HPV self-sampling has been widely supported by the scientific community following a strong body of literature on the subject. Self-sampling is important in cervical cancer screening as it has been shown to improve participation. It is well documented that HPV-testing has proven superior to cytology with regards to sensitivity in detection of CIN and cancer. The value of self-collected samples is reliant on the quality of the molecular testing performed, as well as the patients’ preference in sampling procedure and compliance to follow up on positive test results. Due to the incompatibility of self-samples and cytology, triage of HPV-DNA positives by testing for molecular biomarkers is highly warranted.Our objective was to compare the detection rate of genital Human Papillomavirus (HPV) infection in self- and clinician-collected samples by a 14-type HPV-DNA test and a 7-type mRNA E6/E7 test.P = 0.19). Overexpression of mRNA E6/E7 from 7 HPV types was 7.1 and 6.3%, respectively (P = 0.71). The difference between HPV-DNA and HPV-mRNA positivity rates were statistically significant in both self-collected  and clinician-collected samples . Overall agreement between the two collection methods was fair, with a concordance rate of 78.2% (390/505), k = 0.34 (95% CI: 0.25–0.44), P < 0.001, for the HPV-DNA test and 92.5% (467/505), k = 0.40 , P < 0.001, for the mRNA test, respectively. 96.8% of the participants reported they felt confident carrying out the self-collection themselves, and 88.8% reported no discomfort at all performing the procedure.Five hundred five women were recruited. Each study participant had two sample collection procedures performed upon the same visit, alternating order in execution of the self-collection or the clinician-taken procedure first or second, 1010 samples in total. HPV-DNA prevalence was 22.8% in self-collected versus 19.2% in clinician-collected samples (This comparative study of two sampling methods reports fair agreement of HPV positivity rates between the self-collected and clinician-collected specimens using Abbott hrHPV and PreTect HPV-Proofer’7 tests. Only one third of HPV-DNA positive women had overexpression of mRNA E6/E7.ISRCTN77337300.The online version contains supplementary material available at 10.1186/s12879-021-06189-2. Cervical cancer is a major public health problem, and the second most common cancer in women living in less developed regions with an estimated 570,000 new cases in 2018. According to the World Health Organization, approximately 311,000 women died from cervical cancer in 2018: more than 85% of these deaths occurring in low- and middle-income countries , 5 and wCurrently, most guidelines recommend cytology in triage of HPV-DNA positives. As PAP reflex testing is not applicable on self-collected material, a new clinician-collected sample is required for the purpose of triage, hereby suffering increased risk of loss to follow up; based on the nature of non-attenders. An alternative to cytology in triage could be detection of molecular biomarkers; being compatible on self-sampled material .Literature describes effects caused by device and specimen processing in terms of various concentration and quality of cellular material that possibly impair test performance , 13 whilThis study was conducted to compare the performance of self-collected samples by using a self-sampling device  versus clinician-collected samples by using the professional-use device  in combination with a 14-type HPV-DNA and 7-type mRNA E6/E7 test as the primary outcome.Patient-reported acceptability of the self-collected samples method was the only secondary outcome.The study was approved by the institutional ethics review board (CI/243/18) at Eduardo Liceaga, Mexico General Hospital, Mexico City, before commencing.http://www.isrctn.com/ISRCTN77337300Trial registration: ISRCTN, ISRCTN77337300. Registered 15 December 2020 - Retrospectively registered, During the period August 2018 to April 2019, study participants were recruited among women attending cervical cancer screening and among health professionals working at the Oncology clinic of Eduardo Liceaga, Mexico General Hospital, Mexico City. Eligible were sexually active women aged 30–65 that responded positively to the invitation and had no history of medical or surgical treatment  for cervical cancer. Excluded were pregnant and breastfeeding women, those who had had sexual activity within 24 h before to the collecting samples procedure, and those who chose not to sign the informed consent. Prior to enrollment, the purpose and nature of the study were explained to each participant to obtain their written consent. The two sampling procedures were performed upon the same day, in alternating order for each participant upon arrival. Since the study was conducted at the clinic for cervical screening, clinicians’ additional training was unnecessary to collect the reference sample. For the self-sampling device, the manufacturer’s written and illustrated instructions for use in Spanish were provided to all participants. The procedure was performed in a separate room for the women’s discretion without assistance by health staff. Finally, after the samples were collected, the participant completed a questionnaire about her experience during the self-collection procedure. Respondents scored items on an 8-point Likert scale, ranging from 1 (“no discomfort”) to 8 (unbearable discomfort”). All women were informed to discuss their test results upon completion with a healthcare professional to decide on further follow-up. Patient sensitive information was anonymized by a 6-digit code and registered using Microsoft Excel 16.16.23 (200615–2016). Only study responsible staff at the hospital had access to the registered information.The participants of this study performed self-collection of cervicovaginal material using the XytoTest medical device , an ergonomically designed device to be inserted into the vagina, with a diameter less than 8 mm and a length of 14 cm [A clinician collected the sample by using the Cervex-Brush® , which is among the most widely used method for cervical cancer screening, commonly used as a reference method for validation of new devices . After sme HR HPV test  is an automated, qualitative multiplex assay based on real-time polymerase chain reaction (PCR) intended to detect 14 high-risk HPV genotypes  and to partially genotype 16, 18 from the other 12 high risk genotypes. Human Beta-globin is detected as an internal control. The test has been clinically validated according to international consensus guidelines and proved to also be accurate for self-collected samples [The Abbott RealTi samples , 18. AllPreTect HPV-Proofer’7  is a diagnostic kit for the qualitative detection and direct typing of E6/E7 mRNA from HPV 16, 18, 31, 33, 45, 52 and, 58. The kit contains an intrinsic sample control (ISC) targeting a human housekeeping gene to assess specimen quality and reveal possible factors that may inhibit amplification. The kit utilizes real-time NASBA technology, an enzymatic one-step amplification process able to amplify RNA under isothermal conditions at 41 °C . SeveralThe sample size was estimated considering a two-tailed hypothesis, with a 95% confidence interval, a statistical power of 80%, and an effect size (Cohen’s d) of 0.12555. With these characteristics, the estimated sample size required was 500 study participants, calculated using the software GPower 3.1.9.2. Microsoft Excel 2016  and IBM SPSS Statistics software package version 21  were used for data collection and evaluation. Cohen’s Kappa coefficients were calculated to evaluate the agreement between the two sampling methods regarding hr-HPV results, applying the most commonly used scale to express the strength of the agreement as follows: 0.00–0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80 and ≥ 0.81 indicated slight, fair, moderate, substantial, and almost perfect, respectively. The Wilcoxon signed -rank test was applied to evaluate the differences between the two sampling methods. A statistically significant difference was defined as a 5% chance of a type I error (α ≤0.05). For comparison of the presence of HPV-16, HPV-18, and 12 other hr-HPV genotypes between the two samples, the following terminology was used: concordant or discordant. A concordant result was determined if results from all three channels  showed at least one identical genotype in both samples, where discordance represented no similarities in genotypes at all.Overall, a total of 505 women participated in the study, everyone within the inclusion criteria. 65.5% of the participants were recruited among women attending the general screening program at the clinic, and 33.5% were recruited among the health professionals working at the Mexico General Hospital. The inclusion of health professionals was partially to learn whether the level of education might affect the quality of the sample evaluated by cellularity and sample integrity if the instructions for self-collection was not sufficiently clearly described as health professionals presumably would have a better understanding of sample taking in general. In total 1010 samples were collected equally distributed between self-collected samples, and clinician collected specimens. All the collected samples by the two procedures were valid for processing. The mean age of the participants was 43.8 ± 8.1 years . Just over two-thirds of the women (69.7%) reported they were older than 18 years when they first had sexual intercourse, and less than a third (30.3%) were under the age of 18 , whilst the positivity rate for HPV mRNA E6/E7 was about one third; 7.1%  versus 6.3%,  respectively (P = 0.71).The prevalence of hr-HPV-DNA varied from 22.8%  to 19.2%  among the self-collected and clinician-collected samples (P > 0.05). Mostly non-HPV16/18 genotypes were detected. The vast majority of the infections identified in self-collected samples were single (85.2%) and multiple infections were found in 17 specimens (14.8%) in line with 84.5 and 15.5% among clinician-collected samples , k = 0.40 (95% CI: 0.25–0.56), P < 0.001, for the HPV mRNA test, respectively. In women with at least one positive HPV-test, the agreement was 31.7% (51/161) for the HPV-DNA test and 28.3% (15/53) for the HPV mRNA test. Kappa for the mRNA test in the 161 women with at least one positive HPV DNA test was 0.30 (95% CI: 0.12–0.48), P < 0.001  had discordant HPV-DNA results. In 46 cases, the women were clinician-collected sample HPV-positive/self-collected sample HPV-negative. Among these, 24 had the clinician-collected sample taken first and 22 women the self-sampling first in order. Sixty-four women were clinician-collected sample HPV-negative/self-collected sample HPV-positive; among those of whom 27 had their first sample by their clinician versus 37 women self-sampling first. Five women were detected as hr-HPV positive in both samples collected, with no similarities in genotypes. Regardless of order of the method used, no significant differences in the agreement rates for self-collected, and clinician-collected sampling techniques were observed, P < 0.001), tested using Wilcoxon signed rank test.All 505 paired samples obtained valid results for the two subsequent HPV amplification tests done, hence no exclusions were made due to low cellularity. The manual cell count using Bürker chamber was done for a random subset of paired samples to evaluate the number of cells collected by each sampling method, 97 pairs in total. Forty-seven of the self-collected samples were done by health professionals, 50 self-collected by women attending screening. The average number of cells per milliliter was calculated following standard protocol and descriptive statistics are presented in Table . The selP = 0.19).Evaluating the cellularity obtained by self-collection performed by the two subgroups of study participants , no difference was observed  is designed to optimize clinical performance, and since no relevant clinical outcome by the means of cytology or histology is available in this study, this will not be possible to discuss. It is well known that PAP test sensitivity  varies dWithin this study population, the hr-HPV detection rate by PCR assay was about 20%, which is comparable to the prevalence (24.7%) found in another study conducted in the Mexican population . ClinicaThe obtained significant higher cellularity per milliliter for the self-collected versus the clinician-collected specimens in this study might be explained by the fact that XytoTest has an area coated with a highly adhesive hypoallergenic elastomer and therefore cells are more readily collected as soon as the device is inserted into the vagina.It is known that molecular biomarkers are of vital importance in relation to the triage of patients in cervical screening programmes . All curThe perspective of implementing HPV mRNA in triage of test positives are among one of the options discussed to represent an improved, highly specific risk-based approach for maximizing screening benefits and minimizing harms –21, 30.Our data confirm 7-type mRNA testing to be applicable for reflex testing on self-collected specimens. Among the study participants, only one third of the HPV infected women had a positive mRNA test, an appealing situation for effective triage reducing the number of colposcopies and biopsies.Recent research has proven the 7 genotypes included in the mRNA test to be the most important to screen for, being responsible for 90% of all cervical cancers , 31, 32.Reliable self-sampling methods and molecular diagnostics may significantly aid prevention of cervical cancer, by simplicity, increased accessibility to screening and accurate diagnostics.By combining HPV-DNA testing and identification of mRNA E6/E7 biomarkers, both high sensitivity and specificity might be maintained for improved patient management , 34.A clear limitation of this study is the lack of clinical data; cytology and a histology examination of samples that were found to be hr-HPV positive or mRNA E6/E7 positive, which would have enabled associations to be drawn between molecular assays with possible morphological changes, and presumably identify progression of cervical lesions. Unfortunately, these data were not available for our review, nor contemplated in the inclusion criteria of this study.Although the acceptability of self-collected samples was found to be high among the women participating in this study, the questionnaires are of limited reliability because the study administered no parallel healthcare provider-reported questionnaire.This comparative study of two sampling methods reports fair agreement of hr-HPV positivity rates between the self-collected and clinician-collected specimens using Abbott hr-HPV and PreTect HPV-Proofer’7 tests. Only one third of HPV-DNA positive women had overexpression of mRNA E6/E7, effectively discriminating women warranted for immediate colposcopy/biopsy from return to follow-up and suggests longer follow-up interval for single HPV-DNA positive women. Such a strategy will inevitably reduce over-referral for colposcopy but needs clinical and cost-benefit assessment in prospective studies.Additional file 1.


Cervical cancer is recognized as a serious public health problem since it remains one of the most common cancers with a high mortality rate among women despite existing preventative, screening, and treatment approaches. Since Human Papillomavirus (HPV) was recognized as the causative agent, the preventative HPV vaccines have made great progress over the last few years. However, people already infected with the virus require an effective treatment that would ensure long-term survival and a cure. Currently, clinical trials investigating HPV therapeutic vaccines show a promising vaccine-induced T-cell mediated immune response, resulting in cervical lesion regression and viral eradication. Among existing vaccine types , deoxyribonucleic acid (DNA) therapeutic vaccines are the focus of the study, since they are safe, cost-efficient, thermostable, easily produced in high purity and distributed. The aim of this study is to assess and compare existing DNA therapeutic vaccines in phase I and II trials, expressing HPV E6 and E7 oncoproteins for the prospective treatment of cervical cancer based on clinical efficacy, immunogenicity, viral clearance, and side effects. Five different DNA therapeutic vaccines , pNGVL4a-Sig/E7(detox)/HSP70, MEDI0457) were well-tolerated and clinically effective. Clinical implementation of DNA therapeutic vaccines into treatment regimen as a sole approach or in combination with conservative treatment holds great potential for effective cancer treatment. Cervical cancer is a largely preventable cancer of the cervix, which is the narrow part of the lower uterus that connects to the vagina. According to the World Health Organization (WHO) statistics, cervical cancer became the fourth most frequent cancer in women in 2018, with 570,000 cases, which represent 6.6% of all female cancers worldwide [Previous studies have established the strong causative association between persistent infection with certain high-risk Human Papillomavirus (HPV) types and the development of cervical cancer . HPV is About 90% of deaths from cervical cancer occurred in low- and middle-income countries largely due to the lack of proper prevention, early diagnosis, and effective screening . In 2018Nowadays, early-stage cervical intraepithelial lesions (CIN) are treated by means of surgical resection of cancerous tissue, which include conization, loop electrical excision procedure (LEEP), and radical hysterectomy . These aTherapeutic vaccines such as TheraCys, PROVENGE, and IMLYGIC used for the treatment of urothelial carcinoma in situ, prostate cancer, and advanced melanoma respectively with promising results . These cCurrently, HPV therapeutic vaccines under investigation include live vector vaccines , subunit vaccines (peptides and protein-based vaccines), plant peptide/protein-based vaccines, nucleic acid vaccines (DNA and ribonucleic acid (RNA) replicon-based vaccines), and cell-based vaccines (dendritic cell-based vaccines and adoptive cell transfer) . Among tThis systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews (PRISMA) statement . The stuArticles were manually searched using databases as PubMed/MEDLINE, Google Scholar, and clinicaltrials.gov published in English from the year 2010.The search was performed using the following keywords: “cervical cancer”, “cervical intraepithelial neoplasia”, “HPV”, “HPV-positive”, “E6 and E7 oncoproteins”, “therapeutic vaccine”, “DNA vaccine”, and “DNA therapeutic vaccine”. We used the medical subject heading (MeSH) term “Uterine Cervical Neoplasms” (MeSH Unique ID D002583) as major topic and “Vaccines” (MeSH Unique ID D014612), “E6 protein, HPV type 18” (MeSH Unique ID C052603) and “E7 protein, HPV type 16” (MeSH Unique ID C059731).The search was narrowed by using “Cervical cancer OR Cervical Intraepithelial Lesion AND DNA therapeutic vaccines”, “DNA therapeutic vaccines AND E6 OR/AND E7 oncoproteins”. The selected studies were independently reviewed for inclusion eligibility by two reviewers (Akhatova and Aimagambetova) using standardized data collection forms. The following data were collected from the studies: the author, year of publication, number of study participants, vaccine administration strategies, and the main outcomes . Any discrepancy in the assessment of articles was resolved by discussion and consensus, as well as input from the third and fourth reviewers (Chan and Azizan).The articles were selected to meet the following eligibility requirements to be included in the study: (1) research article, (2) human subject research, and (3) the study of DNA therapeutic vaccines targeting HPV E6 and E7 oncoproteins. The presence of the following did not allow for the study to be included: (1) reviews and case reports, (2) irrelevance to cervical cancer or CIN, (3) mouse model studies, (4) articles on preventative HPV vaccines, and (5) the use of the inappropriate methodology. Abstracts lacking full information about predefined criteria were excluded without further review.The types of studies included were phase I and phase II clinical trials that were initiated and completed between 2003 and 2017, studying the clinical efficacy of DNA therapeutic vaccines expressing HPV E6 and E7 oncoproteins for the treatment of cervical intraepithelial lesions of grades 2 and 3 both newly diagnosed and recurrent malignancies. The treatment of the lesion may or may not be followed by conization or loop electrosurgical excision procedure. The study population was female patients aged 18 or older with histopathologically diagnosed CIN of grades 2 and 3, known to be caused by HPV 16 and/or HPV 18 based on polymerase chain reaction (PCR) amplification results. The main outcomes of the review were clinical efficacy based on the lesion regression, viral load reduction, immunogenicity, in particular, HPV E6 and E7 specific CD8+ T cell response, and AEs after vaccination.During this study, 120 articles were identified through PubMed/MEDLINE and Google Scholar searching platforms .Eighty-three articles were excluded based on the abstract, representing literature reviews. From the remaining 37 articles, 24 articles were excluded at this stage: 11 studies were mouse model-based, 9 studies were not addressing the study question, 2 studies were regarding the preventative vaccines, and 2 studies included additional oncogenes in the development of therapeutic vaccines. We included 6 studies performed between 2003 and 2017 in our systematic review after the exclusion of 7 articles studying the effects of therapeutic vaccines with either viral, bacterial, or peptide vectors ,15,16,17Therapeutic vaccines were evaluated based on clinical efficacy , viral clearance, immunogenicity, and adverse events after the vaccination. Subjects, female patients aged 18 or older with histopathologically diagnosed cervical intraepithelial neoplasia of grades 2 and 3, known to be caused by HPV 16 and/or HPV 18, received DNA therapeutic vaccines in different dose formulations and were evaluated generally within 20 weeks  for the effects of vaccines mentioned above. Study populations ranged from 10 patients to 167, median age mostly being 21–30, except for Hasan et al. (2020) study, where the median age of participants was 51.50 years old due to more advanced stages of cancer .Clinical efficacy was evaluated according to the histopathological regression to CIN ≤1, which is less than one-third of the thickness of the cervical epithelium, on a colposcopy-guided biopsy 15, 20, or 36 weeks after the first injection. All six studies ,15,16,17GX-188 E in phase I trial by Kim et al.  showed aViral load was measured by means of PCR amplification to assess the clearance of HPV DNA from the cervical biopsy after vaccination. Choi et al. established that HPV clearance was associated with the histopathologic regression as 77% of regressors had no trace of HPV DNA, while only 12% of non-regressors had no viral load in the tissue biopsy . MEDI045ex vivo ELISpot assay with cryopreserved and thawed peripheral blood mononuclear cells (PBMCs) at pre- and post-treatment stages. The vaccine response is considered positive when the increase in T-cell frequency was at least three times greater compared to the study entry measurement. GX-188E both in phase I [6 PBMC, whereas no patients produced similar responses in cohort 2 [Immunogenicity is one of the key features of the therapeutic vaccines as it represents the potential of the vaccine to induce virus-specific T cell response, in particular HPV E6 and E7 specific CD8+ T cell immune response. IFN-γ response was measured by means of  phase I  and phas phase I  studies  phase I ,13. VGX- phase I . On the  phase I . In cohocohort 2 . pNGVL4acohort 2 ,16.Overall, all vaccines were well-tolerated without vaccine-related serious adverse events. The most common adverse events were injection site pain and erythema, as well as constitutional symptoms  ,15,16,17This systematic review summarizes the findings of phase I and phase II clinical trials investigating the treatment of patients with histopathologically diagnosed CIN associated with HPV 16 or/and HPV 18 with DNA therapeutic vaccines. Six studies have demonstrated immunologic response in the form of lesion size regression, viral clearance, and increased T cell response of five different DNA vaccines–GX-188E (phase I and phase II), VGX-3100, pNGVL4a-CRT/E7 (detox), pNGVL4a-Sig/E7 (detox)/HSP70, MEDI0457. Vaccines were plasmid DNA encoding for either non-oncogenic E6/E7 or both, and chaperonin proteins such as HSP 70 and Calreticulin for the enhancement of the uptake by antigen-presenting cells, and MHC class I processing and presentation. MEDI0457  had the Previous reviews have studied various existing therapeutic vaccines including live vectors, plant-based, protein, whole cell, and combinatorial vaccines . This isDNA therapeutic vaccines were also assessed based on their clinical efficacy, i.e., the ability to induce cervical lesion regression. The regression to ≤CIN1 among study participants was observed in all studies with significantly varying degrees. The study of VGX-3100 vaccine with both treatment and placebo groups showed a response rate of 49.5% vs. 30.6%, respectively . MeanwhiIt was established that women, after excision of the cervical lesion, are more likely to have a relapse; therefore, viral clearance is a key factor of vaccine efficacy . VGX-310Nevertheless, there are several limitations to this study. Firstly, limited data exist on the topic of DNA therapeutic vaccines, as not a single therapeutic vaccine against cervical cancer was approved. All these clinical trials were in either the phase I or phase II stage of assessing the efficacy and safety in humans. Secondly, the majority of studies enrolled a small number of participants without masking, stratification, or the control group, which poses a potential risk for bias. As vaccines investigated in this study had different structural designs, it was not feasible to make a statistical analysis of vaccine outcomes; therefore, qualitative analysis was performed overall.As it was stated by WHO, a global strategy to accelerate the elimination of cervical cancer, 90–70–90 targets for prevention, screening, and treatment are the key to success. Preventative bivalent and quadrivalent HPV vaccines have undergone significant advancement in development and implementation. However, these preventative vaccines do not elicit a therapeutic effect but could be used as an adjuvant to surgical treatment. DNA therapeutic vaccines represent a potentially safe and novel approach to cervical cancer treatment. The main goal of this review was to discuss the effectiveness of existing DNA therapeutic vaccines against cervical cancer expressing HPV 16/18 oncoproteins E6 and E7. The idea of DNA therapeutic vaccines is inducing an adaptive immune response and immunologic memory via the expression of tumor antigens and activation of antigen-presenting cells.de novo or boost existing immune responses. Moreover, studies have shown that using femtosecond laser treatment could also improve transfection efficiency administered intradermally and into the lesion in vivo. Thus, continuous efforts to improve the efficacy of DNA therapeutic vaccines and implementation of therapeutic vaccines into a treatment regimen as a sole approach or in combination with conservative treatment may greatly improve the current situation.DNA therapeutic vaccines are currently undergoing clinical trials to improve the potency of therapeutic vaccines and clinical efficacy using strategies as a modifying route of administration, adjuvant therapy, prime-boost regimen, and co-administering with other drugs for a synergistic effect. Nowadays, despite the treatment of locally advanced disease with chemoradiation, patients have a high recurrence rate and a poor 5-year survival rate, estimated at 50% and 70%, respectively. In contrast, the MEDI0457 vaccine, which contained VGX-3100 plasmid coupled with an IL-12 expression plasmid to promote T-cell function, evaluated the disease progression-free survival (PFS) at 12 months, which was estimated as 88.9% overall. These findings strengthen the hypothesis that DNA therapeutic vaccines could effectively induce


To understand how human papillomavirus (HPV) screening results, HPV-related knowledge and attitudes are related to vaccination intention in three cost ranges and the actual vaccination behavior in a community sample of men who have sex with men (MSM).th-month follow-up.MSM aged 20 years of age or older were recruited between October 2015 and May 2016 from community health centers that provide HIV testing and consultation services in Southern Taiwan and on social media. MSM were seen at baseline and again at 6 months after baseline in a cohort study. The baseline study included 253 individuals; 182 of them returned for the 6th-month follow-up. At each visit, MSM were asked to receive HPV screening and filled out a questionnaire. Structural equation modeling was used to test whether attitudinal factors and HPV screening results from the baseline affect their self-reported actual vaccine uptake at the in 6p = .02), and then predict vaccine uptake at the follow-up (p < .001). A positive HPV screening result can predict vaccine uptake at the follow-up (p = .004).Our research included 171 participants from the cohort because they had full information of the study variables (mean ± SD age = 29.21 ± 6.18). Our model showed good model fit using indices such as the comparative fit index  and root mean square error of approximation . HPV knowledge can predict those who have intention to take up HPV vaccine no matter what the price (Our findings highlight the impact of vaccine price and HPV screening results on the intention and uptake of HPV vaccine. It is important to raise awareness of HPV in male populations. Clinicians and health educators should establish a safe and private environment for male patients for inquiring about HPV vaccine and HPV-related cancers.The online version contains supplementary material available at 10.1186/s12889-021-12396-y. HPV vaccines can effectively protect men from penile cancer, genital warts, and anal and oropharyngeal cancers for both men and women . CurrentA policy to encourage providing HPV vaccine for MSM faces some challenges. One is the concern regarding cost effectiveness, although studies in England and Australia have indicated the benefits of HPV vaccinations in MSM , 45. AnoWe used the Health Belief Model (HBM) and the Theory of Planned Behavior (TPB) in understanding the uptake of HPV vaccine in men , 14, 27.Despite the factors derived from risk perceptions and attitudes toward the HPV vaccine having been tested in several studies , 14, 28,HPV vaccine uptake as a type of self-care intervention for sexual health is also influenced by the HPV vaccine prices. Countries with higher income have a higher rate of introducing HPV vaccine with wider coverage . The affth-month data for this study. The baseline study included 253 individuals; 182 of them returned for the 6th-month follow-up. In both visits, participants were asked to receive HPV screening and filled out a questionnaire. Survey questions included assessments of HPV knowledge, HPV vaccine attitudes, and intention to take up HPV vaccine at different prices. Penile and anal HPV screening was used to detect HPV infection involving 37 genotypes . Screening results were emailed to each participant within a month after the baseline, along with an information sheet that explained how to interpret the results; HPV vaccine as a preventive measure was briefly mentioned. Only the screening results from the baseline were used for our study. At the 6th-month follow-up, participants were asked whether they had actually received HPV vaccine. The study was approved by the Ethics Committee of the National Cheng Kung University Hospital (reference number: A-BR-103–075). More details regarding the cohort description and baseline findings are reported elsewhere [Participants were recruited between October 2015 and May 2016 from community health centers that provide HIV testing and consultation services in Kaohsiung City in Southern Taiwan and on social media. Eligible participants were 20 years of age or older, had ever had sex  with another man, and were willing to give written consent to participate in the cohort study. There were no exclusion criteria. We included participants who missed HPV vaccines at the appropriate recommended ages because they can still be exposed to new HPV infection. If the MSM’ clinicians had suggested that they receive HPV vaccine, it was still possible for them to receive the vaccine even though it may not be as beneficial as receiving the vaccine at a younger age. MSM were seen at baseline and again at 6 months. We used baseline and 6lsewhere , 39. In Demographics included age, relationship status, education, occupation and monthly income.HPV knowledge was based on 28 items from the original scale that included questions such as “having many sexual partners increases the risk of HPV infection” and “HPV vaccines are most effective if given to people who've never had sex” developed by Perez et al. to measure HPV knowledge (Cronbach's α: 0.739-0.916) . ParticiHPV-related and HPV vaccine attitude included five constructs: capacity to obtain HPV-related information, perceived severity of HPV disease, subjective norm, perceived benefits of HPV vaccine, and perceived barriers to HPV vaccine. All questions were asked on a five-point scale from 1 “stronger disagree” to 5 “stronger agree.” The mean score represented the level of each construct of HPV vaccine attitude. Details regarding each construct are listed below.Capacity to obtain HPV-related information used three questions that included whether the participants had enough information about HPV vaccine, whether they thought there was enough research about the HPV vaccine, and whether they were comfortable discussing sexual health issues or HPV vaccines with healthcare providers  , 19, 35.Perceived severity of HPV disease was based on three questions that included whether the participants felt that contracting HPV, genital warts, or HPV-induced cancer was a serious problem  , 23, 23.Subjective norm was based on four questions that included whether they believed in the advice from healthcare providers and government regarding HPV vaccine, and whether they felt that recommendations for HPV vaccination from healthcare providers and government were important (Cronbach's α in the present study = 0.877) .Perceived benefits of HPV vaccine were based on five questions that included whether participants thought that the HPV vaccine could prevent HPV, genital warts, or HPV-induced cancer, whether they thought that the HPV vaccine could protect their sexual health, and whether they thought that the HPV vaccine could protect their partners from every type of HPV  , 30, 35.Perceived barriers included eight questions such as concerns regarding the vaccine’s safety and whether the participants thought that it was hard to find a provider or clinic to receive the HPV vaccine  , 12, 35.Intention to receive the HPV vaccine was assessed by one question: whether the participants were willing to take up HPV vaccine within six months given the efficacies in preventing genital warts, penile and anal cancers, conditioned on three price range possibilities: (a) NT$8000 to 12,000 , (b) NT$4000 to 8000 (US$ 133 to 267), or (c) free for a total of 3 shots. This question was revised from previous research but used price ranges that matched the prices in Taiwan . For eacth month follow-up. We asked participants whether they had received any HPV shot in the past six months. As long as they had initiated the first shot, they were considered as having received HPV vaccine in the follow-up.Vaccine uptake was assessed in the 6t-test and χ2 tests to compare the characteristics, HPV knowledge, HPV vaccine attitude, HPV infection, and intention between those who reported having received the HPV vaccine at the 6th-month follow-up and those who had not. We used Pearson’s correlation to compute the constructs between HPV vaccine attitude, HPV infection, intention, and uptake.We used mean and percentage to present the descriptive statistics for participant characteristics, HPV knowledge, HPV vaccine attitude, HPV infection, intention and uptake. Then we used independent Our research established the model that HPV knowledge and vaccine attitude can predict intention to take up vaccine with a different price and that HPV screening results and intention can predict HPV vaccine uptake. Structural equation modeling (SEM) was applied to test the model about HPV vaccine uptake and factors associated with intention among MSM Fig. . Comparath-month follow-up. Out of 171 MSM, 34.5% had anal or penile HPV DNA positivity, 62% were below age 30, 76% had a college degree or below. Almost 60% were single or single but dating; the rest were in committed relationships. About two thirds were employed. Twenty-eight had received HPV vaccine at the 6th-month follow-up (16.4%). As for intention, 45% were willing to take up HPV if the price was NT$8000 to 12,000, 60% if the price was NT$4000 to 8000, and 93% if the vaccine was free.Table th-month follow-up with those who did not, only HPV positive and intention to receive the HPV vaccine were significantly associated . Capacity to obtain HPV-related information was correlated positively with perceived barriers to HPV vaccine (r = 0.431). Perceived severity of HPV was correlated positively with subjective norm (r = 0.405) and perceived benefits of HPV vaccine (r = 0.268). Subjective norm was correlated positively with perceived benefits of HPV vaccine (r = 0.534). Perceived benefits of HPV vaccine were negatively correlated with perceived barriers to HPV vaccine (r = -0.356). We provided results of a regression analysis to determine what percent of the variance of the outcome variable (vaccine uptake) was determined by the different independent variables in Supplementary file 2.R2 are the following: 0.022 for HPV knowledge, 0.064 for those who have intention to take up HPV vaccine no matter what the price is versus those who have no intention even if it were provided free or those who only have intention when the vaccine is free, 0.022 for those who have intention to take up HPV vaccine if the price is below NT$8000 versus those who have no intention even if it were provided free or those who only have intention when the vaccine is free, and 0.113 for HPV vaccine uptake.Our model as shown had good fit to the data . The research use of type I error at 0.05, degree of freedom at 20, sample size at 171, null RMSEA at 0.00, and alternative RMSEA at 0.08, the power of the model was 0.82. The value of p < 0.05). (2) Those who have intention to take up HPV vaccine no matter what the price can predict vaccine uptake at the 6th-month follow-up . (3) HPV screening can predict vaccine uptake at the 6th-month follow-up . One path was marginally significant: from capacity to obtain HPV-related information to HPV knowledge . There were no significant differences between perceived severity of HPV disease, subjective norm, perceived benefits, and barriers to intention to receive the vaccine in the 6th-month follow-up  HPV knowledge can predict those who have intention to take up HPV vaccine no matter what the price , and self-reported HPV screening results or genital warts , 26, 42.The price of HPV vaccine affects an individual’s intention for vaccine uptake . CompareInterventions designed to improve HPV knowledge to increase vaccine uptake in MSM needs to be supported with policies to subsidize HPV vaccine in this population. The insufficient HPV knowledge may result from participants’ low capacity to obtain HPV-related information, a tendency we observed in our data that was only marginally significant. Only 46% of MSM in our sample felt free to discuss HPV vaccine with other people; even fewer (29%) felt free to talk to healthcare providers about sexual health, which may contribute to MSM’s inability to obtain HPV-related information. Because of the major discrimination against MSM  is determined by the different independent variables.Additional file 3. Standardized direct and indirect effects of knowledge, Those who have intention to take up HPV vaccine if the price is below NT$8000 vs. the referencea, Those who have intention to take up HPV vaccine no matter what the price is vs. the referencea and Vaccine uptake in the 6th month follow-up.


Astrocytes are a specific type of neuroglial cells that confer metabolic and structural support to neurons. Astrocytes populate all regions of the nervous system and adopt a variety of phenotypes depending on their location and their respective functions, which are also pleiotropic in nature. For example, astrocytes adapt to pathological conditions with a specific cellular response known as reactive astrogliosis, which includes extensive phenotypic and transcriptional changes. Reactive astrocytes may lose some of their homeostatic functions and gain protective or detrimental properties with great impact on damage propagation. Different astrocyte subpopulations seemingly coexist in reactive astrogliosis, however, the source of such heterogeneity is not completely understood. Altered cellular signaling in pathological compared to healthy conditions might be one source fueling astrocyte heterogeneity. Moreover, diversity might also be encoded cell-autonomously, for example as a result of astrocyte subtype specification during development. We hypothesize and propose here that elucidating the epigenetic signature underlying the phenotype of each astrocyte subtype is of high relevance to understand another regulative layer of astrocyte heterogeneity, in general as well as after injury or as a result of other pathological conditions. High resolution methods should allow enlightening diverse cell states and subtypes of astrocyte, their adaptation to pathological conditions and ultimately allow controlling and manipulating astrocyte functions in disease states. Here, we review novel literature reporting on astrocyte diversity from a developmental perspective and we focus on epigenetic signatures that might account for cell type specification. The principal components of the human brain and spinal cord are neurons and glial cells that form and interact through complex networks. The complexity of these networks relies on the specialized subtypes of neurons and glia, which arise during development as a consequence of highly regulated processes of cellular differentiation and maturation. Among glial cells, astrocytes have become a major focus of interest in the field of central nervous system (CNS) pathology because astrocytes are one key component in the cellular responses to injury ,2,3. AstOne of the main setbacks in understanding and controlling astrocyte response to injury lies in their cellular heterogeneity. Astrocyte heterogeneity after injury may arise, at least partially, in response to extracellular cues, which are specific to the nature and/or the location of the injury, and thus heterogeneity can be a consequence of exposure to different signaling molecules and/or gradients of the latter ,5,11. BaIn regard to development, astrocytes as well as neurons originate from neural progenitor cells (NPCs) and they develop unique and thus distinguishable features in response to extracellular cues. Such cues trigger intracellular signaling cascades which impinge on stable, epigenetic modifications on the chromatin. In general, epigenetic changes are crucial not only for cell lineage differentiation but also for cell type specification . InteresAstrocytes are a very heterogeneous population of cells. A first description discriminated protoplasmic astrocytes in the gray matter from fibrillary astrocytes in the white matter. Later on in humans, at least nine types of distinct astrocyte morphologies have been described ,19,20. A2+ waves. Further, the engrafted astrocytes extended processes forming end-feet that reached blood vessels [Interestingly, when human glial progenitor cells were engrafted into the mouse neonatal forebrain, spontaneous differentiation led to functional astrocytes, which enhanced synaptic plasticity and integrated into resident astrocyte networks through coupled gap junctions that propagated Ca vessels . These o2+ transient properties, and one subtype had neurogenic properties. Another study analyzed 33 brain regions of 4 different primate species including humans. One finding of interest for this review is that human astrocytes are even more heterogeneous when compared to nonhuman primates [The morphological distinction of astrocytes in combination with expression of specific marker genes allowed correlating structure and function ,24,25. Hprimates . This fiprimates .Chrdl1 (Chordin like 1). Astrocytes expressing Chrdl1 are located in neuronal layers 2–3 and 4; however, this pattern varies across regions suggesting astrocyte arealization across the cortex. Of particular note, astrocyte layers’ positioning occurs during brain development and lineage commitment, resembling the spatial distribution of neurons. As the spatial distribution of neurons is to some extent under the control of epigenetic mechanisms, it is tempting to speculate that these processes also impact astrocyte diversity and distribution [Novel technological approaches allowed construction of a Large-area Spatial Transcriptomic (LaST) map, a working pipeline for the quantification of single cell gene expression in situ . The autribution ,29,30.When experimentally addressing astrocyte response to injury through histological methods such as immunohistochemistry using astrocyte markers such as Glial Fibrillar Acidic Protein (GFAP), several features of the phenotype are shared between different insults, including hypertrophy and overlapping of astrocyte 3D domains . Using aWhen addressing astrocyte response to injury at a transcriptional or proteomic level, differences other than morphological variations are being uncovered. Zamanian et al. 2012) analyzed the transcriptome of astrocytes after brain ischemia or after systemic administration of the pro-inflammatory stimulus LPS (lipopolysacharide). Here, astrocytes were analyzed as one unique population of cells responding to injury. Both insults elicited the expression of pan-astrocyte markers; however, each injury promoted the expression of a specific set of genes. For example, LPS stimulation promoted the expression of genes involved in inflammation, and the authors suggested a detrimental role, while astrocytes in ischemia showed a protective profile of gene expression. Further works have linked the pro-inflammatory pathological phenotype to the expression and secretion of complement protein C3, which, once released, engages C3 receptors on neurons and microglia promoting toxicity and activation, respectively ,37,38,392 analyzeAnderson et al. (2016) analyzed the transcriptome of astrocytes that formed glial scars after spinal cord injury using a Ribo-tag technique for specific purification of mRNA undergoing translation in astrocytes. Here, the authors report that glial scar-forming astrocytes express multiple genes involved in supporting axonal growth . These works revealed astrocyte heterogeneous response  following different CNS insults . However, since astrocytes were analyzed in bulk and not at the single cell level, it was not possible to distinguish between astrocyte subpopulations. In this regard, more recent findings using single cell transcriptomic analysis of astrocytes in experimental autoimmune encephalomyelitis (EAE) suggested that different astrocyte subtypes  coexist in the same cellular response .The authors of the studies above suggested that acquisition of different reactive phenotypes of astrocytes depended to a great extent on the extracellular milieu and microenvironment. However, as we propose in this article, intrinsic astroglial properties, which might be acquired during CNS development and confer cell-autonomous heterogeneity, might also have to be considered in any attempt to understand and describe heterogeneity in reactive astrogliosis.Astrocyte diversity that originates as a result of development is likely to be a source of heterogeneity in reactive astrogliosis when combined with a microenvironment caused by a specific insult or pathology. We consider that it further becomes relevant to understand how astrocyte diversity is achieved during differentiation and maturation.Neural cells, namely neurons and glia, including both astrocytes and oligodendrocytes, arise from one multipotent progenitor population named neural progenitor cells (NPCs) ,40,41,42During CNS development, multipotent NPCs will first differentiate to neurons, directly or through intermediate neuronal progenitors . After eAs cells differentiate following intrinsic and extrinsic cues, their differentiating potential is gradually restricted becoming more committed to a specific cell population ,45,46,47Cell type specification and fate commitment gradually occur in a time-dependent manner, giving rise to a stable conversion which generally is not spontaneously reversed, even after the external cues that induced differentiation are no longer available. Such cell identity-conferring stability can be mediated through specific epigenetic mechanisms ,30,41. We will briefly comment on the major ligands and signaling pathways that impinge on astrocyte differentiation and maturation, and highlight some of these pathways that involve downstream epigenetic mechanisms. in vitro model of human induced pluripotent stem cell differentiation, PDGF (Platelet-Derived Growth Factor) also activated JAK-STAT signaling, which was followed by astrocyte differentiation [The signaling pathway associated prominently with astrocyte differentiation is the JAK (Janus Kinase)-STAT  pathway that is activated by cytokines, including LIF (Leukemia Inhibitor Factor), CNTF (Ciliary Neurotrophic Factor) and Cardiotrophin 1  after enntiation . Activatntiation . Interesntiation ,50,51. Apart from JAK/STAT-signaling, the NOTCH-activated pathway controls astrogenesis. NOTCH-1 is a transmembrane receptor that binds to DELTA-LIKE and JAGGED ligands. Ligand binding promotes two consecutive cleavages of NOTCH, which releases its intracellular domain (NICD) into the cytoplasm. Cleaved NICD translocates to the nucleus where it binds to different cofactors, including transcription factors . During Bone Morphogenic Protein (BMP), a member of the TGF-β (Transforming Growth Factor-β) cytokine family that activates SMAD transcription factors, is a key factor of development , and it Although the above mentioned signaling pathways affect astrogliogenesis individually, they can also act in concert. For example, a crosstalk between STAT3 and NOTCH pathways has been reported in which HES1 and 5, transcription factors upregulated by NOTCH signaling, interact with JAK2 and STAT3, and facilitate STAT3 phosphorylation and thus activation . In thisMost of the currently published articles that address astroglial differentiation refer to astrocytes as a unique population of GFAP-expressing cells, and only few studies analyzed the origins of astrocyte heterogeneity. A work published by Bonaguidi et al. (2005) suggests that BMP and LIF signaling generate different kind of astrocytes with different features including different morphologies and cell division properties. Here, exposure to LIF promoted the differentiation of bi-tripolar astrocytes with proliferative capacities, while BMP promoted a stellated morphology and reduced cell divisions . RecentlAlthough the activation of transcription factors is normally transient, their activity might have a long term impact on the regulation of gene expression programs. Therefore, understanding the downstream events controlling transcription factor networks beyond signaling pathways might be of importance. Therefore, in the next section we will comment on different epigenetic mechanisms that were linked to signaling pathways and transcriptional control involved in astroglial differentiation.The term “epigenetics” is used to describe stable alterations in gene expression which may be triggered cell-autonomously or in response to environmental cues . The seqPioneering experiments by Frantz and McConnell (1996) in ferrets have shown the importance of cell-intrinsic mechanisms in neural cell fate restriction. Here, cortical progenitors at E42 (Embryonic day 42) that were developmentally primed to generate upper layer neurons were transplanted into an E32 cortex, in which progenitors generate mostly deep-layer neurons. The authors observed that the transplanted progenitors from the later embryonic stage were restricted towards generating upper layer neurons. Even when transplanted “late” progenitors were exposed to a change in the extracellular environment and were confronted with upper layer instead of deeper layer cues, they were incompetent to differentiate into deep layer neurons. At that time, intrinsic, yet undefined molecular mechanisms were proposed to be in place and responsible for restricting developmental potentials of progenitors with advanced age ,69. HeteIn the following we will address how DNA methylation and histone modifications, i.e., acetylation and methylations impact on NPC differentiation towards astrocytes and how they might contribute to astrocyte heterogeneity. The role of other epigenetic mechanisms, i.e., noncoding RNAs such as microRNAs, that play a pivotal role in astrocyte differentiation, was recently reviewed elsewhere . A summaDNA methylation is the conversion of a cytosine to 5-methylcytosin (5mC) by covalent transfer of a methyl group to the C-5 position in the pyrimidine ring ,72. MethGfap promoter and activated Gfap expression. However, accessibility of STAT3 and binding to the Gfap promoter were abolished if the respective genomic regions contained high levels of DNA methylation [S100b gene promoter and allowed activation of transcription [In the context of astrocyte differentiation, it has been recognized that following STAT3 activation, this transcription factor bound the hylation . S100B ihylation ,80. Unlihylation . The gencription .Dnmt1 is crucial for correct timing of astrogliogenesis. Its deletion in NPCs resulted in DNA hypomethylation and accelerated JAK-STAT pathway activation, which was accompanied by precocious astrogliogenesis of cortical NPCs [Dnmt1 deletion in NPCs also promoted astrogliogenesis of hippocampal dentate gyrus granule progenitors [cal NPCs . Similargenitors .Gfap, favors differentiation into astrocytes [Tet2 expression, indirectly suppressed astroglial lineage differentiation.Thus, generally, DNA methylation in NPCs is essential to preserve neuronal lineage differentiation by keeping astrocytic genes in a permissive silenced state. Further support for this view comes from a recent report in which the authors demonstrated using NPCs that DNA demethylation, conferred by TET2, of astrocyte lineage genes, including trocytes . InteresTogether with DNA methylation, different histone modifications have been reported to impact astrocyte differentiation. The transcriptional outcome of histone modifications varies and depends on the different types of post-translational modifications that include, for example, acetylation, methylation, phosphorylation, and ubiquitination, and locations of modifications within different histones and residues within the respective protein. We will focus mainly on histone acetylation and methylation, which are the most studied modifications as of yet that impact astrocyte differentiation.Histone acetylation, independently to which lysine residue it is located within a histone protein, leads to an open state of the chromatin and thus actives transcription . The enzS100b gene expression as mentioned above. Here, the promoter of the S100b encoding gene was demethylated upon astrocyte differentiation and it gained simultaneously acetylated H3 [Gfap encoding gene gained also acetylated H3 after exposing NPCs to retinoic acid, which promoted astrocyte differentiation [Gfap promoter and the presence of HATs changed the chromatin configuration to the open state that allowed STAT3 binding and transcriptional activation.Of importance for astrocyte differentiation is the study of lated H3 . Similarntiation . RetinoiGfap promoter. These modifications facilitated transcriptional activity, which was shown using the NTera-2 cell line that differentiated into an astrocyte-like lineage [STAT3 interacts directly with HATs as it is recruited by cAMP response element-binding protein (CREB) binding protein (CBP) and the transcriptional coactivator EP300. CBP/EP300 have acetyltransferase (HAT) activity ,93 and t lineage .Gfap gene product in human astrocytes, showing that even after differentiation, HDAC activity regulates Gfap expression [The ablation of HDAC3 in glial progenitor cells promoted astrogenic differentiation with concomitant loss of oligodendrocytes . This fipression . It is, TGF-β signaling has been linked to astrocyte differentiation through the activation of SMAD proteins ,59,97. HRegarding histone methylations and transcriptional outcome the picture is much more complicated, as several marks are associated with active transcription such as methylations of histone 3 at lysines 4 or 36 , while methylations at other residues in H3 confer gene silencing, for example H3K27me and H3K9me. Further, lysines can be subjected to mono-, di- or trimethylation  with different outcomes in regard to transcription .Gfap promoter [NTera-2 cells that differentiated into an astrocyte-like lineage, had not only higher levels of acetylated H3 through STAT3-recruited HATs but also increased levels of the activating mark H3K4me3 at the promoter .Ngn1) occurs in the late stages of cortical development, when progenitor activate their astroglial potential, whereby neurogenesis is repressed and astrogenesis promoted [Polycomb group (PcG) proteins modulate the chromatin structure and generally repress transcription . PcG propromoted ,104.Ezh2 in cortical progenitors in early development during their neurogenic phase using an Emx1-Cre mouse driver line, accelerated gliogenesis and glial differentiation towards GFAP-positive astrocytes [However, the actions of PcG-mediated control of lineage determination of NPCs might be highly context-dependent, especially in a temporal dimension, as the conditional depletion of trocytes .Gfap promoter, and reduced neurogenesis by enhancing astrocyte formation [H3K9me is a repressive mark known to promote heterochromatinization by recruitment of heterochromatin protein 1 (HP1) and it forms one barrier for cellular reprogramming ,107. Hisormation , and thuBdnf (Brain Derived Neurotrophic Factor) gene at the same time that they removed the activating mark H3K36me3 from the Gfap gene body in NPCs. Together, this epigenetic remodeling promoted neuronal differentiation. Further, KDM4A/C depletion facilitated astrocyte differentiation at the expense of neuronal differentiation [Histone demethylases KDM4A (JMJD2A) and KDM4C (JMJD2C) removed H3K9me from the promoter of the neurotrophic factor ntiation . Here, ontiation .Some of these findings might seem contradictory since the ablation of methyltransferase ESET and demethylase KDM4A for H3K9me led both to an increase in astrocyte differentiation. However, these studies did not address global changes in astrocyte transcriptome after enzyme ablation. The expression of other genes involved in the neurogenic/gliogenic switch might be affected by these genetic manipulations and are being neglected. To analyze a broader gene network, new studies will benefit from mRNA sequencing and concomitant profiling of the epigenetic landscape.Gfap promoter. KDM5A knockdown in NPCs promoted H3K4me3 at the Gfap promoter and concomitant astroglial differentiation, which suggested that KDM5A demethylase activity repressed astrogenesis [Histone demethylase KDM5A removes the activating mark H3K4me3 from the ogenesis .Histone ubiquitination also plays a role in astrocyte differentiation. Ring finger protein 20 (RNF20), also known as an E3 ligase, catalyzes the monoubiquitination of histone 2B at lysin 120 (H2BK120ub). RNF20 in cooperation with MOF  acetyltransferase, which acetylates H4K16, promoted transcriptional activity of STAT3 favoring astrocyte generation .In some cases, the deposition of a histone modification might alter the deposition of a second one, a phenomenon known as histone-to-histone crosstalk . InteresA recent work conducted by Tiwari et al. (2018) used a combinatorial approach to analyze transcriptional regulation of differentiating astrocytes at a genome wide level. The authors implemented mRNA-seq and ChIP-seq at different differentiation stages of the astroglial lineage. This allowed the study of different epigenetic marks such as H3K27ac and H3K4me1, found at active or activatable enhancers, and the correlation of the marks with the expression of downstream genes . In thisWhen all of the described aspects are taken together, it becomes clear that the switch from neurogenesis to astrogenesis depends on epigenetic mechanisms occurring at the chromatin level, that regulate gene expression programs involved in astrocyte fate commitment. However, much less is known when it comes to our current understanding, which mechanisms regulate lineage subtype specification in relation to astroglial function. It might be possible that astrocyte subpopulations are under the control of a combination of different epigenetic mechanisms triggered by specific location/environment and activity during maturation .A very interesting and recently discovered source of astrocyte diversity has its basis in parental inheritance. In a way which is not fully known, specific alleles in astrocytes are decorated with epigenetic marks conferring a parental-specific gene expression. This imprinted expression pattern may lead to a diversity of astrocytes across brain regions with genes carrying either maternal or paternal imprinting and differential gene activity . Some ofPrevious works have addressed the role of specific epigenetic mechanisms mainly during the neurogenesis/gliogenesis switch by deleting or inhibiting enzymes and addressing the relevance of certain epigenetic marks at the promoter of genes involved in astrocyte differentiation. Although new studies were able to analyze astrocyte subpopulations by addressing transcriptomic programs ,14, to dSingle cell RNA-seq and analysis allows clustering of isolated astrocytes and potentially correlating each cluster to a specific function, based on the transcriptional signature. Further, it is possible to analyze after single cell ATAC (Assay for Transposase-Accessible Chromatin)-seq the chromatin landscape in diverse astrocyte clusters to determine open and closed regions, especially enhancers that are used to establish or maintain cellular and functional heterogeneity ,119. ThiUsing single cell analysis followed by ChIP-seq with antibodies against different epigenetic marks, could allow the assignment of a histone code to the astrocyte clusters mentioned above. Single cell ChIP-seq has been used either when starting from cultured cells  or from in utero electroporation could represent a promising approach for conducting gain/loss of specific epigenetic marks in astrocytes.In the above mentioned approaches astrocytes are isolated from their microenvironments. Although the analysis of gene expression may be highly indicative of the function of each cluster, astrocyte origin is lost. It is possible then to obtain samples (astrocytes) directly from the tissue by conducting spatial transcriptomics that allow, through a combination of different approaches, regional positioning of cell clusters obtained from the single cell RNA-seq . Fan et Analysis of astrocyte epigenetic diversity can also be carried out from human biopsies or necropsies. However, in such cases the homeostatic astrocyte phenotype has to be addressed since aging and clinical conditions of the human donors might change the epigenetic signature of astrocytes. A promising approach involves the use of human induced pluripotent stem cells and generation of organoids. However, in such case it is still difficult to recreate the conditions and architecture of a healthy human brain, as the epigenome might be altered as well during extended cultivation periods and differentiation paradigms ,130. FurAstrocyte basal diversity contributes to the heterogeneity of reactive astrogliosis, which is still a major focus of interest in the field of neurodegeneration. Different populations of astrocytes display different transcriptional programs, which are probably epigenetically regulated and, although several epigenetic mechanisms have been described as intrinsic regulators of astrocyte differentiation, the epigenetic signatures of astrocyte subtype specification still remain unknown.Basal epigenetic diversity can be acquired during embryonic development when astrocytes differentiate and functionally maturate in a “healthy” microenvironment. Knowing the epigenetic signature of each astrocyte subtype might be relevant to fully restore homeostatic capacities of surviving astrocytes in an injured brain or spinal cord.


The ability to obtain tumor material from cells in the blood of cancer patients provides a significant benefit over the use of tumor tissue as a diagnostic to make treatment decisions. However, the traditionally defined circulating tumor cell (CTC) has been shown to be useful only in some cases. A recently identified type of circulating stromal cell, which appears to be more frequent than CTCs, was found engulfing tumor material at the tumor site and then entering the blood stream. These cells were defined as cancer-associated macrophage-like cells (CAMLs). Together, CTCs and CAMLs may be able to provide information for cancer detection and diagnosis, without the use of tissue. CTCs and CAMLs have many clinical applications, three of which are summarized in this review: for prognosis, as companion diagnostics, and for residual disease monitoring. Cancer encompasses numerous subtypes and is the second leading cause of death in the United States. Worldwide, cancer deaths in 2020 were about 10 million ,4,5. HowPathologically defined circulating tumor cells (CTCs) are actual tumor cells that have entered the blood stream ,7,8,9. WIn addition to CTCs, a circulating cell population that is a specific type of phagocytic cell has been identified in the tumor stroma. This particular type of stromal cell is a macrophage that engulfs tumor cells and debris within the tumor microenvironment and then enters the blood. These cells were named Cancer-Associated Macrophage-Like Cells (CAMLs), as they are CD14-positive and phagocytose tumor material ,28,29,30To date, CAMLs have been found in patients with 20 types of solid tumors, i.e., breast, esophageal, lung , prostate, pancreas (adenocarcinoma and acinar carcinoma), renal cell carcinoma (adeno and sarcomatoid), hepatocellular carcinoma, head and neck (numerous types), neuroblastoma (numerous types), sarcoma (numerous types), melanoma, neuroendocrine tumors, PNET, cholangiocarcinoma, ovarian, colon, uterine, urothelial, bladder, and endometrial tumors. Very likely CAMLs can be found in all solid tumors. CAMLs have also been found in cancers at all stages of development. However, CAMLs are not found in healthy individuals.This review describes the type of information CTCs and CAMLs can provide to support prognosis, companion diagnostics, and monitoring of residual disease by a blood test.TM microfilter is a size-exclusion device with 7.5 µm-diameter pores, 180,000 pores distributed uniformly within a 9 mm-diameter area on a strong, low-autofluorescence, 10 µm-thick polymer. Images of the microfilter are shown in Reference [Size-exclusion methods are ideal for isolating CTCs and CAMLs from the blood stream. The CellSieveFiltration by size is a suitable method to consistently capture multiple types of tumor-associated cells in the blood, both CTCs and CAMLs. Filtration can be performed under low pressure using a syringe pump or a vacuum pump .For cell isolation, 7.5 mL of whole blood, collected in a CellSave Preservative Tube, is prefixed in 7.5 mL of prefixation buffer. The 15 mL sample is passed through the filter for 3 min. The microfilter removes all red blood cells and 99.9% of white blood cells ,11,12. TMost invasive carcinomas are known to express cytokeratin filaments (CK) 8, 18, 19, while not expressing the white blood cell marker CD45. Typically, pathologically defined CTCs (PDCTCs) exhibit filamentous cytokeratin patterns. as shown in CAMLs, shown in The low auto-fluorescence background of the filter material enables the determination of medium and low expression levels of the marker of interest and provides the ability to accurately measure CAML size.Companion diagnostic assay development usually starts using cell lines that provide negative, low, medium, and high expression levels of the markers of interest and are screened against multiple antibodies for the marker of interest. Marker intensity on CAMLs and CTCs are validated using patient samples, and adjustment of reagents and expression levels is required to finalize the scaling criteria.p < 0.001), and significantly shorter overall survival (OS), with Hazard ratio of 3.6 , compared to patients with CAMLs <50 µm. Both patients and oncologists are very interested in the prognosis of a patient’s disease. CAMLs can potentially provide broader prognostic information than CTCs. The number of CAMLs in a sample provides prognostic information, but the unusual property of CAML size provides even greater prognostic accuracy. In a presentation at the 2017 Annual ASCO meeting, 293 patients with six types of cancers—breast, prostate, pancreas, esophageal, lung, and kidney—were evaluated for 24 months . The datp < 0.001) and significantly worse OS .For patients with pancreatic cancer, similar findings of PFS and OS were also shown in a paper published in 2021 . The papp < 0.0001), (b) for lung cancer, n = 31 patients had CAMLs < 50 µm, and n = 28 patients had CAMLs ≥ 50 µm , and (c) for prostate cancer, n = 47 patients had CAMLs < 50 µm, and n = 27 patients had CAMLs ≥ 50 µm .In this review, we present data from 192 patients according to cancer, stage, and CAML size, including three different cancers: breast (n = 59), lung (n = 59), and prostate (n = 74), expending the number of patients in those three cancers from the 2017 ASCO presenation. This group comprised both treated and untreated patients. p = 0.0077), (b) lung cancer , and (c) prostate cancer . The data support that CAMLs are suitable for clinical applications involving patients with breast, lung, and prostate cancers.p = 0.0172), (b) Stage 2, with (n = 36) patients with CAMLs < 50 µm, and (n = 14) with CAMLs ≥50 µm , (c) Stage 3, with (n = 18) patients with CAMLs < 50 µm, and (n = 29) with CAMLs ≥ 50 µm , and (d) Stage 4, with (n = 17) patients with CAMLs < 50 µm, and (n = 43) with CAMLs ≥ 50 µm .p = 0.9298), (b) Stage 2 , (c) Stage 3 , and (d) Stage 4 .CTCs are found primarily in patients with late-stage breast, prostate, colorectal cancers, and with small cell lung cancer. If one CTC is found in 7.5 mL of peripheral blood, then the prognosis is poor. An even worse prognosis is associated with the presence of one or more CTCs in mitosis .p = 0.00011. For OS, HR = 3.1 CI95% 1.6–5.9, p = 0.00124. As the p values indicate, the presence of a single CTC indicated poor prognosis. p < 0.00001. For OS, HR = 4.9 CI95% 2.9–8.2, p < 0.00001. As the p values indicate, CAML size is more informative than the CTC number.The same 192 patients were analyzed for the presence of CTCs in relation to PFS and OS, combining all stages, as shown in Many therapies targeting markers on a tumor have been FDA-approved, and an even broader spectrum of promising therapies targeting different tumor markers are under development and in clinical trials. To determine the applicability of a particular therapy for a patient, the patient’s tumor must express the drug target at a meaningful level. A companion diagnostic is a clinical diagnostic test that matches a patient’s tumor drug target to a specific drug or therapy and is usually performed prior to intervention with the therapy.The majority of companion diagnostics for the treatment of solid tumors rely on tumor tissue obtained by biopsy ,3,4,5,6.Recently, a number of blood-based companion diagnostic tests and complimentary diagnostic tests have been approved by the FDA, such as the FoundationOne Liquid CDx and Guardant 360 CDx tests. However, these diagnostics are only applicable for highly specific genetic changes found in cell-free DNA in patient blood ,5. TheirCell-based liquid biopsy can deliver companion diagnostics for therapy markers as well as mutations of the cancer. The assay can deliver tumor DNA in the CTCs and CAMLs for sequencing for genetic information, though sequencing will not be covered in this review paper. The method for providing companion diagnostics for drug targets on the surface of tumor cells is described below.Typically, three fluorescent channels are adequate to identify CTCs and CAMLs. There is at least one additional fluorescent channel available for use to measure a drug marker.We have developed assays for more than 20 drug targets on CTCs and CAMLs, such as PD-L1, PD-L2, CCR5, HLA-DRB3, CXCR4, HER2, p-ERK, PRAME, HHLA2, FGFR1, FGFR2, FGFR3, and others. Clinical validations backed by a large number of patients with a variety of cancers are important and are on-going.For carcinomas, cytokeratins are used to identify CTCs. For sarcomas, vimentin is used to identify CTCs, as sarcomas are not epithelial in origin and thus do not express cytokeratins .We describe two liquid biopsy companion diagnostic examples, PD-L1 and CCR5, which illustrate how the expression of these markers and their variation provides useful clinical information.PD-L1, expressed on the surface of tumor cells, suppresses immune recognition by binding to its receptor PD-1, found on activated T cells, B cells, and myeloid cells. Blocking the PD-L1–PD-1 axis with a number of FDA-approved cancer immunotherapies activates the immune system to unleash CD8 T cells to kill the tumor. High PD-L1 expression on tumor cells is desirable for immunotherapy ,33,34,35® (nivolumab) of BMS, Keytruda® (pembrolizumab) of Merck, Tecentriq® (atezolizumab) of Genentech, Imfinzi  of AstraZeneca, and Libtayo® (cemiplimab-rwlc) of Regeneron and Senofi-Aventis. All three FDA-approved companion diagnostics for those immunotherapies are based on tissue: Dako® PD-L1 IHC 22C3 pharmDx, Dako® PD-L1 IHC 28-8 pharmDx and Ventana® PD-L1 SP142 [Currently, five immunotherapy drugs have received multiple FDA approvals for a wide spectrum of cancer therapies: OpdivoL1 SP142 .While PD-L1 is detected on the surface of tissue and CTCs, it is also found inside CAMLs, because CAMLs engulf tumor cells and tumor debris. PD-L1 is in CAML cytoplasm. Chemoradiation therapy can change PD-L1 expression, which can enhance the response of some patients who receive subsequent immunotherapy. For those patients, the best time to perform PD-L1 companion diagnostics for immunotherapy is after chemoradiation therapy .The PD-L1 assay has been extensively validated for many types of cancers in more than 2000 patient samples, especially for non-small cell lung cancer (NSCLC) and breast cancer. The data include patients treated with four different immunotherapy drugs , as well as patients treated with immunotherapy–drug combinations.CCR5, also known as C–C chemokine receptor type 5, is predominantly expressed on T cells, macrophages, dendritic cells, eosinophils, microglia, and cancer cells. Approximately 50% of human breast cancers express CCR5. CCR5 has been found to be upregulated in aggressive breast cancer, especially in triple-negative breast cancer. CCR5 can also be expressed on a variety of other solid tumors.CCR5 usually appears in pools or as small dots on the surface of tumor cells, when CCR5 becomes activated by chemokine C–C Ligand 5 (CCL5)  . ActivatCCR5 appears in pools on the surface of CTCs, as shown in The determination of CCR5 expression in CTCs and CAMLs is very different from that of PD-L1 expression. For CCR5, clinical data show that the aggressiveness of a cancer is related to the number of CCR5 dots. A manuscript has been submitted for publication on this phenomenon . CurrentSince CAMLs are easy to identify by their shape and features, it is possible to eliminate CD45 in the assay to allow two or more targeted markers in the same assay. It is also possible to first stain for cytokeratin and C45 to identify the cells and then quench the cytokeratin and CD45 after imaging, so that the cells can be re-stained for three different drug targets. The quenching technique can be repeated to analyze 12–15 markers on the same cells . With adAs observed in tissue biopsies, the expression of the marker of interest is variable among the cells. Similarly, for CAMLs, a marker expression is not uniform in all the CAMLs in a blood sample. The scoring also varies across different tumor types. The establishment of a scoring indicating high, medium, and low expression of the marker needs to be validated by analyzing a sufficient number of patient samples for each type of cancer, with known patient clinical information.Companion diagnostics based on cells captured on the microfilter can be performed at desirable time points and can be easily repeated. This flexibility is useful, because some treatments can affect the expression of markers over time.Treatment is necessary to improve the survival of cancer patients. However, oncologists have few options to determine if a tumor has been eradicated at the end of standard therapy. For example, a surgeon may not know if surgery has removed all of the malignant tissue. Chemotherapy is frequently administered after surgery to reduce the chance of residual disease. The existence of residual disease is commonly regarded as an indicator of poor prognosis in all cancers. Initial studies on tracking CAMLs have found that the number and size of CAMLs at the end of treatment confirm the existence of residual disease, as well as its aggressiveness, as described earlier.Precision Oncology in 2021 [An example is given in  in 2021 . Though  in 2021 .In summary, prognosis, companion diagnostics, and residual disease determination by blood test would be beneficial to patients and oncologists. CAMLs in combination with CTCs as a liquid biopsy provide a source of tumor material from the patient. CTCs are found in patients with SCLC and late-stage breast, prostate, and colorectal cancers, while CAMLs are consistently found in patients with all 20 types of solid tumors analyzed and in all stages of disease progression; therefore, CAMLs are able to provide reliable tumor material. In addition, CAML size provides very useful information in addition to CAML number.We presented data from many studies of PFS and OS of patients followed up for 24 months (1) combining patients with breast, prostate, pancreas, esophageal, lung, and kidney cancers, (2) breast, prostate, and lung cancers analyzed as individual cancers, (3) analyzing the use of CTC number versus CAML size, and (4) analyzing the effect of cancer stage. In summary, the common conclusion is that the presence of one CAML ≥ 50 µm in 7.5 mL of whole blood is a predictor of poor prognosis.Many cancer drugs are under development to target specific markers expressed by tumor cells on their surface. The response of the patient to the therapy depends on whether the patient’s tumor expresses those markers. CTCs can be used to develop companion diagnostics, but the problem of solely depending on CTCs is that they are not always present in 7.5 mL of whole blood. This paper showed that CAMLs can be used to determine whether a patient will benefit from a given drug. The drug target can behave very differently. PD-L1 marker staining is diffuse inside CAMLs and can be helpful to determine the response to immunotherapy. The intensity of PD-L1 fluorescent staining in CAMLs provides information about PD-L1 expression. The CCR5 marker, however, appears as dots inside or on the surface of the CAMLs. The level of CCR5 expression is based on the number of CCR5 dots. Companion or complementary diagnostics can be developed by a blood test utilizing markers in CAMLs.At the end of therapy, the patient and the oncologist need to know if the patient still has cancer as well as the aggressiveness of the disease. Again, CAMLs can provide this information through a comparison of CAML number and size before and at the termination of therapy. An example is given in this review, showing that the decrease of CAML number at the end of therapy correlated with longer PFS.Cell-based liquid biopsies have broad capabilities beyond those described in this paper based on our clinical data; they can: (a) predict patients’ response to a new therapy after the initial treatment, (b) monitor the response to therapy over time, (c) detect cancer recurrence in patients in remission, (d) allow cancer screening for a single cancer or multiple cancers, and (e) deliver whole-tumor DNA for sequencing.Liquid biopsies combining CTCs and CAMLs can also provide approaches for basic research on cancer and cancer biomarkers not requiring tissue and for drug development.


Head and Neck cancer (HNC) is a fatal malignancy with poor prognosis. Human Papillomavirus (HPV) infection is becoming the prominent cause of HNC in the western world, and studying the molecular mechanisms underlying its action in cancers is key towards targeted therapy. To replicate, HPV regulates the host DNA damage repair (DDR) pathway. SMAD4 is also involved in the regulation of the DDR machinery and likely plays important role in maintaining cell viability upon genotoxic stress. In this study, we investigated the role of HPV in the upregulation of SMAD4 to control the DDR response and facilitate its lifecycle.SMAD4, Rad51 and CHK1 expression was assessed in HPV-positive and HPV-negative HNC using TCGA data, a panel of 14 HNC cell lines and 8 fresh tumour tissue samples from HNC patients. HPV16 expression was modulated by E6/E7 siRNA knock-down or transduction in HPV-positive HNC cell lines and Human Primary keratinocytes respectively. SMAD4 half-life was assessed by cycloheximide treatment in HNC cell lines, together with βTRCP1-dependent SMAD4 ubiquitination. SMAD4 siRNA knock-down was used to determine its role in HPV-mediated regulation of DDR machinery and to assess cisplatin sensitivity in HPV-positive HNC cell lines.We found that HPV increases SMAD4 expression is both HPV-positive HNC tumours and cell lines, impairing its degradation which is mediated by the E3 ubiquitin ligase βTRCP1. SMAD4 expression highly correlates with the expression of two main players of the DDR pathway, CHK1 and Rad51, which expression is also upregulated by the presence of HPV. In particular, we demonstrate that HPV stabilizes SMAD4 to increase CHK1 and Rad51 expression. In addition, SMAD4-deficient HPV-positive cells have increased sensitivity to cisplatin treatment.Our results give a clear molecular mechanism at the basis of HPV regulation of the DDR pathway. In particular, we show how HPV stabilizes SMAD4 to promote DDR protein expression, which may be used to facilitate viral replication and HNC onset. Moreover, we found that SMAD4 silencing in HPV-positive HNC cell lines increases sensitivity to cisplatin treatment, suggesting that HPV-positive HNC with low SMAD4 expression may be preferentially susceptible to similar treatments.The online version contains supplementary material available at 10.1186/s13046-022-02258-9. SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 (Deleted in Pancreatic Cancer-4) is a central mediator of TGF-β signalling by forming complexes with receptor-activated SMADs (SMAD-2 and -3). The SMAD complexes then translocate to the nucleus and regulate the expression of genes involved in many cancer-related processes such as proliferation, apoptosis and inflammation .Reduced SMAD4 expression is common in tumours and occurs through a combination of mutations, copy loss and transcriptional downregulation , 3. ReduThe loss of SMAD4 plays a crucial role in the response to DNA damage leading to increased genomic instability. This is very prominent in squamous cell carcinoma of the skin and of the Head and Neck cancer (HNSCC or HNC) and suggests a distinct role of SMAD4 in the progression of various types of tumours , 10.HNC is the sixth most common cancer worldwide  with a 5The tumours are different at the molecular level impacting on the clinical outcome: in general, HPV-related HNC has a more favourable prognosis . Loss ofThe productive life cycle of high-risk HPVs is dependent on epithelial differentiation and on activation of host DNA repair pathways that also play critical roles in the development of many cancers. Many oncogenic viruses express proteins that affect the cell cycle and DNA damage repair regulatory pathways. Several studies have shown that during genome amplification, HPV proteins interact with different components of the cellular DNA repair machinery to activate or downregulate the expression or activity of factors of the ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) pathways. Importantly, many proteins from ATM and ATR pathways colocalize with HPV replication sites, further supporting the role of these factors in the HPV life cycle , 16.Here, we report that HPV-positive HNC cell lines and tumours show increased expression of SMAD4 compared to the HPV-negative ones. This increased expression seems to be due to deficiency in SMAD4 degradation in HPV-positive cell lines, due to impaired ubiquitination by the βTRCP1 E3 ligase. Moreover, we demonstrate how HPV-mediated activation of the DNA damage regulatory pathway is dependent on SMAD4 expression and affects DNA-damaging drug sensitivity.HNC cell lines, acquired from different sources , were acFresh tumours and adjacent normal tissue samples from HNC patients were separately collected upon surgery under IEO Biobank approval and preserved at -80C degrees. Samples were then snap-frozen in liquid nitrogen and processed for RNA and protein extraction using AllPrep DNA/RNA/protein kit (Quiagen) following manufacturers’ instruction. Samples were collected from 5 HPV-negative and 3-HPV-positive HNC patients, were all derived from primary tumours except for one relapse, and from male patients except for one female patient. Samples were collected mainly from the oral cavity and the tongue, except for one that was collected from the pharynx.All encoding exons and flanking intronic sequences of SMAD4 were analysed by Sanger sequencing. Genomic DNAs (40 ng per reaction) from each cell line  were amplified with primers designed for amplification of all encoding exons and around 50 base pairs of flanking intronic sequences of SMAD4. Amplified genomic DNA fragments were directly sequenced utilizing the same forward or reverse primers used in the original PCR amplification. All sequencing was performed on the Applied Biosystems TM 3500 Dx Genetic Analyzer capillary automated sequencers at the Cogenthech facility (Milan).pLXSN HPV16 E6/E7, pLXSN HPV11 E6/E7 and empty vector were previously described , pSuper 3VO4 and protease inhibitors (Sigma). After lysis, an equal amount of protein for each sample was resuspended in denaturing sample loading buffer, separated on SDS-PAGE gel and immunoblotted with the indicated antibodies. The following antibodies were used: SMAD4, CHK1, p-CHK1 and βTRCP1 , p63, pH2Ax, Flag and GAPDH (Abcam), HPV16 E7, p53, H3 and Rad51 (Santa Cruz Biotechnology), and Vinculin (Merck Millipore). Membranes were then incubated with the appropriate horseradish peroxidase (HRP) secondary antibodies and the signal was acquired with Chemidoc (Bio-Rad). Densitometric analysis of the intensity of the protein bands were preformed using ImageJ software , and standardized to the housekeeper levels.Cells were lysed in either a sodium dodecyl sulfate (SDS) lysis buffer: a 1:3 mixture of buffer I  and buffer II  or Urea 8 M, containing 0.5 mM N-ethylmaleimide (NEM), 0.5 mM NaF and 2 mM sodium NaRSEM normalized expression data was extracted from the TCGA PanCancer Atlas on the cBioPortal , 25. PatRNA was extracted from cells with the Quick-RNA MiniPrep kit (Zymo research). cDNA was generated by reverse transcription-PCR with Reverse Transcriptase (Promega). Relative levels of specific mRNAs were determined with the Fast SYBR Green detection chemistry system (Applied Biosystem). All PCR reactions were performed with a QuantStudio 6 Fast Real-Time PCR system (Thermo Fisher Scientific). Ribosomal Phosphoprotein (RpP0) was used as a house-keeper gene for normalization.3VO4) for co-immunoprecipitation and in SDS buffer to detect SMAD4 ubiquitination. Immunoprecipitation was performed in E1A buffer  using protein extracts incubated with the indicated antibodies o/n at 4 °C. SDS Samples were loaded on SDS-PAGE gel and immunoblotted with the indicated antibodies. Total extracts (input) were loaded as control.HNC cell lines were lysed in E1A lysis buffer  and 24 h later were treated with 10uM Cisplatin for 24 h. Cell viability was then assessed using CellTiter-Glo Luminescent Cell Viability Assay and luminescence was measured using a Glomax Discover plates reader (Promega).Statistical differences were evaluated using Tukey’s multicomparison analysis after one-way ANOVA analysis to compare multiple samples or unpaired t test to compare only two samples using GraphPad Prism v9.0 .Previous analysis of TGCA data , which wTP63 locus, leading to the expression of a truncated 25-kDa protein at the carboxyl terminus of p63 [Thus, we hypothesised a direct regulation of SMAD4 by HPV16 E6/E7 oncoproteins. By modulation of E6/E7 expression upon knock-down in HNC cell lines or HPV16 E6/E7 retroviral transduction in primary human keratinocytes (HKs), we demonstrated a direct effect of high risk HPV16, but not low risk HPV11 on the expression of SMAD4 protein level Fig. . Interess of p63 . ΔNp63α,s of p63 . As shows of p63 . Since ps of p63 , we hypoWe then assessed the stability of SMAD4 protein by cycloheximide (CHX) treatment. As shown in Fig. We then analysed βTRCP1 expression in HNC cell lines and human tumours. As Fig. HPV is known to modulate the DDR response to promote its life-cycle . SMAD4 m, upon modulating E6/E7 expression either through knock-down in HNC cell lines (Fig. Moreovernes Fig. D and E ones Fig. F and G, nes Fig. , SH. Intnes Fig. E and G, nes Fig. H. Interenes Fig. I. All thSMAD4 was shown to regulate DDR genes  and lackListeria monocytogenes impairs the SUMO pathway, thus decreasing the stability of SMAD4 [TGFβ is well-known for its anti-proliferative activity and malignant progression is typically associated with the loss of responsiveness to TGFβ-induced cell growth inhibition. The cell-cycle machinery of proliferating cells is necessary for HPV replication. TGFβ profoundly suppresses epithelial cell proliferation. Studies in cervical cancer show that HPV-encoded proteins bind to TGFβ receptors and signal transducers, resulting in their degradation and promoting the proliferation of infected cells . Interesof SMAD4 , which iof SMAD4 . Here weof SMAD4 . Here weof SMAD4 , 41 and of SMAD4 . CHK1 isof SMAD4 , leadingSMAD4 expression is deeply linked to the expression of genes involved in the DNA damage response  and reguOur data provide a rationale for the establishment of a new signature associated to SMAD4 expression in HNC, and in particular in HPV-positive HNC. Detection of SMAD4 levels can be used as a predictive marker to select patients that would benefit from new targeted therapies, based on DNA repair checkpoint inhibitors alone or in combination with DNA-damaging agent, to increase therapy efficacy, de-escalate cisplatin treatment and reduce the incidence of treatment resistant tumours.Additional file 1.