human studies protocol, enrolling patients, obtaining cervical tumor biopsies, prescribing and performing CRT, and prospectively collecting clinical outcome data. MDM and CAM performed HPV genotyping on the Washington University cohort tumor samples. FJR, MI, JZ, SM, NG, MG, RR, NM, and JKS conceptualized the study, designed experiments, and performed analyses. FJR, MI, and JZ performed data analysis of HPV genotypes, transcript expression, and integration status using Washington University and TCGA patient data. FJR, NM, and NG performed in vitro experiments. FJR and JKS prepared the manuscript. All authors contributed to final review of the manuscript.


The human papilloma virus (HPV) high-risk variants (HPV-HR) such as HPV16 and HPV18 are responsible for most HPV related cancers, including anogenital and head and neck cancers. Here, we present two patients with HPV-HR-associated gynecological malignancies who, after failing radiation therapy, were treated with experimental salvage immunotherapy regimen resulting in complete, durable responses in both patients. Each patient was diagnosed with recurrent, radiation-refractory, HPV-HR positive, squamous cell carcinoma of the lower genital tract. Patient A was a 90-year-old, African American, with metastatic vulvar cancer to the right inguinal-femoral triangle and pulmonary metastases. Patient B was a 41-year-old, Caucasian, with a central-recurrence of cervix cancer. Each patient received at least two intratumoral quadrivalent HPV-L1 vaccine (Gardasil™) injections and daily topical TLR-7 agonist (imiquimod) to the tumor surface 2 weeks apart. This combination of intratumoral vaccinations and topical TLR-7 agonist produced unexpected complete resolution of disease in both patients. The importance of radiation therapy, despite being considered a treatment failure by current definitions, cannot be understated. Radiation therapy appears to have offered a therapeutic immune advantage by modifying the tumor microenvironment. This immune protocol has potential to help patients with advanced HPV-HR-related malignancies previously considered incurable. The human papilloma viruses (HPV) are responsible for ∼30% of viral-induced cancers. There are at least fourteen known oncogenic or high-risk HPV (HPV-HR) types. Prophylactic HPV vaccines have proven effective in preventing benign and malignant manifestations of this virus in both men and women. Unfortunately, these vaccines require at least 10–20 years “lead-in” time before showing an epidemiological-effect in cancer prevention. Despite the proven successes of these vaccines, society has neither embraced the significance nor adequately utilized this medical advancement. In addition, immunotherapies to effectively treat HPV-HR cancers have been elusive. We present two patients with HPV-HR positive gynecological malignancies who recurred following radiation therapy. Both patients were desperate and facing dismal prognoses. We describe the “off-label” use of two FDA-approved immune drugs consisting of intratumoral vaccinations of quadrivalent HPV-L1 vaccine (Gardasil™) and daily application of topical imiquimod resulting complete resolution of radiation refractory disease in both patients.These are the only two patients treated with this combination of intra-tumoral vaccinations of quadrivalent HPV vaccine and topical imiquimod after presenting with radiation refractory disease. Each female patient had personal and logistical issues limiting travel for inclusion into regional/national research studies. Both patients requested alternative treatment options that could be administered during their current treatment choices. Following Bioethics Committee review and approval, of each patient was consented and received the immune regimen of intratumoral injections of quadrivalent HPV-L1 vaccine (Gardasil™) and topical imiquimod . DemograPatient A was a 90-year-old African-American female with recurrent, HPV-HR positive, squamous cell carcinoma (grade 1) of the vulva with metastatic disease to the right inguinal-femoral triangle (11 × 11 cm tumor) and bilateral pulmonary metastasis. Previously, this patient presented with a 4 cm peri-clitoral, grade 1, squamous cell carcinoma treated by radical hemi-vulvectomy and bilateral inguinal sentinel node biopsies. The vulvectomy specimen demonstrated no lymph-vascular space invasion and the specimen’s margins were clear. Bilateral inguinal sentinel lymph nodes, consisting of two nodes on the left and three nodes from right, were negative for malignant cells. No further therapy was required, and the patient never returned for follow-up until presenting 3 years later with 11 × 11 cm squamous cell carcinoma of right groin causing a right lower extremity deep vein thrombosis. Chest x-ray revealed a “snow storm” appearance from bilateral pulmonary metastasis . The pat3  followed by two low-dose-rate cesium brachytherapy implants . By the second tandem and ovoid application, the cervical tumor had completely resolved. Four weeks after her second cesium application, Patient B presented to the office complaining of vaginal discharge. Exam found a 3–4 cm recurrent, grade 1, squamous cell carcinoma,  treating 90-year-old women with multiple cutaneous, basaloid, squamous cell skin cancers [There are several studies describing vaccine-based immunotherapies to treat malignant tumors. A complete review of therapeutic vaccines can be found elsewhere , most de cancers . Though  cancers .in-situ vaccine.” [Radiation therapy is vital in changing the tumor’s well-established, immunosuppressive microenvironment . Fractioaccine.” , 12, 14 Radiation therapy, when use as a single treatment modality, has not consistently produced therapeutic responses outside of the radiated field known as “abscopal effects” without the addition of adjuvant immunotherapies , 12. UnfFrom a safety standpoint, this immune protocol produced no discernible toxicities in either patient. Our observations suggest that combination of imiquimod and quadrivalent HPV vaccine is likely less toxic and more tolerable than systemic cytotoxic chemotherapies. Most immune therapies do not result in cross-resistance with conventional therapies , 17. TheWe describe two female patients presenting with radiation-refractory, HPV-associated, squamous cell carcinomas of the lower genital tract facing death, patient A, or pelvic exenteration, patient B. Each patient was treated with an experimental therapy consisting of intratumoral injections of the preventative, quadrivalent HPV-L1 vaccine and topical application of imiquimod within 4 weeks following radiation therapy. Each patient demonstrated complete resolution of the treated tumor in addition to patient A’s clinical abscopal response of her pulmonary metastases allowing her to live an additional 9 months 2 weeks after starting this combination immune therapy. Currently, Patient B is alive with no evidence of disease 8 years following the modification of her surgery to a less radical operation due to resolution of her central recurrence 8 days after her second cycle of intratumoral therapy. We believe radiation therapy followed by intratumoral vaccinations and topical TLR-7 agonist resulted in a “prime-boost” response facilitating an appropriate cytotoxic antitumor response. If this combination of cancer therapies can be validated in appropriate clinical trials; this therapy could offer an urgent and unmet need for patients with treatment-refractory, HPV-related malignancies. Additionally, both medications in this report are FDA-approved for other conditions and have established safety profiles. The combination of radiation therapy followed by sequential intratumoral vaccinations and topical imiquimod could conveniently translate to the clinic setting and offers potential cost savings.


The human papillomavirus (HPV) vaccination rate in Japan has fallen to nearly zero since the suspension of governmental proactive recommendations in 2013, owing to the development of purported adverse events.This study aimed to evaluate the effects of a brief web-based educational intervention using the theory of behavioral insights on the willingness of adults to consider the HPV vaccine for their daughters and sons.We recruited 1660 participants aged 20 years or older in March 2018 via a webpage and provided them with a 10-item questionnaire related to the following aspects: awareness regarding HPV infection and vaccination, willingness for immunization, and actions for prevention. We randomly stratified participants based on sex and age with or without a brief educational intervention involving scientific information presented in an easy-to-read format.Only 484 (29.2%) of the respondents were aware of the benefits of HPV vaccination. Although only 352 (21.2%) of the respondents displayed a willingness for immunization of their daughters, there were 40 (4.8%) more respondents in the intervention group with this willingness . In a subanalysis, the willingness toward vaccination for daughters in men was significantly higher in the intervention group . However, such a difference was not observed among women .This study suggests that a brief web-based educational intervention increases the willingness of adults to consider the HPV vaccine for their children, especially among men. Thus, providing adequate information to men may be a useful strategy to improve the currently low rates of HPV vaccination.UMIN Clinical Trials Registry UMIN000049745 (UMIN-CTR); https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000049745 In Japan, the human papillomavirus (HPV) nonavalent vaccine was approved by the Pharmaceuticals and Medical Devices Agency (PMDA) for girls aged 9 years or older in July 2020 . The corAccording to the strategy of the World Health Organization devised in 2019 for controlling CC , by 2030In addition to HPV-related cancers, vulvar, anal, and throat malignancies may be prevented by the vaccine ,10. A JaSpecific information regarding the effects and adverse events of HPV vaccines needs to be fully disseminated to the Japanese population. Considering this, approximately 40% of the population is willing to be vaccinated . TherefoVaccine awareness programs are necessary, and campaigns through the media and social network services can play significant roles ,4. In anWe recruited a total of 1660 participants in March 2018 via a specially designed webpage for this study. These were registered members of the research panel owned by Macromill Inc . The participants were 20 years old or above as on March 12-13, 2018. They were recruited until the target sample size was fulfilled. We randomly assigned each participant to respond to an identical questionnaire after (intervention group) or prior to (control group) providing behavioral insights material  featuring brief scientific information presented in an easy-to-read format  to each group. The intervention was performed using an automatic web-based allocation system stratified by the sex  and age  of the participants. Randomization was performed by the web-research system of Macromill Inc. Participants and investigators were blinded to the distribution (double-blinded). Once the upper limit of each stratum was reached, new participants could not be added to the web system. This ensured uniform distribution of the stratification factors. In the intervention group, we provided the BI-material prior to answering questions related to preventive awareness, following consent for the online study. The control group was provided with the same material as that provided to the intervention group after all responses were completed.The participant demographics included age, sex, marital status, number of children, sexual experience, household income, medical background, education, and tobacco use. For female participants, we also collected information regarding HPV vaccination history and previous pap screening. Medical background was defined as follows: a respondent who had a history of working as a medical professional  or who attended medical school.The respondents completed a 10-item questionnaire. The first half determined HPV awareness as background information, and the second half addressed willingness to receive HPV vaccines and undergo screening tests. The respondents were instructed to answer “Yes” or “No” for each question.The awareness questions were as follows:Q1. It is possible to detect both cancer and precancerous lesions through CC screening.Q2. Sexual experience is associated with HPV infection.Q3. CC screening is necessary for women even after vaccination.Q4. I have heard of the benefits of the HPV vaccine.Q5. I have heard of the adverse events associated with the HPV vaccine.The willingness questions were as follows:Q6. If you have/had a daughter, do/would you consider getting her vaccinated against HPV?Q7. If you have/had a son, do/would you consider getting him vaccinated against HPV?Q8. Would you consider undergoing a pap smear? If male, will you want your family or partner to undergo a smear test?Q9. Would you undergo the cancer screening tests recommended by the government?Q10. Do you plan informing family members, friends, or others about cancer prevention and screening ?t test, the chi-square (χ2) test, and multiple regression analyses. These were performed using SPSS, version 27 (IBM Corp).Statistical evaluation comprised the Student P value of .05. P values less than .05 were regarded as significant. The hypothetical baseline willingness rate in the control group was determined based on our previous study [The sample size was calculated as 80% powered to detect a 10% effect in the intervention group (increased from 40% in the control group to 50% in the intervention group) with a two-sided us study . The samThe adjusted odds ratios (aORs) related to the background knowledge level from the binominal logistic regression were analyzed to assess differences in both groups. In the subanalysis, questions Q2 and Q4, for which the responses showed a significant difference in the rate of awareness between the two groups (data not shown), were included as covariates in the aOR in the analysis for men. Questions Q1, Q2, and Q5, for which the responses showed a significant difference in the rate of awareness between the two groups (data not shown), were included as covariates in the aOR in the analysis for women.The study protocol was approved by the Institutional Research Ethics Committee of Yokohama City University School of Medicine (A180200004). The trial registration number is UMIN000049745. We received research funding from the Japan Agency for Medical Research and Development (grant number 15ck0106103h0102). The survey was outsourced to Macromill Inc.P=.96), children (P=.84), household income (P=.58), sexual experience (P=.26), education (P=.44), medical background (P=.50), and tobacco use (P=.64)  .For questions Q1 to Q5 regarding HPV awareness, the recognition rate for Q2 was significantly higher (by 7.8%) in the control group than in the intervention group. For questions Q4 and Q5, the values in the intervention group were significantly higher  than those in the control group .Only 484 (29.2%) of the respondents were aware of the benefits of HPV immunization (Q4), whereas 543 (32.7%) were aware of the adverse effects (Q5).Only 352 (21.2%) of the respondents displayed a favorable attitude toward HPV immunization for their daughters (Q6). However, an additional 40 (4.8%) participants responded affirmatively in the intervention group  compared to those in the control group . For Q7,P=.37) or to communicate HPV-related issues (Q10) did not increase (P=.17) (The number of respondents expressing a desire to receive a screening test (Q8) ( (P=.17) .P=.02; P=.22; Differences were identified in Q6  and Q7 . The willingness to consider vaccination for daughters in men was significantly higher in the intervention group , and the willingness to consider vaccination for sons was also higher in men than in women .In an overall comparison between men and women irrespective of intervention, the willingness to consider vaccination for daughters in men was significantly higher than that in women  and Q7 . While in the control group, differences were not identified between men and women for Q6  and Q7 .In the intervention group, higher rates were identified in men than in women for Q6  to assess the benefits of BI-material employing brief scientific information and its ability to motivate individuals to consider the HPV vaccine for their children. Our results showed that providing brief scientific information could increase the willingness to consider HPV vaccination for daughters and sons. This effect was observed typically among male participants. Similar minor interventions may potentially modify mindsets favorably. However, such brief digital information failed to affect the mindset in women. A possible reason why the intervention was more effective among men than women is that women had a more negative image toward HPV vaccination. There was a significant difference between men and women in the awareness level. Overall, 45.7% of women responded that they know about the adverse events of HPV vaccination, while this rate was 19.8% in men (data not shown). Such awareness might have influenced the difference in the intervention effect.In terms of the COVID-19 pandemic, a difference in awareness of prevention strategies was observed between men and women ; therefoAccording to a systematic review regarding the effect of the pedagogical approach on social awareness or action, more reliable and validated studies are required to change the perception or mindset of the target population . This paA systematic review revealed that general communication about childhood vaccination resulted in a positive change by 20%; however, this excluded the HPV vaccine . In contA US RCT reported that education through social media is effective for improving general awareness regarding vaccination. However, the study was limited to a sample of 58 participants . A phaseWe recognize several limitations in this study. First, the sustainability of effective change was not evaluated. Typically, with respect to health issues, taking action requires time. Therefore, a study should assess not only a change in mindset, but also the appropriate course of action. We have already performed an RCT (UMIN000039273) assessing the sustainability of general acceptance and concrete behavior for HPV vaccination. Second, the impact of the study on the behavioral outcome was unclear; specific vaccination functioning needs to be tracked. Third, selection bias was present as the respondents were Japanese individuals enrolled by an internet survey company.Based on the sex-wise subanalysis, improved information in male participants may be the key to improve the rate of HPV vaccination in Japan. Additionally, video-based content with patient feedback is expected to result in a broader impact. Thus, a varied approach for men and women may be required.Our study revealed a positive outlook toward HPV vaccination following a brief web-based educational intervention, especially among men. Such an approach is extremely effective to overcome challenges related to communication and information overload.A brief web-based educational intervention based on the theory of behavioral insights increases the willingness of Japanese adults to consider the HPV vaccine for their daughters and sons.


Human Papillomaviruses have co-evolved with their human host, with each of the over 200 known HPV types infecting distinct epithelial niches to cause diverse disease pathologies. Despite the success of prophylactic vaccines in preventing high-risk HPV infection, the development of HPV anti-viral therapies has been hampered by the lack of enzymatic viral functions, and by difficulties in translating the results of in vitro experiments into clinically useful treatment regimes. In this review, we discuss recent advances in anti-HPV drug development, and highlight the importance of understanding persistent HPV infections for future anti-viral design. In the infected epithelial basal layer, HPV genomes are maintained at a very low copy number, with only limited viral gene expression; factors which allow them to hide from the host immune system. However, HPV gene expression confers an elevated proliferative potential, a delayed commitment to differentiation, and preferential persistence of the infected cell in the epithelial basal layer, when compared to their uninfected neighbours. To a large extent, this is driven by the viral E6 protein, which functions in the HPV life cycle as a modulator of epithelial homeostasis. By targeting HPV gene products involved in the maintenance of the viral reservoir, there appears to be new opportunities for the control or elimination of chronic HPV infections. Human papillomaviruses (HPV) comprise more than 200 types that are grouped into five genera  with tropisms for skin and mucosal epithelial sites. Infection leads to characteristic HPV type-specific disease pathologies, ranging from benign hyperproliferative lesions to inapparent or asymptomatic infections that can, in certain individuals, progress to high-grade neoplasia and invasive cancer. As a group, these viruses have evolved to infect and thrive at many different body sites by adapting to local epithelial conditions, which vary not only between cutaneous and mucosal epithelia, but also amongst epithelial appendages, such as hair follicles and sweat glands, with each of these sites providing distinctive epithelial niches and mechanisms of site-specific immune regulation ,2.Studies on the “high-risk” HPV (hrHPV) types, which are implicated in the development of cervical cancer, have, in the last few decades, led to the development of HPV nucleic acid tests useful in cervical screening, as well as to the development of prophylactic vaccines . The widDespite the differences in their epithelial tropisms and pathogenicity, all HPV types have a similar genome arrangement and gene functions . SeveralOne advantage of developing treatments is that HPV infects the squamous epithelium that covers the surface of the body, which means that any potential anti-HPV drug candidate can be administered topically. Topical administration is considered an effective method of drug delivery, especially for HPV-associated lesions, because it also avoids first-pass effects in the liver, which makes it possible to administer drugs whose side effects are too severe to be administered systemically. In fact, the anticancer drugs 5-FU and Bleomycin have been used topically or by local injection in cases of verruca vulgaris . For conHPVs primarily infect stratified epithelial tissue and cause chronic infections. To maintain a persistent infection for years or decades at stratified epithelial sites, HPV needs to infect basal cells and form a reservoir of infected cells in the epithelial basal layer. Infected cells must remain in the basal layer for prolonged periods of time alongside their uninfected neighbours, and maintain the viral genome in those cells without detection. HPVs are thought to confer such properties on the cells they infect as a result of expression of the viral E2 and E6 proteins  Figure , and becE2 is a transcriptional repressor that directly regulates E6 and E7 gene expression by binding to the viral promoter region, but E2 also modulates viral genome replication and partitioning  during persistent infection . In cellThe primary function of the E7 protein is to drive cells’ S-phase cell cycle re-entry in the upper epithelial layers, which is important for viral genome amplification in differentiated cells ,54. The Epithelial development and maintenance require cellular attachment to the basal lamina, cell–cell contact inhibition as a way to regulate cell density and proliferation, and in due course, delamination of cells from the basal layer that are destined to differentiate. Understanding epithelial homeostasis and how papillomaviruses modulate it are thus of considerable importance for the development of effective treatments. Contact inhibition classically regulates proliferation in multicellular colonies, a process whereby cultured cells stop dividing when they become confluent and fully occupy a 3D space . In monoThe host machinery for epithelial homeostasis is broadly conserved among different epithelial sites. HPVs have co-evolved with their host and become adapted to their respective niches, and their E6 proteins now target similar pathways in infected cells in subtly different ways . The α-HBy contrast, the way that other cutaneous lrHPV E6 proteins  affect basal cell commitment to differentiation is not as direct as seen with the α-HPV types, which modulate total Notch receptor levels via p53. LrHPV types from outside of the α-group modify the host cellular environment through the Notch-MAML-dependent pathway . The E6/To date, anti-HPV therapies that target E6 have been evaluated with respect to their cytotoxicity and their role in stimulating apoptosis in HPV-associated cancer cell lines . AlthougUndoubtedly, the availability of prophylactic vaccines and the implementation of screening programmes have successfully reduced the burden of hrHPV infections and associated diseases, including cervical cancer. However, these efforts do little for those who are infected by non-vaccine HPV types or who have not been vaccinated, or for individuals infected by the prevalent lrHPV types, which continue to impose a considerable economical and medical burden on the public. The development of anti-HPV therapies remains a pressing issue if HPV-associated morbidity and mortality are to be eliminated.A primary role of the E6 protein during productive infection is to interfere with normal epithelial homeostasis to facilitate viral persistence. In normal stratified epithelium, basal cells respond to contact inhibition of proliferation to maintain an appropriate and balanced basal cell density. Cells that are more sensitive to mechanical deformation by crowding stress will be extruded towards the apical epithelium, signalling the cytoskeleton of the adjacent cell to contract, and for ongoing neighbouring cell proliferation to force its loss from the epithelial basal layer. The delaminating cell must commit to differentiation by activating the Notch signalling pathway in order to maintain the stratified epithelium. To retain infected keratinocytes at the basal layer, HPV encodes its E6 protein to alter the process of contact inhibition of proliferation, cellular junction formation, and commitment to differentiation. For clearance of HPV infection, we suspect that the mechanisms that allow persistence of viral reservoir must be understood at a molecular level, and in due course, targeted by anti-virals. Specific in vitro models of the basal and spinous layer of the epithelium open the way for future investigation of how HPV regulates normal epithelial homeostasis, and will facilitate the development of “true” anti-HPV therapies.