The Human Papillomavirus (HPV) has different strategies for persist in the cells. This characteristic has led us to consider the presence of the virus in tissues of the oral cavity that had no clinical signs of infection. The aim of this study was to detect the presence of DNA-HPV at multiple sites of the oral cavity. A case-control study was designed: Oral Squamous Carcinoma Group (OSCG), healthy n=72 and Control Group (CG), n=72, healthy volunteers paired by sex and age with OSCG. Four samples were taken from OSCG: saliva, biopsy, brush scraping of lesion and contralateral healthy side. In CG a saliva sample and a scratch of the posterior border of tongue were collected. HPV was detected by PCR using Bioneer Accuprep genomic DNA Extraction kit, and consensus primers MY09 and MY11. Chi square test was applied. 432 samples were obtained from 144 individuals. DNA-HPV was detected in 30 (42%) of OSCG subjects and 3(4%) of CG. Two or more positive samples were obtained in 67% of the OSCG, 67% in saliva and 60% in biopsy; in CG 100% of the individuals were positive in the two samples. HPV is frequently present in oral cavity as a multifocal infection, even without the presence of clinical lesions. Key words:HPV, Oral cavity, Saliva, Oral cancer. The first cells to become in contact with the human papillomavirus (HPV) in the oral cavity, as in the genital mucosa, are undifferentiated cells of the basal layer of the epithelium. This contact implies the previous existence of ulceration or micro-wounds on the epithelial surface that allows its entry -3. ViralWe do not find in the current literature at our disposal, studies on the presence of simultaneous HPV infection in different tissues of the oral mucosa. Nevertheless, some authors reported the presence of HPV in different sites of the anogenital tract without clinical symptoms of infection. These studies report cases of simultaneous infection of the oral and genital mucosa -11. StudThe objective of this work was to detect the presence of DNA-HPV in multiple sites of the oral cavity.A case-control study was designed and conducted between 2010 to 2017, which was approved by the Institutional Committee of Ethics in Health Research from the School of Dentistry, Universidad Nacional de Córdoba (CIEISN°6/2010). All the subjects signed an informed consent.- Study groupsTwo groups were considered:a- Oral Squamous Carcinoma Group (OSCG): 72 patients with diagnosis of oral squamous cells carcinoma; b- Control Group (CG):72 clinically healthy at the oral cavity, which means, with any oral clinical lesions at the time of this study, paired by sex and age (+/- 5) with the OSCG. This group was relieved from a population study titled “Survey of oral health parameters in the adult population from Córdoba city 2014–15” done in the city of Córdoba, Argentina. A multistage cluster sampling was conducted, with random selection of census tracts, probability proportional to size, blocks, households, and persons; the same number of individuals per tract was selected. The included subjects were asked to attend a health center in the area. Pregnant women and subjects under age 18 were excluded. The results were informed by phone or via mail to patients and healthy controls and they were also clinically controlled.- Data collectionClinical history of all patients was recorded, including personal data, personal and hereditary records, risk factors and oral health state. Subjects were inspected by personnel trained for clinical diagnosis. OSCG were subjected to biopsy to obtain diagnostic certainty. The data on tobacco consumption was made according the number of pack year smoked; a subject that smoked fifteen or more pack years in twenty years was considered a smoker . One alc- Sample collectionFour samples per patient were obtained from the OSCG: 1-unstimulated whole saliva collected in sterile tube; 2-cytobrush of lesion; specimens were placed in sterile tube containing 2 ml phosphate buffer solution with antibiotic and antifungal agents; 3-cytobrush of healthy contralateral mucosa to the lesion site, as in 2; and 4- biopsy of lesion; samples were placed in sterile tube in fresh conditions. All samples were preserved at 4°C until DNA extraction. The CG group was subjected to saliva sampling and cytological analysis of the posterior border of the tongue, using the same technique as in OSCG. In this group, we chose this area for being one of the most exposed to micro-wounds that would facilitate the entry of the virus.- Sample processingDNA-HPV was detected via PCR using Bioneer AccuPrep genomic DNA Extraction kit, and consensus primers MY09 and MY11. These primers amplified a fragment of approximately 450 base pairs corresponding to the highly conserved L1 region of the virus genome. The method involved a 50 μl reaction mix containing 10 μl DNA, 1.5 mM MgCl2, 200 μM of each dNTP , 10 pmoles of each oligonucleotide primer and 1 U Taq DNA polymerase . The temperature profile used for amplification constituted an initial denaturation at 94°C for 3 min followed by 35 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min which was extended for 5 min in the final cycle. Typing was performed by means of the analysis of the amplified product using the enzyme restriction fragment length polymorphism (RFLP) with the following restriction enzymes: Bam H1, Dde I, Hae III, Hinf I, Pst I, Rsa I, and Sau3 AI. A mixed of PCR product and buffer was prepared to each enzime separately for subsequent addition of individual restriction enzymes. The mix included 6.5 µl PCR product, 2 µl buffer, 1.5 µl restriction enzyme, followed by incubation 40ºC for 60 min. The result was a characteristic banding pattern visualized with UV light . Subject- Statistical analysisData were expressed as relative frequencies; data association was analyzed using the chi square test, and OR was used to reveal the intensity of the association.Two study groups were formed with 144 subjects, corresponding 72 to each group. The total of samples obtained from both was 432. p<0.0001  patients of OSCG and 3 (4%) in subjects of CG. The analysis and comparison of the results of OSCG with controls showed a highly significant difference, with an OR of 16.43 (CI: 5.10-52.94), p<0.0001 .- DNA-HPV at multiple sites: 67% of the HPV+ patients of OSCG had two or more positive samples. In the case of CG, 100% of the cases were positive in both samples When positive patients in two or more sites in the OSCG were analyzed and was related with the tumors site, we observed that the 65% were located at the border of the tongue and oropharynx.- Genotypes: The genotypes were 16/ 6/ 11/ 31/ 33/ 45/ 52/ 53/ 69, with 16/ 6/ 11 being the most frequent. In nine samples of the OSCG it was not possible to identify the genotype. In the OSCG, 12 patients (40%) had high-risk genotypes: 16/ 52/ 45/ 31/ 69 and 33, with 50% of these being HPV 16. Of the remaining positive types, 9 patients (30%) were of low risk (6/11); one of these patients had combination of genotypes 6 and 11, and of the remaining nine patients (30%) were not genotyped. In CG all genotypes were of low risk (11/84).The results of the present work show the prevalence of positive samples in more than one site of the oral cavity in DNA-HPV positive subjects of the groups studied. We also observed that in healthy mucosa the virus was present despite the lack of clinical signs of infection. In fact, 100% of controls and 43% of patients with oral cancer were positive in healthy mucosa.Some authors assume that HPV could present as a multifocal infection in different mucous membranes -19. Our et al. studied the HPV in a risk group of sexually transmitted diseases, and evaluated the presence of the virus at different sites with no clinical signs of infection. They took samples from the oral, genital and anal mucosa. The highest values were recorded in genital samples, and 37% of HPV positive values were obtained in the oral cavity. This result is similar to the values found in the samples taken in the present work in the carcinoma group. It should be noted that although they obtained similar percentages in both sexes, 51% of HPV positive males in the oral cavity had simultaneous genital infection, resulting in a significant association, with an OR of 3.63 (CI 1.37-9.58)  in this group and at this site, and HPV 16 was the most frequent. In oral mucosa, HIV positive cases had a higher percentage of high-risk genotypes than HIV negative cases . subjects . In a sisubjects  where stve cases ,22. In ove cases . Table 4ve cases .We believe important that these results of virus detection and the expression of predominant genotypes in both sexes are useful to support educational campaigns and the need to apply HPV preventive vaccines. Although the World Health Organization recommends the inclusion of males in HPV vaccination strategies, only in some countries like Argentina these are applied preventively since 2017, in both sexes .et al. analyzed HPV 16 in saliva of patients with head and neck carcinoma and compared it with a healthy control group; they found significant differences between groups (Regarding our findings of a high positive DNA-HPV percentage in saliva, similar to that in tissue subjected to biopsy, several authors have discussed the utility of detecting the virus in this fluid. Zhao n groups . In our Oral cavity HPV often occurs as a multifocal infection, even without the presence of clinical lesions. The current vaccination scheme in Argentina prevents against the genotypes circulating in our medium. Future studies are necessary to assess the clinical and epidemiological implications of the virus presence in saliva.


To adopt HPV self-sampling in Japan, we assessed the concordance between self- and physician-collected human papillomavirus (HPV) samples from Japanese patients and examined the performance of HPV self-sampling for cervical intraepithelial neoplasia grade 2 or worse (CIN2+).Patients who had previously tested negative for intraepithelial lesions or malignancy/HPV-positive, and patients with atypical squamous cells of undetermined significance or worse (ASCUS+) cytology were eligible for this cross-sectional study. Participants performed HPV self-sampling using an Evalyn brush, which was submitted at the Fukui Prefectural Health Care Association. The Evalyn brush heads were stored in ThinPrep vials. The physician, however, performed HPV and cell sampling using an endocervical brush and immediately stored the brush heads in ThinPrep vials. All participants underwent colposcopy and biopsy. Histopathological diagnoses were made by pathologists at Fukui University Hospital. HPV infection was confirmed using a PCR-based Cobas 4800 HPV DNA test. Cytological analysis was performed at Fukui Prefectural Health Care Association.κ = 0.76). HPV16/18 showed higher agreement rates than other HPVs . Both groups showed 100% sensitivity to CIN2+, but specificity was 57.0 and 58.1%, respectively.HPV-positive rates for physician-collected samples and self-collected samples were 51 and 50%, respectively. The perfect match rate of HPV type between the groups was 88% (For HPV typing, a good concordance rate was seen between self- and physician-collected samples. Self-sampling showed high sensitivity for CIN2+. Self-sampling using the Evalyn brush and Cobas 4800 may be feasible for screening Japanese individuals. Japan has a higher incidence of age-adjusted cervical cancer than the United States and Australia, where screenings are organized . The morEpidemiological studies using molecular technology show that persistent infection with high-risk HPV (hrHPV) is strongly associated with development of cervical intraepithelial neoplasia (CIN) and cervical cancer . TherefoHPV self-sampling is a screening method, where HPV sampling is performed by the screening participant and displays high HPV test concordance with HPV physician sampling in various referral and screening populations . HPV selSeveral HPV self-sampling devices and detection methods are available . HPV selWe hypothesized that HPV self-sampling with the Evalyn brush and Cobas 4800 PCR-based HPV testing would show good HPV-type agreement between physician sampling and self-sampling along with high detection sensitivity of CIN2+ in the Japanese population. To test this hypothesis, we compared HPV self-sampling using the Evalyn brush and Cobas 4800 with physician sampling performed in a Japanese referral population that included NILM/HPV patients.We conducted a cross-sectional study comparing the results of HPV self-sampling with physician sampling at the University of Fukui Hospital from January 2019 to July 2019. Previous studies report that referral populations sampled by physicians showed an hrHPV-infection rate of ~20 to 50%, resulting in colposcopy, regardless of cytology results . To obtaParticipants received instruction on how to submit samples after HPV self-sampling but not details concerning use of the Evalyn brush for sample collection. Participants were instructed to read the instructions describing use of the Evalyn brush before self-sampling, with these instructions created under supervision of the Japan Cancer Society. These instructions were verified that Japanese people could read and understand before this study. Participants performed HPV self-sampling in the bathroom at the hospital and then submitted the brushes. Immediately after HPV self-sampling, HPV physician sampling and cytology were performed. The Rovers Cervex brush  was used for physician sampling. After sampling, the Cervex brush was immediately placed in ThinPrep vials , and colposcopy and biopsy were performed. All physicians involved in sampling were gynecologic oncology specialists. The Evalyn brush and physician-sampled ThinPrep vials were stored at room temperature and transferred to the Fukui Health Care Association on a fixed day of the week. Cytologists examined the cytology samples using the LBC method at the Fukui Health Care Association. Two pathologists at Fukui University Hospital examined the colposcopic biopsy tissue. Cytologists and pathologists were not informed of HPV test results prior to diagnosis.The Evalyn brush was processed after transfer to the Fukui Health Care Association. The Evalyn brush was placed into ThinPrep vials and stirred to release the cells. Both physician- and self-sampled ThinPrep vials were stored at room temperature until measurement. HPV testing was performed using the Cobas 4800 system. HPV-16 and -18 as well as 12 other HPV genotypes, including -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68, were measured. Previously reported methods were used for all measurements . HPV test test was used to compare continuous variables. Agreement rates of perfect matches for HPV typing between the self- and physician-collected samples were examined, as were agreement rates for HPV-16/-18 and HPV others. HPV concordance between paired samples was assessed using the Kappa statistic  and defined as “Poor” (κ ≤ 0.20), “Fair” (0.21 ≤ κ ≤ 0.40), “Moderate” (0.41 ≤ κ ≤ 0.60), “Good” (0.61 ≤ κ ≤ 0.80), or “Very good” (κ ≥ 0.81). The sensitivity and specificity of HPV self-sampling were calculated using HPV physician sampling as the standard , and specificity of 90% (95% CI 78‒97%). HPV-16/-18 showed a higher agreement rate than HPV others . Additionally, HPV-16/-18 showed higher sensitivity and specificity than HPV others .Table p = 0.461). The number of days from self-sampling to HPV testing in all cases was 12.7 ± 6.9 days for HPV test-matched cases and 14.2 ± 7.3 days for HPV test-unmatched cases, although the difference was not significant (p = 0.482).Table Table κ: 0.76), with HPV-16/-18 showing a higher match rate and κ than HPV others. The detection sensitivity for CIN2+ by self-sampling was 100%. The results showed that HPV self-sampling using an Evalyn brush and a Cobas 4800 PCR-based method might be feasible for cervical cancer screening in Japan.We investigated whether HPV self-sampling with an Evalyn brush and Cobas 4800 demonstrated HPV-type agreement with physician sampling and high detection sensitivity for CIN2+ in a Japanese referral population. The HPV-type concordance rate between sampling methods was 88% (κ: 0.76), consistent with a meta-analysis using a referral and screening population, regardless of detection methods and devices  [κ: 0.70) [κ: 0.64) [In HPV self-sampling, participants collect the samples, making sample validity dependent on the patient. Therefore, it is important to compare HPV test results with those obtained from physician-collected samples considered the gold standard. The results of the present study showed a complete agreement rate for HPV typing of 88% (κ: 0.66)  and a Jaκ: 0.66) . Anotherκ: 0.70) , whereasκ: 0.64) . In the κ: 0.64) . Therefoκ: 0.64) . These fCases showing different results between sampling methods complicate the introduction of HPV self-sampling in screening. Most discrepant HPV tests involved NILM, and our results showed for the first time that discrepant cases using the Evalyn brush and Cobas 4800 in a Japanese population did not involve dysplasia. Inconsistencies in these cases associated with HPV testing between self- and physician sampling might be explained by self-sampling detecting HPV only in the vagina with no infected hrHPV in the cervix . A previThe sensitivity of CIN2+ detection by self-sampling was 100% and the same as physician sampling. A previous study evaluating sampling using the brush and PCR-based hrHPV testing by women referred for colposcopy due to abnormal cervical smear and/or post-coital bleeding with normal cytology showed a CIN2+-detection sensitivity of 93% for self-sampling and 91% for physician sampling . AnotherPrevious studies indicated that CIN2+-detection sensitivity by self-sampling exceeded that of cytology in a referral population ; howeverHere, samples collected with the Evalyn brush remained dried until transfer to a ThinPrep vial. Our result showed that the number of days from self-sampling to HPV testing did not influence discrepancies in HPV testing. The Evalyn brush is reportedly stable for the measurement of HPV DNA PCR results for up to 32 weeks , suggestPrevious studies of HPV self-sampling targeted possible applications for cervical cancer screening; however, the high NPV value associated with HPV self-sampling for CIN2+ indicated that self-sampling might be useful for following up potential high-risk groups previously identified as HPV+. A previous study following HPV+ women reported an NPV for HSIL or worse was 98.8% (95% CI 91.6–99.8%) in self-sampling and support self-sampling for exclusion of the disease during follow-up of HPV-positive women . In the This study has some limitations. We examined a small number of referral cases, and sampling was not performed in a screening setting; however, to ensure the feasibility of HPV self-sampling using the Evalyn brush and Cobas 4800, it was necessary to evaluate match rates and CIN2+-detection sensitivity in a Japanese referral population. Therefore, studies in a Japanese screening population are needed. Additionally, HPV self-sampling was performed in a hospital and not at home, which would be the case in a screening setting. HPV-infection status can be affected not only by recent sexual acquisition or re-infection but also from recurrent detection of a controlled or latent infection . TherefoIn conclusion, HPV self-sampling using an Evalyn brush and Cobas 4800 showed good agreement with physician sampling in a Japanese population. Moreover, the sensitivity of CIN2+ detection by self-sampling was as high as that for physician sampling using Cobas 4800 in a Japanese population. These results suggest that HPV self-sampling using the Evalyn brush and Cobas 4800 PCR-based testing methods might be efficacious for cervical cancer screening in Japan.


Infection with human papillomavirus (HPV) is one of the main causes of malignant neoplasms, especially cervical, anogenital, and oropharyngeal cancers. Although we have developed preventive vaccines that can protect from HPV infection, there are still many new cases of HPV-related cancers worldwide. Early diagnosis and therapy are therefore important for the treatment of these diseases. As HPVs are the major contributors to these cancers, it is reasonable to develop reagents, kits, or devices to detect and eliminate HPVs for early diagnosis and therapeutics. Immunological methods are precise strategies that are promising for the accurate detection and blockade of HPVs. During the last decades, the mechanism of how HPVs induce neoplasms has been extensively elucidated, and several oncogenic HPV early proteins, including E5, E6, and E7, have been shown to be positively related to the oncogenesis and malignancy of HPV-induced cancers. These oncoproteins are promising biomarkers for diagnosis and as targets for the therapeutics of HPV-related cancers. Importantly, many specific monoclonal antibodies (mAbs), or newly designed antibody mimics, as well as new immunological kits, devices, and reagents have been developed for both the immunodiagnosis and immunotherapeutics of HPV-induced cancers. In the current review, we summarize the research progress in the immunodiagnosis and immunotherapeutics based on HPV for HPV-induced cancers. In particular, we depict the most promising serological methods for the detection of HPV infection and several therapeutical immunotherapeutics based on HPV, using immunological tools, including native mAbs, radio-labelled mAbs, affitoxins (affibody-linked toxins), intracellular single-chain antibodies (scFvs), nanobodies, therapeutical vaccines, and T-cell-based therapies. Our review aims to provide new clues for researchers to develop novel strategies and methods for the diagnosis and treatment of HPV-induced tumors. HPVs bend mouth . Epidemind mouth .6 Daltons. It consists of 72 shell particles comprising three-dimensionally symmetrical icosahedrons with a diameter of about 55 nm. It has a lipoprotein-free membrane, a core, and a protein capsid. The HPV genome has four parts (The HPV genome has approximately 7800 to 7900 base pairs (bp) and a molecular weight of 5 × 10ur parts Figure 2ur parts , 13. TheCervical cancer, the fourth most common type of cancer in women worldwide, is one of the most preventable HPV-induced cancers. Unfortunately, cervical cancer remains a major public health problem affecting middle-aged women . Aside fINK4A protein, the immunohistochemical overexpression of which may be a useful screening test for HPV-induced cancers, is one of the important downstream factors of E7. In normal cells, p16 can be negatively transcriptionally regulated by active pRb. However, in HPV-positive cells, the pRb is inactivated by E7, thus resulting in the significant overexpression of the p16-encoded protein in HPV-positive cancer cells  method. This method can only be used to detect HPV DNA and HPV types and cannot be used to accurately predict HPV-positive cancers , 21. Forer cells . Althouger cells . The poser cells .Over the decades, many kinds of HPV protein antibodies, including both polyclonal and monoclonal antibodies (pAbs and mAbs) against various types of HPV proteins, have been developed for both diagnosis and therapy of HPV-positive carcinomas , 29–31. There are many kinds of sample types including cervicovaginal, oral and serum samples for HPV detection in cervical cancers. However, a study shows that immunoglobulin-A and -G (IgG and IgA) responses to HPV16 in different kinds of sample types of women with cervical intraepithelial neoplasia (CIN) function independently of one another . It is tTumor tissues are mainly used for validation of the detection methods based on antibodies or antigens, which can be used for prognosis, tumor typing, and medication instruction after surgery. For instance, two mouse mAbs induced by a recombinant HPV16 E7 oncoprotein were used to establish a chemiluminescent immunoassay based on a labeled streptavidin-biotin (LSAB)- enzyme-linked immunosorbent assay (ELISA) method and a luminol detection system that was suitable for the detection of HPV16 E7 oncoprotein in HPV16-positive cervical carcinoma tissues. In this method, an IgG2a-type anti-HPV16 E7 mAb was used as the detection antibody, and an IgM-type anti-HPV16 E7 mAb was used as the capture antibody . A RabbiIn addition to oncogenic early proteins, many other kinds of anti-HPV antibodies, such as HPV L1 and L2, were also developed for diagnosis, evaluation of vaccines, or basal biomedical research. Two mAbs, specifically recognized as HPV18 L1 and VLPs, were used to develop an ELISA kit based on HRP-conjugated antibodies, and this method displayed good linearity, repeatability, and sensitivity for detecting HPV18 L1 pentamer and HPV18 VLP . AntibodThe antibodies developed underpin the basis for tumor typing, diagnosis, prognosis, and further development of the therapeutic tools. However, it is assumed that detection of oncogenic antigens in tumor samples may be an auxiliary method for pathological identification and diagnosis. Besides, there are many limitations for early-stage detection or prediction of an HPV-positive tumor since the samples are acquired after the tumor size is large enough. They can be obtained only for a more accurate pathological analysis. Besides, it is difficult to acquire the valid tumor samples without an operation.Interestingly, the exfoliated cervical cytological examination may be used for detection in the early stage of HPV-positive tumors, such as cervical cancers of the Ia stage. It can be used as an adjuvant method for cervical cytological tests. For instance, a mouse mAb panel against a C-terminal conserved sequence polypeptides of human papillomavirus (HPV) L1, a major capsid protein, by using the IHC method used for the detection of HPV in exfoliated cervical cells. The detection rate was comparable to that obtained using the commercial HPV genotyping kit currently used in clinical practice . Anotherin vivo. As an antibody mimetic, affibodies contain three helical bundles with 58 amino acids in structure  were screened from a phage-displayed peptide library and used for molecular imaging in tumor-bearing mice. Biosensor binding analysis showed that the four affibody molecules were bound to HPV16 E7 with very high affinity and specificity. Affibody ZHPV16E7384 was conjugated with Dylight755 and showed great potential to be used for in vivo tumor imaging and diagnosis of HPV-induced cancers . An increased level of pretreatment log-unit E6 titer was significantly associated with increased risk of disease recurrence of HPV-OPC . After tHigh serological positivity of anti-HPV antibodies may be one cause of HPV infection, but this leads to two different results for carcinogenesis. One is that patients with higher serological positivity of anti-HPV antibodies may be caused by too much virus load in the body, resulting in high-risk of carcinogenesis. The other is that patients with higher responses of anti-HPV antibodies may be due to better patient immune systems, resulting in a quicker elimination of viruses in the body and a decreased possibility of carcinogenesis. For instance, serological positivity for IgA and IgG in the patients with cervical neoplastic lesions showed higher titers than those in the normal group . This coin vitro-translated E6 and E7 proteins of HPV-16 were also used and showed high serological positivity in the sera of a large amount of patients with cervical carcinoma generated , which are similar to native virions in structure but have no viral genome, were firstly used for reaction with the IgA and IgG responses in the sera. For example, VLPs were tested by ELISA for detecting antibodies in the sera of HPV 16 in women with cervical cancer and CIN, and the results showed that this assay using HPV-16 VLP may be useful as a diagnostic tool to supplement cervical cytological tests . Howeverenerated , indicatenerated .In addition to VLPs that similar with natural proteins, chimeric particles containing HPV-16 L1 protein fused with E6 and E7 seroreactive epitopes show a much better effect for detection of IgG antibodies in the sera of patients HPV16-positive CIN1 than those obtained with VLPs containing only the HPV-16 L1 protein . A recomIntriguingly, some biomimetic peptides are also used in serological detection of anti-HPV antibodies. Recently, a nonapeptide (16L1) derived from the HPV-16 major capsid protein was used in an ELISA system to detect potential cross-reaction of serum IgG and cervical IgA antibodies HPV-associated low-grade squamous intraepithelial lesions (LSIL) and cervical cancer patients. The results showed that the 16L1 peptide is a high-risk epitope that induces cross-reactive antibodies in patients with high-risk, but not low-risk, HPV-induced LSIL, indicating that this method is suitable for distinguish high- and low-risk infected women in the stage of low grade .In summary, the selection of valid natural HPV antigens and design of biomimetic peptides are important for better detection of HPV protein antibodies. It is therefore urgent to explore how the antibodies circulating in the blood are induced by the HPV proteins, and the epitope profiles should be widely studied for a better understanding of the immunological responses upon HPV infection and what the differences in immunological responses during carcinogenesis are compared with those of the non-tumorigenic HPV infection.The magnifying and accurate detection system used also accounted for the application of HPV antibodies detection. Currently, many detection methods for the diagnosis and prediction of HPV-associated tumors are tried in basic research. For instance, a luciferase-based detection method for determining serum antibodies is possible for the diagnosis of HPV-associated head and neck squamous cell carcinoma . A new LHowever, the ELISA assay allows only one antigen to be evaluated at a time per well. An assay based on electrochemiluminescence (ECL) was developed by using multiplex technology from Meso Scale Diagnostics  with maltose-binding protein (MBP)-tagged E6 and E7 oncoproteins. This is a high-throughput method, but it requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV E6 and E7 oncoproteins .Collectively, the present detecting methods are mainly based on chemiluminescence and ECL that can magnify the signals of immunological reactions. The success of novel detection systems also depends on the clear understanding of the biomarkers in HPV-induced cancers. The current biggest obstacle of immunodiagnosis of HPV-induced cancers is therefore the appropriate specific biomarkers.via interacting with multiple signaling pathways and regulatory modes that participate in the modulations of intraepithelial neoplasia (IN), tumorigenesis, cell cycle progression, cell survival, and metastasis   pathway –93. HPV  pathway . HPV16 Eigenesis , 96. E6 ase E6AP . Recentlase E6AP  and is sl models .These oncoproteins are potential drug targets for the therapeutics of HPV-associated cancer. Among them, immunotherapy-based on HPV is very important. From current studies, we can see there are many kinds of immunotherapeutical methods or immunotherapy-like methods based on HPV studied in basal research, including anti-HPV protein mAbs, radioimmunotherapy, affitoxins, single-chain antibodies (scFvs), nanobodies, therapeutic vaccines, and T-cell-based therapies.via intraperitoneal or intratumoral injections in mice models significantly inhibited cervical tumor growth (via necrosis (Anti-HPV E6 and HPV E7 mAbs r growth . Anotherr growth . Other mr growth –105. Thenecrosis . In contvia the “cross-fire” effect produced by radiation in 360° spheres (Radioimmunotherapy (RIT) is a kind of therapeutic method that systemically administers radiolabeled mAbs to bind to specific tumor-associated antigens , 106. Co spheres Figure 5 spheres .188Re) attached to HPV E6, making them selective mediators of tumor destruction even in experimental cervical tumors expressing low levels of E6  are equally effective for radioimmunotherapy based on an anti-HPV E6 mAb in HPV-positive cervical cancer , epidermal growth factor receptor (EGFR), and insulin-like growth factor type 1 (IGF1R) , 110. Threatment , 111. ThEscherichia coli. Applications of scFv against fibroblast growth factor receptor (FGFR) and fibroblast growth factor 1 (FGF-1) have shown a great anti-tumor effect both in vitro and in vivo  antibodies are the smallest immunoglobulins, but they have a high antigen‐binding affinity Figure 6 in vivo –114. Rec in vivo . Functioe models , 117. Anoprotein . Besidesr effect . Two scFr effect . These rHH, Camelidae family (E. coli) or yeast (Pichia pastoris). Recently, Nbs also function important therapeutic approaches for cancer treatment  are another kind of small antibodies (approximately 15 kD) that are naturally devoid of light chains, the antigen-binding fragment of which consists of only one single domain referred to as nanobody (Nb) or the heavy chain antibody variable region VHH, Figure 6e family . Nb is mreatment . Nbs thareatment . Importareatment . These svia major histocompatibility complex class I and II (MHC I/II), leading to CD8+ cytotoxic T-cell or CD4+ helper T-cell responses. This method has also been trialed for cancer therapy , where these antigens are processed and digested into short peptides by proteasome, which can stimulate antigen presentation  therapy . Recentl therapy . There a therapy .Listeria monocytogenes, Lactobacillus lactis, Lactobacillus plantarum, and Lactobacillus casei, or viruses, such as adenoviruses, adeno-associated viruses, alphaviruses, and vaccinia viruses. They can infect macrophages and stimulate immune responses that kill tumor cells. Listeria-based E7 vaccines were reported as a promising method for the treatment of HPV E6/E7-expressing autochthonous solid tumors in mice , which can make the bacteria escape from phagosomal lysis and decrease the CD4+FoxP3- and CD8+ regulatory T cells, resulting in HPV vaccine-mediated tumor regression  coat protein. This vaccine candidate was then challenged in a new luminescent animal model based on a C3-Luc cell line, and a strong cellular immunity was induced, resulting in eliciting strong anti-tumor effects . Unlike ex vivo and subsequent deliver DCs to the patient  (Dendritic cell- and tumor cell-based vaccines are two main kinds of whole cell-based vaccines. DC-based HPV vaccines load the DCs with HPV antigens  patient . A phase patient . Tumor c (GMCSF) , 151. BuOver the last decades, tumor immunology has expanded our understanding on the relationships between the tumor cells and tumor environment where immune cells, such as cytotoxic T cells, helper T cells, and regulatory T cells, are located. These cells are final effectors of immune response that can induce tumor regression. Recently, there are several types of T-cell based therapeutics also known as T-cell-based therapeutic vaccines or adoptive T-cell therapies (ACTs), such as tumor-infiltrating cells (TILs), engineered T-cell receptor (TCR) T cells, and chimeric antigen receptor T (CAR-T) cells, that has been tested in experimental research and clinical trials .ex vivo, and then infused back to patients to regress the tumor development. This method has been used in the treatment of melanoma, gastrointestinal cancer, and lung cancer  for the treatment of several types of hematological malignancies. Although this method is less successful in solid malignancies, a phase I/II clinical study (NCT03356795) is performing for intervention of CAR-T against cervical cancer positive to GD2, PSMA, Muc1, Mesothelin or other markers. If this method is efficient, then CAR-T against HPV oncoantigens may also be promising therapeutics for HPV-positive cancers.in situ or detect the antibody in the internal body fluid, such as sera, or external body fluid, such as saliva and vaginal wash. The immunotherapeutical methods including mAbs, radiolabeled mAbs, affibodies, nanobodies, therapeutic vaccines, and T-cell based therapies are developed to target the HPV oncoproteins, including HPV E5, E6, and E7. For the immunodiagnosis, detecting anti-HPV protein antibodies may be of promise because this method is relatively easier and more accurate. However, the method should be combined with other diagnostic methods, such as HPV typing, based on HPV, DNA, or other molecular targets, such as p16, which may be more accurate. For the immunotherapeutics, the T cell-based and novel antibodies-based therapies may be a promising method for the therapeutics. However, because of the heterogeneity of the HPV-induced cancers, it is very difficult to find a universal way to cure all HPV-induced cancers. It is therefore urgent to perform a more comprehensive study to develop a better molecular typing method that is based on HPV antigens. Although it is not easy, it will hold a great promise for the use of immunodiagnosis and immunotherapeutics based on HPV for HPV-induced cancers.From above, we can conclude that immunodiagnosis and immunotherapeutics has been rapidly developed for HPV-induced tumors. The immunodiagnostic methods are exploited to detect the antigen in tumors or cells The accuracy of immunodiagnostic and immunotherapeutic methods is based on the specific antigen and antibody that are specifically associated with the pathological progression of HPV-associated tumors. From current studies, HPV E6 and E7 are the two most important protein targets for both diagnosis and therapy. However, the biggest problem is that the specificity of detecting these proteins and their corresponding antibodies need to be further optimized. More basal research should thus be performed to explore the profiles of the expression, functions, and regulations of these HPV oncoproteins in HPV-induced oncogenesis. Afterwards, more clinical trials should be done to validate the results obtained from the basal research. It is believed that immunodiagnosis and immunotherapeutics based on HPV will hold great promise for the prevention of people with HPV infection and therapy for patients with HPV-associated tumors.ZD and RH reviewed the literature, designed the tables and figures, and wrote the manuscript. YD, LT, JD, LL, LB, and YM collated the literature and proofread the manuscript. HC scrutinized and revised the text. All authors contributed to the article and approved the submitted version.We are grateful for funding support from the National Natural Science Foundation of China , the Fundamental Research Funds for the Central Universities (Nos. SWU120009 and XYDS201912), the National Key Research and Development Program of China (Nos. 2016YFC1302204 and 2017YFC1308600), the Experimental Technology Development Fund Project of Chongqing University of Technology (No. sk201812), the Opening Project Funding from the State Key Laboratory of Silkworm Genome Biology (No. sklsgb161718-5) and the Natural Science Foundation of Chongqing (No. cstc2019jcyj-zdxmX0033).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this paper, the dynamic model of the rigid space stepped-pipe strings system is derived with Lagrangian method to represent the system dynamic behaviors which enriches the analysis method of longitudinal vibration of stepped-pipe strings. The stepped-pipe strings is constructed of pipes with different diameters and lengths, the physical properties of which mainly depends on the axial force and the depth of deep-sea mining. Based on lumped element method, the heave compensation system with dynamic vibration absorber is designed for longitudinal vibration suppression of the stepped-pipe strings. The analytical solution is obtained by modal analysis method when the mining ship is subjected to sea breeze excitation. The proposed method is easily implementable for rigid space stepped-pipe strings system with complex multi-degree-of-free deep-sea mining dynamic model. Furthermore, the optimal combination of mass ratio, spring coefficient and damping ratio is shown to have a better vibration suppression performance. Finally, numerical simulations on the stepped-pipe strings system with or without dynamic vibration absorbers are provided to demonstrate the effectiveness of the proposed method. Eq (Substituting Eqs –6 into E)}={f(t)}where  and [Kr] are diagonal matrices,and the r-order modal mass and stiffness are {U(r)}T[M]{U(r)} and {U(r)}T[K]{U(r)}, respectively.According to the concept of modal matrix and the orthogonality of modal vectors, the modal mass matrix and modal stiffness matrix can be obtained, which are denoted asAccording to Eq , combineβ and λ are the multiplier of mass matrix and stiffness matrix respectively, also called Rayleigh coefficients.In general, modal vectors generally have no weighted orthogonality to damping matrices, then Rayleigh damping model is used to diagonalize damping matrices in this paper. Rayleigh damping model was proposed by Rayleigh in1877, which is a convenient and simple uncoupling method of the equations of motion in the case of linear response . AssuminSubstituting Eq  into 1414, The dr-order damping ratio can be deducedThus, the i- and j-order)can be obtained,then the β and λ can be calculated as:According to the reliable test data estimation and calculated natural frequency, the two appropriate damping ratios = UNTf(t) is the normalized excitation corresponding to the generalized coordinates.Substituting Eqs  into 7)7), the nModal equations are all decoupled, and each equation is an independent equation with corresponding modal coordinates, which is completely equivalent to a single degree of freedom. Therefore, no matter what the initial conditions are, the modal equation can be solved according to the theory of single degree of freedom.In this section, numerical simulations on a stepped-pipe strings with DVA in harmonic excitation are carried out to demonstrate the effectiveness of the proposed lumped parameter modal. The stepped-pipe strings model combined with two DVAs is shown in The development of explicit optimal absorber parameters is challenging for a damped structural system since the fixed points no longer exist in the frequency response curve . The lonμ values are given in Let μ value that the damping effect is getting better as the mass of DVA increases. However, the larger mass of DVA increases the axial stress of the stepped-pipe and the dynamic requirements of the mining ship. Generally, the mass ratio μ depends on the specific engineering requirement, the value range of μ1 or μ2 is within  in the system.It is clearly shown that the amplitude of each stepped-pipe is decreased with the increase of the μ and k (spring stiffness constant).There is an optimum combination of the mass and spring constant for the dynamic vibration absorber to minimize the resonance amplitude and the maximum axial stress of the stepped-pipe string . Fig 5 sμ value has little effect on vibration reduction if the value of spring stiffness is very small or large. We can obtain that the amplitude is smaller in case of the absorber with larger mass when the spring constant of dynamic vibration absorber varies between 0.2x105–0.6x105N/m.It can be found that the change of ξ). ξ form 0.02 to 0.11.In addition, the longitudinal vibration response of the stepped-pipe strings system has also been compared for five Rayleigh damping coefficients , spring coefficient (k) and damping ratio(ξ) near the first natural frequency.It is important to note the m to 1.59m, and the maximum amplitude of buffer position decreases from 5.85m to 4.15m. And the natural frequency value changes from the original natural frequency to two natural frequency points.The simulation results show that the maximum value of longitudinal vibration amplitude decreases sharply after attaching with dynamic vibration absorber at the pump and buffer. The maximum amplitude value of the second pump position decreases from 2.25From the above analysis, it can be seen that the longitudinal amplitude of the buffer at the bottom of the stepped-pipe strings is the largest, followed by the position of the second pump at the first order resonance. If the longitudinal amplitudes of these two positions are suppressed, the amplitudes of the whole stepped-pipe system will be improved correspondingly.F of 12kN and the excitation frequency Ω of 2rad/s. The exciting force column vector can be expressed as:The mining ship is simplified as a particle on the top of the stepped-pipe strings. The heave motion of the mining ship caused by wave fluctuation is simplified as simple harmonic motion, which produces an exciting force on the stepped-pipe strings. The exciting force was represented by a cosine wave excitation with the peak magnitude Therefore, the normalized modal generalized excitation force vector in the 6-degree-of-freedom stepped-pipe strings system is written as:Using modal superposition method, Eq  is expanTherefore, the output of forced longitudinal vibration of the stepped-pipe strings is determined byrad/s.To illustrate the application of the proposed method to the vibration analysis of stepped-pipe strings, we focus on the longitudinal displacement, velocity and acceleration of the stepped-pipe strings in Figs In Figs A unified and rigorous mass lumping scheme is proposed in this paper, which can be applicable to the longitudinal vibration analysis of stepped-pipe strings. Based on the Lagrangian equation and the background theory on vibration analysis of stepped-pipe strings for DSM, the multi-degrees-of-freedom dynamic model of the system was established. Modal testing has been carried out by sea breeze excitation. Out of the tested excitation, the stepped-pipe strings yields the accurate natural frequencies, and the good mode shapes are obtained. In the modal analysis of stepped-pipe strings without DVA, it is found that the vibration amplitude of the fourth part is the larges tin the first mode, followed by the second part in the second mode. After attaching DVA in the second and fourth parts, the vibration amplitude decreases obviously. The compensation rate  of the fFuture research may take into account effects of other vibrations on the stepped-pipe strings and the parameters optimization of DVA in order to increase accuracy.S1 Data(TXT)Click here for additional data file.S2 Data(TXT)Click here for additional data file.


The antisense transcript, emanating from the opposite strand to a protein-coding or sense strand, has been reported to have critical roles in gene regulation. The perturbation of an antisense RNA can alter the expression of sense messenger RNAs. In this study, a long noncoding RNA TTN-AS1 (lncRNA-TTN-AS1), which is transcribed in the opposite direction of the human titin (TTN) gene, has been identified and explored in skin cutaneous melanoma (SKCM). We found that the expression of TTN and lncRNA-TTN-AS1 had a significantly positive correlation in SKCM cells. Functionally, ectopic expression of TTN and lncRNA-TTN-AS1 promoted SKCM tumorigenesis and metastasis both in vitro and in vivo. Moreover, knockdown of TTN partially abrogated lncRNA-TTN-AS1 induced SKCM tumorigenesis. Mechanistically, hypomethylation of transcription initiation site was responsible for lncRNA-TTN-AS1 high expression levels. LncRNA-TTN-AS1 facilitated SKCM progression by promoting TTN expression at both transcriptional and posttranscriptional levels. As detailed, lncRNA-TTN-AS1 had a significant effect on the increase of TTN promoter activity. Besides, lncRNA-TTN-AS1 also induced the accumulation of TTN in cytoplasm by increasing the stability of TTN mRNA. Clinically, we found that high TTN and lncRNA-TTN-AS1 expression were positively correlated with poor overall survival of SKCM patients, and may be considered as novel biomarkers and drug targets for SKCM patients. Emerging evidences indicate that majority of patients in China tend to be younger. Although more and more oncogenes have been revealed to play critical roles in SKCM2, the molecular mechanism underlying SKCM carcinogenesis and progression is still not well documented. Hence, it is necessary to have a deep comprehension of SKCM mechanism.Skin cutaneous melanoma (SKCM) is the most malignant tumor type in skin cancers and the 5-year overall survival (OS) rate of SKCM patients is still unsatisfactory4, among which the antisense transcript was once considered to be transcriptional noise. The antisense transcript, emanating from the opposite strand to a protein-coding or sense strand, were recently shown to play a vital role in gene regulation of the corresponding sense transcripts6. For example, ANRIL is transcribed from the opposite direction of INK4A-ARF-INK4B gene cluster and may exert its oncogenic action in melanoma by suppressing the expression of INK4A and INK4B7.Long noncoding RNAs (lncRNAs), a class of noncoding RNAs, has been reported to have abundant functions in development of cancers8. In this study, we identified that the expression of lncRNA-TTN-AS1 was positively correlated with TTN in SKCM. High TTN and lncRNA-TTN-AS1 expression levels resulted in tumor progression and poor OS of SKCM patients. Further studies revealed that lncRNA-TTN-AS1 exerted its carcinogenic effect in SKCM by promoting TTN expression at both transcriptional and posttranscriptional levels. Taken together, we assumed that the interaction between TTN and lncRNA-TTN-AS1 played a pivotal role in SKCM carcinogenesis and progression.In previous research, we reported an antisense transcript lncRNA-TTN-AS1 (ENST00000589434), which is transcribed in the opposite direction of the human titin (TTN) gene. We found that lncRNA-TTN-AS1 upregulates actin-binding protein fascin homolog 1 (FSCN1) by sponging miR-133b, and combines directly with the mRNA-stabilizing protein HuR to stabilize FSCN1 mRNA, resulting in the epithelial-mesenchymal transition cascade in esophageal squamous cell carcinoma (ESCC)https://portal.gdc.cancer.gov/). In situ hybridization of 198 human SKCM tissue samples was performed to detect TTN expression. The SKCM tissue samples were divided into two groups (high and low) according to the scores of TTN expression level. Moreover, in situ hybridization of 165 human SKCM tissue samples was performed to detect lncRNA-TTN-AS1 expression, and the samples were also divided into two groups (high and low) according to the scores of lncRNA-TTN-AS1 expression level. Survival curves calculated by Kaplan–Meier and log-rank tests revealed that SKCM patients with higher TTN and lncRNA-TTN-AS1 expression had shorter OS than those with lower TTN and lncRNA-TTN-AS1 expression  in ESCC14, whereas high methylation of its gene promoter leads to decreased expression of p53-induced lncRNA TP53 target 1 (TP53TG1) in human cancer15. In this study, we found that hypomethylation of transcription initiation site was responsible for lncRNA-TTN-AS1 overexpression.In previous research, we found that lncRNA-TTN-AS1 upregulates FSCN1 by competitively binding miR-133b and combines with HuR to stabilize FSCN1 mRNA, which further promotes ESCC invasion cascades16. It was reported that the transition from N2BA to N2B was coupled to specific changes in the expression pattern of TTN17. Recently, lncRNAs were shown to process with alternative splicing, which enabled a single gene to produce multiple messenger RNA variants and distinct protein isoforms6. Hence, we speculated whether lncRNA-TTN-AS1 could also manipulate TTN with alternative splicing, which is worthy of further investigation.TTN, as known as Titin, is encoded by a single gene with a molecular weight of 3000–3800 kDa. The two major TTN isoforms are co-expressed resulting from multiple alternative splicing steps: the smaller N2B and the larger N2BA isoform18. For instance, lncRNA AB074169 regulates CDKN1a (p21) mRNA stability by interacting with KH-type splicing regulatory protein (KHSRP) in papillary thyroid carcinoma19. To determine the underlying mechanism of lncRNA-TTN-AS1 and TTN interaction involved in SKCM tumorigenesis, RNA pull-down and LC/MS-MS assays were constructed to unravel the lncRNA-TTN-AS1-related proteins and signal cascades in our study. As shown in Fig. + probe, and 64 of them were upregulated whereas the rest 115 proteins were downregulated. After differential comparison of these proteins, 40 proteins were found to be the most associated with lncRNA-TTN-AS1 in B16F10 cells . GO (http://bioinfo.cau.edu.cn/agriGO/) and KEGG (https://www.genome.jp/kegg/pathway.htmL) analyses were explored by online tools. As expected, lncRNA-TTN-AS1-binding proteins mainly participated in the molecular function regulation of RNA binding, antioxidant activity, and cytoskeletal protein binding  to participate in a variety of biological progresses, such as chromatin remodeling, transcriptional regulation, and RNA degradationing Fig. , which wton Fig. . As prevIn summary, our study first demonstrates the concordant regulation of TTN/ lncRNA-TTN-AS1 pairs in SKCM. LncRNA-TTN-AS1 facilitates SKCM tumorigenesis and metastasis by maintaining TTN expression at both transcriptional and posttranscriptional levels. Our findings further elucidate the pathogenesis of SKCM and facilitate the development of targeted diagnostic and therapeutic approaches for this disease. The combination of lncRNA-TTN-AS1 and TTN may serve as a prognostic biomarker panel of SKCM patients.2 and were used during their logarithmic growth phase. The small hairpin RNA (shRNA) of TTN and lncRNA-TTN-AS1 were synthesized and cloned into the lentiviral expression vector piLenti-siRNA-GFP and scramble siRNA GFP piLenti-vector, which was used as a negative control . The full-length lncRNA-TTN-AS1 were synthesized and cloned into the lentiviral expression vector plenti-GIII-CMV-2A-Puro-GFP (plenti-GIII-CMV-2A-Puro-GFP-lncRNA-TTN-AS1) and an empty lentiviral vector as a control . Cells were plated at 70% confluence in a 24-well plate and transfected with 0.2 μg above mentioned vectors using Effectene Transfection Reagent  according to the manufacturer’s instructions. Stably transfected cells were selected with 3 μg/mL Puromycin  for 4 weeks.Seven SKCM cell lines, including B16, B16F10, A375, SK-MEL-1, SK-MEL-5, SK-HEL-1, and Malme-3M, were used in this study. B16, B16F10, and A375 cells were purchased from Chinese Academy of Sciences Shanghai Cell Bank . SK-MEL-1, SK-MEL-5, SK-HEL-1, and Malme-3M were purchased from ScienCell Research Laboratories . All cells were maintained in RPMI1640  with 10% FBS  and recently authenticated and tested for mycoplasma contamination. Cells were cultured at 37 °C in an atmosphere containing 5% COTM Kit . Transcription levels were measured in duplicate by PowerUpTM SYBRTM Green Master Mix (Invitrogen). Relative expression levels of lncRNA and mRNA were normalized to GAPDH expression. The primer sequences were shown in Table Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s specifications. cDNA was synthesized from the total RNA using PrimeScriptAfter transfected with GFP-labeled TTN and lncRNA-TTN-AS1, B16F10 cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and DAPI was used to label the cell nucleus. The localization of TTN and lncRNA-TTN-AS1 was observed with AiryScan LSM800 axiophot photomicroscope . Nuclear/cytoplasmic fractionation assay was performed using the PARIS Kit  following the manufacturer’s instructions.The biotin-labeled lncRNA (both wild type and mutant type) and the antisense RNA were in vitro transcribed with a Biotin RNA Labeling Mix  and the T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche) and purified with an RNeasy Mini Kit (Qiagen). Primers for in vitro transcription were listed in Table For cell apoptosis assay, cells were labeled with Annexin V-APC and propidium iodide (PI) according to the manufacturer’s instructions of cell apoptosis assay kit . For cell cycle assay, cells were stained with PI according to the manufacturer’s instructions of cell cycle assay kit . Cell cycle and apoptosis were determined by flow cytometry . The data were analyzed by FlowJo v5.7.3 software .All cells were fixed on 12 × 12 mm glass slides with 4% paraformaldehyde for 30 min at room temperature and incubated with a mouse anti-TTN antibody . After incubated with a Cy3-labeled secondary antibody , cells were washed by PBS  twice. Dapi  was used to label the cell nucleus. Fluorescence was analyzed with AiryScan LSM800 axiophot photomicroscope .n = 6) and the cells were xenografted (1 × 107/100 μl) into mice after harvested from cell culture plates. The investigator was blinded to the group allocation during the experiment. The weight of mice and tumor volume were measured every 6 days. Twenty-five days later, the mice were killed and the tumors were stripped for further analysis. Immunohistochemistry was performed to describe Ki-67 and TTN distribution.Female C57BL/6 mice  were cared according to Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. The procedures of all animal experiments were complied with Institutional Animal Care and Use Committee regulations. All animal experiments were approved by the Ethics Committee of China Pharmaceutical University Permit Number: SYXK2016–0011. For tumor xenograft assay, B16F10 cells were stably transfected with shRNAs and scramble plasmids. Subsequently, mice were randomly grouped  and 1 × 106 GFP-labeled B16F10 cells were intravenously injected into female C57BL/6 mice. Tumor metastasis was observed by in vivo bioluminescent imaging using IVIS Lumina series III  once every 10 days. Mice were sacrificed after 1 months. Lungs and kidneys were removed for examination. HE staining was used to observe metastatic lesions and immunohistochemistry was performed to describe Ki-67 and TTN distribution.For tumor metastasis assay, mice were randomly grouped  Kit.The cell viability, colony formation, transwell migration/invasion, and wound healing assays were performed as described previouslyhttp://www.urogene.org/methprimer/). Genomic DNA was extracted from cancer cells according to the QiAmp DNA blood Mini Kit (Qiagen) manufacturer’s specifications and then was bisulfite-modified using EZ DNA Methylation-Gold Kit . 100 mg of eluted bisulfite-modified DNA was amplified with primers specific to methylated or unmethylated DNA sequences. The primer sequences for MS-PCR were shown in Table Methylation-specific PCR was used to analysis the methylation of lncRNA-TTN-AS1. Bisulfite sequencing primers were designed on MethPrimer website . 293T cells were transfected with the promoter fragments, which were inserted into the pGL3 plasmid and pRL-TK (Hanbio biotechnology). Firefly (Fluc) and Renilla (Rluc) luciferase activities were determined after 48 h.LncRNA-TTN-AS1 was knocked down or overexpressed in B16F10 cells. After 24 h, the adherent cells were incubated with 50 μg/mL α-amanitin  to block new RNA synthesis. The cells were harvested after 48 h and total RNA was extracted. The RNA stability was measured by qRT-PCR at each indicated time point after blocking new RNA synthesis and normalized to 18s rRNA.The cell lysates were homogenized in radio-immunoprecipitation assay buffer . The proteins were separated by 12% sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis and transferred onto polyvinylidene fluoride membranes , which were subsequently incubated with primary antibodies overnight at 4 °C. Following incubation with the specific HRP-conjugated Goat Anti-Rabbit IgG, chemiluminescence signal was detected using BeyoECL Plus . Antibodies against Cyclin D1 (# WL01435a), CDK2 (# WL01543), CDK4 (# WL01711), Bax (# WL01637), pro-caspase 3 (# WL02117), cleaved-caspase 3 (# WL02117), pro-caspase 9 (# WL03421), and cleaved-caspase 9 (# WL03421) were purchased from Wanleibio . Antibody against Titin/CMD1G (# bs-9861R) was purchased from Bioss . Antibody against β-actin (# 70-ab36861–050) and HRP-conjugated Goat Anti-Rabbit IgG (# 70-GAR007) were purchased from Liankebio . Protein expression was normalized to the β-actin level.t test (two-tailed) between two groups or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. Survival analysis was performed using the Kaplan–Meier test, and the log-rank test was used to compare the differences between patient groups. Statistics with P value < 0.05 were considered as statistically different .All experiments were performed at least three times. Data are presented as the mean ± standard deviation (SD) based on three independent experiments. All statistical analyses were carried out using GraphPad Prism 6 . All data were in a normal distribution, and variance was similar between the groups that are being statistically compared. Statistical evaluation was performed using Student’s Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary figure legendsSupplementary tables


TP53, HPV−OPSCC had significantly more protein‐altering mutations than HPV+OPSCC. The number of mutations was similar in HPV−OPSCC of patients with and without LDR with the exception of FAT1, which was mutated more frequently in patients without LDR. In HPV+OPSCC, HRAS, PIK3R1, STK11 and TP63 were more frequently mutated in patients with LDR compared to patients without. HPV+OPSCC of patients with LDR have a similar mutation pattern as HPV−OPSCC, except TP53, which was mutated to a significantly lower extent. In conclusion, HPV−and HPV+OPSCC with LDR have similar mutation counts in the analyzed genes. We suspect that the number of mutations is not causal for disease progression, rather specific mutations could be important.Patients with HPV‐driven (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) have a significantly improved overall survival compared to patients with HPV‐negative (HPV−) OPSCC. Nevertheless, 13%–25% of patients with HPV+OPSCC develop local/distant recurrence (LDR) and have a course of disease similar to HPV−OPSCC. We hypothesize that HPV+OPSCCs of patients with LDR have a mutation frequency and pattern similar to HPV−OPSCCs, which is associated with severe outcome. We performed targeted next‐generation sequencing using a customized gene panel and compared data from 56 matched HPV+and HPV−OPSCC of patients with/without LDR regarding protein‐altering variants. Despite improved overall survival of patients with HPV+OPSCC, those who develop LDR show a strongly reduced survival rate that is similar or even worse compared to HPV−OPSCC patients. Overall, the number of mutations was similar in OPSCC of patients with and without LDR. In total and with respect to  A subgroup of 13–25% of HPV‐driven (HPV+) OPSCC patients develop local or distant recurrence (LDR) and a severe course of disease similar to that of patients with HPV‐negative (HPV‐) OPSCC. Regarding the genes analyzed, we found that the HPV+OPSCC of patients with LDR carried a higher number of mutations than HPV+OPSCC of patients without LDR, with a similar mutation frequency as the HPV−OPSCC of LDR patients. Surprisingly, HPV−OPSCC of patients who did not develop LDR showed the highest number of mutations, suggesting that not the number but specific driver mutations are dominant factors for recurrence and that tumor‐specific neoantigens may play a role in a supportive antitumor immune response after therapy. On the other hand, HPV−OPSCC often carry TP53 variants.It is known that the HPV early proteins E6 and E7 have a transforming potential.HRAS, PIK3R1, STK11, and TP63 was elevated.Despite the generally good response to therapy and the good prognosis, approximately 13–25% of patients with HPV+OPSCC develop local or distant recurrence (LDR) within 2 years after current standard treatmentWe hypothesize that mutation numbers and patterns in OPSCC are similar in patients with LDR irrespective of HPV status. Therefore, we determined the total number of LDR in patients diagnosed with HPV+and HPV−OPSCC at our hospital between 2000 and 2018. We matched respective patients with HPV−OPSCC with/without LDR to cases of a previously analyzed cohort with HPV+OPSCC and compared frequency and patterns of protein‐altering mutations by targeted next‐generation sequencing using a customized gene panel.22.1INK4A) and detection of high‐risk HPV‐DNA as previously described.n = 139) except two cases , which were not included in our analysis. Among all patients with HPV+OPSCC, 21 (15.1%) patients were identified who developed LDR. Of those, primary tumor tissue meeting our inclusion criteria for sequencing analysis (see below) was available for 14 cases. Patients with established distant metastases at time of primary diagnosis were excluded from the targeted NGS analysis. DNA quality and/or quantity of FFPE tissue samples of five patients were insufficient for sequencing. To each of the remaining patients, we assigned corresponding cases with HPV+OPSCC without LDR and patients with HPV−OPSCC with and without LDR according to N‐stage, risk factors , T‐stage, and gender  and survival analysis, we excluded those patients diagnosed after November 2014 in the group without LDR to ensure an event‐free follow‐up of at least 5 years. HPV association (HPV+) was defined by overexpression of CDKN2A  following manufacturer's protocol. DNA quantity was determined with the Qubit™ 1.0 fluorometer  and the High Sensitivity DNA Kit . Subsequently, 10 ng DNA per sample was prepared following the protocol for the Ion AmpliSeq™ DNA and RNA Library Preparation kit (Thermo Fisher Scientific). Sequencing was performed on the Ion Torrent PGM (Thermo Fisher Scientific).DNA isolation and library preparation were performed as described previously.2.3TP53 (exon 2–exon 11) and 90 target regions in 22 genes . Overall survival (OS) was calculated from date of initial diagnosis (date of routine biopsy confirmed by histology) to the date of death due to any cause or to the last known date when the patient was still alive. OS rates were calculated by the Kaplan‐Meier method and the significance of differences was calculated by log‐rank test. Qualitative and quantitative data between all cohorts were compared using Kruskal–Wallis test, and between distinct cohorts using two‐sided Mann–Whitney 33.1INK4A and high‐risk HPV‐DNA; 15.1% of those patients developed LDR within 5 years after treatment. In contrast, patients with HPV−OPSCC developed LDR in 28.0% of cases . Patients with LDR and HPV+OPSCC have a substantially reduced OS (5y‐OS: 16.2%), which is even worse than that of patients with HPV−OPSCC independent of LDR (p = 0.056).The OS of patients by HPV and LDR status is shown in Figure 3.2TP53, the average base coverage was 3328 reads, with 2999 reads in HPV+and 3659 reads in HPV−OPSCC. Each gene was mutated in at least one primary tumor of both HPV−OPSCC subgroups with and without LDR. In HPV−OPSCC, tumors of patients with LDR harbored 129 mutations  altogether, while tumors of patients without LDR harbored 335 mutations  irrespective of the LDR status. No TP53 mutation was detected in 2/14 (14.3%) patients in each HPV−OPSCC subgroup. HPV−OPSCC of patients without LDR had altogether 64 TP53 mutations compared to 31 variants in HPV−OPSCC in patients with LDR; however, the median number of TP53 mutations per tumor is similar and the comparison did not reach significance . In HPV−OPSCC of patients without LDR, the most frequent mutated genes were TP63 and FAT1 with aberrations in seven samples (50.0%) each. The individual mutation counts of the listed genes between patients with and without LDR of HPV−OPSCC were similar  nor within HPV−(p = 0.415) OPSCC . HPV−OPSCC of patients without LDR presented a higher number of mutations than HPV+OPSCC of patients with LDR , further differences in the mutation number were found in FAT1 (p = 0.096), KRAS (p = 0.045), NOTCH1 (p = 0.071), NRAS (p = 0.045), and RB1 (p = 0.045). Divided into the four subgroups, we observed that HPV+OPSCC of patients without LDR had the least number of mutations, followed by HPV−and HPV+OPSCC of patients with LDR, whereas the highest number of mutations was found in HPV−OPSCC of patients without LDR  compared to HPV−OPSCC with LDR  and HPV−OPSCC without LDR  (Table TP53 (p ≤ 0.001), RB1 (p = 0.024), HRAS (p = 0.043), and FAT1 (p = 0.040), and trending toward significance were PIK3R1 (p = 0.070), TP63 (p = 0.060), and NOTCH1 (p = 0.085)  Table . Signifi4We observed tremendous differences in OS rates not only between patients with HPV+and HPV−OPSCC, but especially with regard to recurrence status Figure . We hypop = 0.079, Mann–Whitney U test; data not shown). Additionally, age at primary diagnosis could play a role in this context since the HPV+OPSCC patients with LDR were older than patients in the other groups , but only in 2.8% HPV+HNSCC.TP53 mutation frequency of 3/20 (15%) HPV+OPSCC from patients with LDR.TP53 mutations in our study could be a consequence of the composition of the cohort, that is, the enrichment of patients with LDR who have an increased number of TP53 mutations  cases, we detected higher FAT1) were elevated in HPV−OPSCC of patients without LDR compared to HPV−OPSCC of patients with LDR (p = 0.094). The human FAT1 regulates cell growth, migration, and polarization with a dual role as tumor suppressor and oncogene.FAT1 has been reported to inhibit tumor growth and epithelial–mesenchymal transition,FAT1 mutations and an improved OS in patients with HPV−but not in HPV+HNSCC patients.FAT1 negatively affect tumor progression in HPV−OPSCC patients.Mutations in the gene FAT atypical cadherin 1  or whole genome sequencing, which requires high‐quality DNA. As above, this is an unavoidable drawback related to the type of available samples. Hence, our data revealed interesting information, but underline the fact that prospective multicenter studies are needed, including frozen tissue biobanking and in‐depth analysis of gene variants in these clinically relevant subtypes of OPSCC. WES was applied in a previous study showing that metachronous HPV‐related OPSCC shares a genomic landscape with HPV‐unrelated cancers and that HPV‐related OPSCC which recurred is genetically similar to non‐recurring HPV‐related OPSCC.TP53 variants do not seem to influence the course of disease in HPV−OPSCC. However, their role for development of recurrence in HPV+OPSCC remains open. Although our study is focused on protein‐altering variant in a selected number of genes, one could hypothesize that specific driver mutations, rather than the number of mutations, are dominant factors in recurrence. Additional whole‐genome‐based studies are needed to investigate this speculation. In addition, it can be speculated that a higher number of tumor‐specific neoantigens is caused by a higher number of mutations in HPV−OPSCC.In summary, we found that HPV+OPSCC of patients with LDR carried a higher number of mutations in the analyzed genes than HPV+OPSCC of patients without LDR, with a mutation frequency similar to that of HPV−OPSCC of LDR patients. Surprisingly, the primary tumor tissue of patients with HPV−OPSCC who did not develop LDR had the highest number of mutations. Furthermore, All authors declare that they have no competing interests.The study was approved by the local ethics committee (reference number: 95/15).Figure S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.


Background: High risk human papillomavirus (hr-HPV)-associated oropharyngeal cancers (OPCs) are characterized by significantly better therapy responses. In order to implement a de-escalated treatment strategy for this tumor entity, it is highly crucial to accurately distinguish HPV-associated OPCs from non-HPV-associated ones. Methods: In this prospective study, 56 patients with histologically confirmed OPC were evaluated. A commercially available sandwich ELISA test system was used for the detection of hr-HPV E7 oncoprotein targeting the genotypes 16, 18 and 45. Results were presented as optical density. Positivity for HPV DNA and p16 immunohistochemistry (IHC) was taken as the reference method. Results: E7 positivity was significantly associated with the reference method (p = 0.048). The sensitivity, specificity, positive predictive value and negative predictive value for the E7 oncoptotein was 60.9% (95% CI 38.5 to 80.3%), 66.7% (95% CI 46% to 83.5%), 64.2% (95% CI 49.4 to 77.4%) and 63.01% (95% CI 48.9–75.2%), respectively, for the cutoff provided by the manufacturer. Conclusions: We found a significant association between E7 oncoprotein detection and the currently used combination. We believe that the use of the ELISA based E7 antigen test could be a valuable addition in cases of ambiguous findings and may be used in combination with other techniques to distinguish between HPV-driven and non-HPV-driven OPCs. However, the low sensitivity of the assay coupled with the small sample size in our study may represent a limitation. We recommend that future larger studies elucidate the diagnostic value of the E7 brush test.  An increase in oropharyngeal cancer (OPC) was first observed in the United States at the beginning of the 21st century ,4, whileThis highlights the need to incorporate a diagnostic surrogate marker which precisely distinguishes between a transient and a transforming hr-HPV infection. The biomarker currently in use for the diagnosis of HPV-driven OPC is the copresence of HPV DNA and overexpression of the cellular marker p16 protein ,12.p = 0.006) [1-S cell cycle transition and uncontrolled cellular replication [P16 is highly expressed in tissues undergoing cell cycle deregulation, suggesting that the detected hr-HPV DNA in the tumor tissue may be the cause of OPC. However, being merely a cellular marker, p16 is also overexpressed in lesions with no HPV association. Rasmussen and coworkers, for example, observed in a cohort of 1243 OPC patients a group of p16-positive but HPV DNA-negative patients. These patients experienced a significantly higher hazard ratio (HR) for metastatic recurrence as compared to HPV+/p16+ patients (HR = 2.56) (= 0.006) ,14. The lication ,16.A recently published study evaluated ELISA-based detection of hr-HPV E7 oncoprotein as a screening method in cervical samples of healthy women. Agorastos and coworkers found in this study that E7 oncoprotein detection might be a promising marker for precisely distinguishing transformation-relevant hr-HPV infections from transient ones . The ELIConfronted with the increasing need to establish a de-escalated therapy strategy explicitly for patients with HPV-driven OPC, we questioned whether this ELISA-based E7 oncoprotein test could be a reliable option in accurately distinguishing between HPV-driven and non-HPV-driven OPC.During the study period, 56 patients with OPC were included. Of the study population, 46 (82.1%) patients were male, the mean age at diagnosis was 65.4 (standard deviation ± 10.12) years and 26 (46.4%) patients were positive for the E7 oncoprotein . The meaIn 23 (41.1%) OPC patients, HPV DNA was detected, and the most common genotype was HPV 16 (83%) .p = 0.04) and smoking was inversely and significantly associated with E7 oncoprotein positivity (p = 0.009). Further clinico-pathological parameters are shown in The E7 oncoprotein was detected in 26 patients (46.4%) and was associated with the American Society of Anesthesiologists (ASA) classification (p < 001) (p = 0.048) with the reference method, including 50 patients with concordant results for p16 and HPV DNA  positivity was detected. There was a high association between p16 and HPV DNA (p < 001) . Patient HPV DNA . The twop = 0.031)  and 95% CI of the E7 oncoprotein ELISA was shown to be significantly higher among patients who were positive for the reference method  (median OD = 0.15) as compared to those who were negative for the reference method  (median OD = 0.04) (= 0.031) . In addition to the cutoff value provided by the manufacturer, we also analyzed the performance of E7 oncoprotein using other arbitrarily selected cutoff points. The best agreement with the reference method is achieved when using a cutoff OD value of 0.2. With this cutoff value, the E7 oncoprotein ELISA shows the highest specificity (and the highest % agreement) as compared to the cutoff value provided by the manufacturer .Sensitivity, specificity, positive predictive value and negative predictive value for the E7 oncoptotein was 60.9% (95% CI 38.5 to 80.3%), 66.7% (95% CI 46% to 83.5%), 64.2% (95% CI 49.4 to 77.4%) and 63.01% (95% CI 48.9–75.2%), respectively. The percent agreement between the standard approach and E7 method was 64%. Concordant results of the three test methods are shown in To our knowledge, this is the first study evaluating the role of E7 oncoprotein detection in patients with OPC for a precise distinction of HPV-driven from non-HPV-driven OPCs with a simple tumor surface brush sample. This test was initially developed to triage HPV-positive women being screened for cervical cancer . A positp = 0.048), consisting of concordant HPV DNA and p16 results. Despite the low sensitivity and specificity of the assay, E7 oncoprotein detection was significantly associated with the reference method (p = 0.04), indicating the lower rate of comorbidities [p = 0.009). This is in accordance with previous studies describing HPV-positive patients to be more commonly non-smokers, whereas smoking and alcohol consumption are the pathogenic mechanisms in non-HPV-driven OPCs [E7 oncoprotein positivity was also associated with the ASA score .Data from cervical cancer studies show that, in some tumors, although definitely caused by HPV, tests are negative for HPV DNA ,28. A plp = 0.0095) and disease-free survival (p = 0.03) in comparison to class I and class II OPC patients [Moreover, in this study, a certain proportion of patients  positive for p16 IHC and negative for HPV DNA was observed. The importance of additional HPV DNA testing to identify HPV-positive OPCs was described in several studies ,30,31. Apatients . Detection of the HPV oncoproteins in the clinical routine of OPC patients is still challenging. The brush test is very feasible and easily applied; however, further studies based on our results and further developments may be necessary. The brush is designed for the cervix, so, in the oropharyngeal region, a smaller brush would be more applicable in order to ensure that the tumor surface can be brushed more precisely without touching the surrounding tissue. A major limitation of the study is the lack of data on E7 mRNA expression. Taken by some as the gold standard for the classification of transcriptionally active hr-HPV, results of mRNA PCR may have supplemented the low sensitivity of this ELISA-based antigen assay. However, we used the combination of HPV DNA and p16 detection as a reference method, since this combination is the standard method for the diagnosis HPV-driven tumor according to many guidelines ,12. The This was a prospectively designed study, and patients presenting with OPC between January 2018 and June 2020 at the Department of Otorhinolaryngology, Medical University Innsbruck, Austria were included. Each patient who agreed to participate in this study gave informed consent. The study was conducted in full accordance with the principles expressed in the Declaration of Helsinki and was approved by the ethics committee of the Medical University Innsbruck. The respective reference number was 1147/2018. Patients were included only when the histology confirmed squamous cell carcinoma of the oropharynx and the patient’s age was at least 18 years. Patients with other diagnoses than OPC in the histopathological examination were excluded. With the exception of 3 patients, all patients had a primary tumor in the oropharyngeal regions, and 3 patients presented with a recurrence or second primary in the oropharynx. In all patients, the E7 brush test was performed before treatment.® HC2 DNA Collection Device, Qiagen, Hilden, Germany and ThinPrep® PreservCyt Solution, Hologic, Manchester, UK) by gently brushing the tumor surface. The brushes were then placed in a sterile container and sent to the Institute of Virology, Medical University Innsbruck. Furthermore, tumor biopsies were obtained and fixed in formalin for routine histopathological examination at the Department of Pathology, Medical University Innsbruck. Additionally, a tumor biopsy for p16 IHC detection was kept in cell culture medium and immediately sent to the Laboratory for Molecular Biology and Oncology, Department of Otorhinolaryngology.Details of specimen harvesting and handling has been described in a previous work . In shorDetection of E7 oncoprotein for genotypes of HPV 16, 18 and/or 45 was conducted using a sandwich ELISA test system  developed and validated (CE-labelled) initially to support diagnostic and therapeutic decisions in cervical cancer screening . The ELI®, Padova, Italy) [For HPV DNA detection, real-time PCR was used based on the amplification of the L1 open reading frames (ORF). As internal control for the availability of cellular material, a PCR for the housekeeping gene beta globin was performed. The HPV DNA was considered positive if the fluorescence signal appeared before the fortieth cycle . Further, Italy) . With th® Histology V-Kit, Roche diagnostics, Basel, Switzerland). The staining was completed by using a universal secondary antibody solution, the DAB MAP Kit and hematoxylin counterstaining (both Ventana products). One experienced observer evaluated the tumor cell areas, and specimens were considered p16-positive if ≥66% of the cells in the tumor areas revealed immunohistochemical reaction products. First, five-micrometer thin paraffin sections were dewaxed and then antigens were retrieved in an automated staining system . For p16 detection, a commercial diagnostic assay was used  was obtained. Combined hr-HPV DNA positivity and p16 positivity served as the reference method. Contingency tables were analyzed with Fisher exact test or Pearson chi-square. Diagnostic accuracy parameters including sensitivity and specificity were calculated using the diagnostic test routines of MedCalc. For data analysis, SPSS Statistics 24 software  was used.We do not have strong evidence that confirms that ELISA-based E7 oncoprotein replaces the reference method for diagnosis of HPV-associated OPC. However, looking at the significant association with the reference method of diagnosis, it could be a valuable addition in the case of ambiguous findings. We strongly recommend further investigation and optimization of the test in large prospective studies.