E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.Human papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes ( These circular double-stranded DNA viruses have a genome size close to 8 kilobases that includes open reading frames (ORF) encoding E1–E7 proteins involved in the transactivation of transcription, transformation, replication and viral adaptation into the host cells3. HPVs are involved in the development of cutaneous or genital warts (or condyloma) and of benign or malignant tumors4. Currently, more than 200 HPV types (https://pave.niaid.nih.gov/) are described and categorized in two groups, namely low-risk (LR) and high-risk (HR) HPVs according to their capacity to promote cell transformation that leads to cancer development. According to the Center for Disease Control and prevention, more than 90% of anal and cervical cancers and 50% of vaginal, vulvar, and penile cancers are due to HPVs5. HPV16 and 18 are frequently associated with anal cancer (87%), cervical, vulvar or penile cancer (70%), vaginal cancer (64%), but also head and neck cancer (85%)6.Human papillomaviruses (HPVs) are non-enveloped icosahedral particles with a size of 50–60 nm diameter8. Indeed, a productive viral replication is associated with a low-grade intraepithelial lesion9. The progression to precancerous and cancerous lesion up to cervical intraepithelial neoplasia and eventually to cervical cancer occurs with a persistent HR-HPV infection and the integration of the HPV genome into the host chromosome mediated by host DNA replication and recombination10, the loss or disruption of E2 protein and the subsequent upregulation of E6 and E7 oncogenes expressions11. These oncoproteins are involved in malignant transformation by altering cell cycle regulation and telomere maintenance via the telomerase activation and the degradation of the tumor suppressors p53 by E6 and retinoblastoma protein (Rb) by E714. In contrast, LR-HPV oncogenes are less able to interfere with p53 and Rb functions than E6/E7 proteins from HR-types15.HPVs infect basal epithelial cells of the skin (cutaneotropic) and of the mucosa of the anogenital and the oropharyngeal tracts (mucosotropic). Cancerous lesions are due to persistence of HPVs that are not cleared in the epithelium by the human immune system17 but detection of HPV DNA is becoming the first line strategy for cervical cancer in women older than 30 years20. Commercially available molecular tests are mainly based on the detection of HPV DNA such as Hybrid Capture 2 assay (QIAGEN)21, Cervista HPV HR22 or HPV 16/1823 (HOLOGIC) Care HPV (QIAGEN)24 and Cobas HPV (ROCHE DIAGNOSTICS)25, are mainly based on the detection of HPV DNA. These allow detecting the presence of the virus but not evaluating its oncogenic activity. In cervical infections HPV load and its variations have been shown to predict the persistence and progression of HPV infections and the severity of the lesions27. In 2010, a study showed that mRNA expression levels of E6 and E7 oncogenes were correlated with the severity of cervical lesions28, but so far, only few HPV tests target viral mRNAs. They are qualitative (e.g. HPV-Proofer (PRETECT))29 and do not discriminate among HPV types (Aptima HPV Assay (HOLOGIC))30.Nowadays, HR-HPV screening or diagnosis mostly involve cytological techniques that have lowest specificity and sensitivity than molecular techniquesE6 and E7 oncogene DNA levels normalized by the cellular GAPDH gene level. Multiplex detection of these three genes in a single assay enables reduction in cost, time and labor as compared to separate detection methods. This technique allows for an extra support of traditional HPV tests to validate and quantify specifically the presence of HPV16 and/or HPV18 infection. Once persistent infection is confirmed, it could be used for a follow-up of patients at risk of cervical cancer, by correlating the activation of oncogenes to the severity of the lesions.The main aim of the present study was to develop a rapid quantitative detection test of HPV16 and HPV18 infections by measuring E6 and E7 genes were obtained using the NCBI reference genomes of HPV16 (accession number: NC_001526.4) and HPV18 (accession number: X05015.1). The primers and probes were designed to amplify and detect specifically E6 and E7 oncogenes of HPV16 and HPV18 using PrimerQuest Tool software with default parameters. The specificity of the primers and probes was checked using the online NCBI BLASTn tool against the human genome (https://blast.ncbi.nlm.nih.gov/). Human GAPDH primers were obtained from previous study31.Target sequences of E6, E7 and GAPDH were labeled on the 5′-end with FAM, VIC and CY5 fluorescent dyes, respectively, and on the 3′-end with a TAMRA compatible quencher for E6 and E7 and BHQ (Black Hole Quencher) for GAPDH.The hydrolysis probes of The sequences of primers and probes, nucleotide positions on HPV16 or HPV18 reference genomes and amplicon lengths are presented in Table 2. PCRs were performed according to the manufacturer recommendations with AmpliTaq Gold 360 Master Mix kit (THERMOFISCHER) in a 25 µL volume containing 12.5 µL of 2X AmpliTaq Gold Mix, 0.5 µL of each primer at 10 µM and 2 µL of template DNA.HPV DNAs were extracted using a BioRobot EZ1 instrument (QIAGEN) from ATCC HeLa cells for HPV18 (CCL-2) and ATCC SiHa cells for HPV16 (HTB-35) cultured in a MEM medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 1% non-essential amino acids 100X (GIBCO), 1% glutamine 100X (GIBCO), 1% sodium pyruvate 100X (GIBCO) at 37 °C with 5% COd-thiogalactopyranoside (EUROMEDEX) and 80 µg/ml X-gal (EUROMEDEX). Cells were incubated at 37 °C for 24 h or during the weekend at room temperature. Transformants were analyzed by PCR using SP6 (5′-TATTTAGGTGACACTATAG-3′) and T7 primers (5′-TAATACGACTCACTATAGGG-3′) according to the following program: 10 min at 95 °C; then 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 20 s at 72 °C; and a final extension step of 7 min at 72 °C. Positive E6 and E7 clones for each HPV genotype and GAPDH clones were placed into 7 mL of LB broth containing 100 µg/mL of ampicillin then incubated overnight at 37 °C under shaking (~ 200 rpm). Plasmid DNA was extracted using the Smart Pure Plasmid Kit (EUROGENTEC). Purified plasmids were linearized by digestion with the NcoI restriction enzyme during 15 min at 37 °C according to the manufacturer’s instructions. Digestion products were run on a 1% agarose gel and the band was purified using the QIAquick Gel Extraction (QIAGEN). The plasmid concentration was quantified by Nanodrop 2000 (THERMO SCIENTIFIC) for HPV16 and by Multiskan GO (THERMO SCIENTIFIC) for HPV18. The copy numbers (copies/µL) were determined with the following formula:Amplicons were produced by standard PCR in a Mastercycler nexus gradient (EPPENDORF) according to the following program: 10 min at 95 °C; then 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C; and a final extension step of 7 min at 72 °C. PCR products were analyzed by electrophoresis on a 2% agarose gel and purified by QIAquick PCR purification (QIAGEN) before being cloned into a PGEM-T easy vector (PROMEGA). The ligation reaction was conducted overnight at 4 °C according to the manufacturer recommendations. JM109 competent cells (PROMEGA) were transformed by heat shock, before being spread with beads onto Luria–Bertani (LB) (BD DIFCO) agar plates containing 100 µg/ml ampicillin (SIGMA), 0.5 mM isopropyl-β-10 copies/µL (GAPDH), 1.44 × 1010 copies/µL , 9.9 × 109 copies/µL , 1.54 × 1010 copies/µL  and 1.58 × 1010 copies/µL . Serial dilutions (from 107 to 100) of the control plasmids were aliquoted and stored at − 20 °C until further use. All steps of qPCR development were run with 2 µL of plasmid solution.Plasmid copy numbers were 1.59 × 105 plasmid copies and using the following steps: 2 min at 50 °C; 5 min at 95 °C; 40 cycles of 10 s at 95 °C; 20 s at Tm; and a final 1 s at 72 °C.Optimization of HPV16 and HPV18 multiplex was run on a CFX C1000 Touch (BIORAD) to determine the most appropriate annealing temperature , primer concentration  and probe concentration  for each gene. According to the manufacturer protocol, qPCRs were performed with a LightCycler 480 Probes Master (ROCHE) kit in 20 µL containing primers and probes (at different concentrations listed above), 10 µL 2X LightCycler 480 probe Master mix, 0.5 µL Uracil-DNA Glycosylase  and 2 µL of DNA template. Multiplex assays were performed in biological and technical triplicates on 105 copies in a multiplex qPCR by using the optimized parameters on a CFX C1000 Touch (BIORAD). Each run consisted in three steps: 2 min at 50 °C, 5 min at 95 °C, then 40 cycles of 10 s at 95 °C and 20 s at 60 °C.The specificities of the primers-probe couples were tested on each plasmid produced as mentioned above at the concentration of 107 to 100 copies). All assays were performed in biological and technical triplicates on a CFX C1000 Touch (BIORAD) with the following program: 2 min at 50 °C; 5 min at 95 °C; 40 cycles of 10 s at 95 °C and 20 s at 60 °C. Assays were validated with a slope of regression curve between − 3.9 and − 2.9 cycles/log10, a R2 > 0.98 and an efficiency range between 80 and 120%32. The limit of detection (LOD) of the assays was defined as the lowest concentration (target gene copy number per reaction) for which ≥ 95% of test runs gave positive results.PCR efficiency and sensitivity of each primers-probe couple with the optimized conditions were determined using a quantitative standard curve , and the coefficient of variation (CV) of both the biological and the technical replicates, using a concentration of 10As reference for HPV detection, we used the diagnostic results obtained at the IHU Méditerranée Infection laboratory using the Xpert HPV Assay (CEPHEID) from 96 clinical materials  collected in liquid-based media   or High Pure Nucleic Acid Large Volume extraction kits (ROCHE) according to the manufacturer recommendations. qPCR on DNA samples (5 µL) were run on a CFX C1000 Touch (BIORAD) according to the optimized parameters determined in this work. qPCR products with Ct values > 38 and PCR products visible on a 2% agarose gel using universal HPV primers were verified by Sanger sequencing using Big Dye Terminator v1.1 Cycle Sequencing Kit (LIFE TECHNOLOGIES) according to manufacturer recommendations on a 3500XL Genetic Analyzer capillary sequencer (THERMOFISHER).GAPDH was determined. The HPV viral load was calculated according to the following formula26:Using the standard curve, the copy number of each HPV18/16 oncogenes and Clinical samples used in this study have been collected in the setting of routine standard clinical management and informed consent was obtained from all subjects. The study was approved by the ethical committee of the University Hospital Institute Méditerranée Infection (No. 2020-03). The study was performed according to the good clinical practices recommended by the Declaration of Helsinki (and its amendments) and all methods were carried out in accordance with relevant guidelines and regulations.33 were calculated to determine the level of agreement of HPV16 and HPV18 detection results between the multiplex systems developed in this study and the Xpert HPV Assay. Kappa values of < 0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80 and 0.81–1.00 were considered poor, fair, moderate, good and very good agreement, respectively.All statistical analyses were performed using the Prism 7 software (GRAPHPAD). Two-way ANOVA and Tukey’s multiple comparison test were performed to analyze the differences between the Ct and RFU values (for primer and probe concentrations) between conditions, with H0 rejected for a p-value < 0.05. Concordance and Cohen’s kappa valueTo optimize the fluorescence rate of each gene in the multiplex system, four primer concentrations  and four probe concentrations  were tested. The optimal concentration for the two multiplex qPCRs (HPV16 or HPV18) was determined as the highest relative fluorescence unit (RFU) for the lowest Ct value  was tested to select the best annealing temperature for the lowest Ct value Table a,b. For E6, E7, GAPDH) separately or pooled were run in qPCR reaction mixes with the pool of primers and probes for HPV16 or HPV18 using the optimal qPCR parameters previously determined. qPCR resulted in a single curve for each specific plasmid run in the HPV16 multiplex mix. This indicated that this triplex was able to detect all the three genes without cross-amplification and non-specific background fluorescence  for all tested genes  were determined as the last dilution of plasmids for which ≥ 95% of the intra- and inter-triplicates gave a positive result. The mean frequency of positives for the multiplex qPCR was 100% at 10The coefficient of variations for the biological (intra-assay) triplicates were all less than 8% and those for the technical (inter-assay) triplicates were all less than 5% Tables  and 3.Concordance between results from our in-house multiplex qPCR systems and from the Xpert HPV Assay was analyzed for 96 clinical samples (Supplementary Table GAPDH gene ranged between 21.07 and 38.48 (median 24.32). The E6 gene showed Ct values between 15.74 and 39.07  and the E7 gene had a Ct range between 15.66 and 39.82 . Seven samples presented Ct > 35 for E6 or E7 genes. For 3/7, only the E6 gene was detected while for 1/7 only E7 was detected. Co-infection with HPV18 was detected in 13 samples . For 2 samples (no. 47 and 48), the GAPDH gene was not detected whereas E6 and E7 oncogenes were early detected (Ct between 15.66 and 20.46). For one sample (no. 2), results were discordant between the Xpert HPV Assay and the multiplex qPCR developed here, as HPV18 was detected instead of HPV16.Among the 46 samples found HPV16-positive by the Xpert HPV Assay, 44 were also positive using the triplex qPCR developed in this study. For these samples, Ct of the GAPDH gene, between 15.18 and 38.90  for the E6 gene and between 16.45 and 39.60  for the E7 gene. Samples that were HPV18-negative were expected to be HPV45 and were verified by Sanger sequencing after conventional PCR using universal HPV primers34. The results confirmed HPV45 in seven cases, but also detected HPV53 in three cases. Co-infection with HPV16 was detected for three samples .Among the 38 HPV18/45 samples detected by the Xpert HPV Assay, 18 were found HPV18-positive using the triplex qPCR. Ct ranged between 21.19 and 30.45  for the E7 gene, 38.02 and 36.67, respectively. HPV16 was also detected in one of the other HR-HPV samples (no. 24).All negative samples (n = 8) were found negative, except one (sample no. 5), for which HPV16 and HPV18 co-infections were detected with late Ct values for Considering the negative controls (negative samples and other HR-HPVs) and the positive samples, the concordance between the Xpert HPV Assay and the triplex qPCRs developed in this study was 93.44% for HPV16 with a Cohen’s kappa value of 0.82  and 73.58% for HPV18 with a Cohen’s kappa value of 0.48 (moderate agreement).E6 and E7 oncogenes for both HPV18 and HPV16 were detected with at least 3 Ct earlier than the GAPDH gene , 6 cells was determined  for HPV16 and from 4.12 × 101 to 7.87 × 108 HPV copies/106  for HPV18. Some samples, particularly those from cervico-vaginal smears, presented a mean value close to the limit of detection previously defined at 101 plasmid copy/µL .Using the standard curves of each gene of interest, the viral load of HPV copies/1036. Between 60 and 90% of worldwide HPV cancers cases are due to HPV16 and/or HPV186 and infection by one of those HPV types has a predictive value of cervical intraepithelial neoplasia grade 3 lesion37. The present study describes the development and validation of two real-time PCR triplexes for fast detection and relative quantification of E6 and E7 DNA oncogenes for HPV16 and HPV18.In most of cases (80–90%), HPV infections are transient as the result of viral clearance by the host immune system whereas viral persistence is necessary to induce HPV related-diseases 2 > 0.99). The difference between the PCR efficiencies obtained for each target gene of the multiplex system never exceeded 15%, which further demonstrated the absence of competition between the different targets38. The low coefficient of variation for both intra- (≤ 7.4%) and inter- (≤ 3.7%) assays showed a good reproducibility of each steps of the multiplex development. The triplex qPCR assays allowed the detection of up to 101 plasmid copies/µL for all genes tested.Amplification efficiencies of each gene of the two multiplex systems ranged between 80 and 120% with good standard curve coefficient of correlation 39. Some of these qualitative HPV tests uses specific technologies such as hybrid capture with signal amplification by chemiluminescence  or hybridization by FRET probe (Cervista HPV HR). Other HPV tests are easier to use but need specific additional laboratory equipment . The two multiplex systems developed in this study allow detecting DNA of two of the most prevalent HR-HPVs and to quantify E6 and E7 oncogenes DNA levels compare to GAPDH housekeeping gene within the same amplification reaction. Moreover, in house probe-based real-time qPCR is a fast, easy to use, specific and reproducible method that only requires a minimal amount of DNA, does not need specific, assay-dedicated laboratory instrument, and can allow testing in the same run multiple sample DNA extracts on 96- or 384-well plates.Currently, diagnosis of HPV infection often uses commercially available tests that target the complete genome, E6 and E7 have a Ct value lower than the GAPDH gene. This could be a signature of an active virus replication and transient infection. In contrast, patients with Ct for oncogenes equal to or higher than the GAPDH gene may reflect persistence under an episomal state or HPV integration to the human genome. In this work, 15 samples with HPV18 and HPV16 co-infections were detected. Detection of HPV co-infections is of particular interest as a combination of HPV16/18 co-infection has been associated with grade 2 and 3 cervical intraepithelial neoplasia (CIN)40. Viral loads per millions of human cells were determined for HPV16 and HPV18 as these could be a marker of CIN2 and be a predictor of lesion progression42. The value measured in this study were within the limits of the standard curves and showed large variations across patients and samples.The multiplexes developed in this study confirmed the clinical diagnosis of the Xpert HPV assay with a concordance of 93.44% for HPV16 and 73.58% for HPV18. Lower concordance and Cohen’s kappa value were observed for HPV18 as the Xpert HPV assay provided a positive signal for both HPV18 and HPV45 whereas our system only targeted HPV18. Using HPV DNA quantification, we noted for some patients that oncogenes E6 and E7 oncogenes are correlated with the severity of cervical intraepithelial neoplasia28. Quantification of mRNA could thus be a novel marker of HPV infection and cervical intraepithelial neoplasia progression43. So far, only few HPV detection test target mRNAs. These tests are qualitative (HPV-Proofer) and do not discriminate HPV types (Aptima HPV Assay). In 2018, a multiplex reverse transcription real-time PCR for E6/E7 mRNA detection of 14 h-HPV without discrimination among types was developed44. This study confirmed the association of E6/E7 mRNA increased levels with the severity of the cervical lesions. Another study showed that the detection of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology45. In this study, we could simultaneously detect and quantify E6 and E7 genes within the same amplification reaction. The triplex qPCR developed in this study could then be used to assess E6/E7 oncogene mRNA levels for a personalized follow-up of patients with confirmed HPV18 and/or HPV16 persistent infections, in order to survey women at risk of developing cervical carcinoma.In 2010, a study showed that mRNA levels of E6 and E7 oncogenes normalized on the GAPDH expression. This could be easily realized simultaneously on many samples (96–384 wells qPCR plate) and could be developed in the future for other less frequent HR-HPVs.To conclude, the multiplex PCR systems developed in this study could be used as extra support of traditional HPV tests to validate specifically the presence of and quantify per million cells HPV16 and/or HPV18 DNA. It may also be used for research in second intention for the follow-up of patients with confirmed infection who are at risk of developing lesions by quantifying expression levels of Supplementary Legends.Supplementary Table 1.Supplementary Figure 1.


Cervical cancer is a serious global health problem. The objective of this study was to provide a suitable cytology-based cervical screening method in women of different ages in primary hospitals.This study was a retrospective cohort study that included 9765 women who underwent primary cytology-based cervical screening and were grouped by age . Patients with abnormal cytology on the primary cervical thin-prep cytologic test (TCT) were advised to undergo triage human papillomavirus (HPV) test. Furthermore, patients with positive outcomes of the 2 indices underwent cervical tissue biopsy. The positive rate of TCT and HPV was compared among the 3 defined age groups. The sensitivity, specificity, and positive predictive value of TCT and HPV were assessed.P = .003), and the positive rate of HR-HPV was similar (P = .299). The sensitivity of initial TCT testing to detect intraepithelial neoplasia was higher than that of triage HPV testing, whereas the specificity, the positive predictive value of triage HPV testing was higher than that of TCT. The Youden index of HPV testing was higher than that of TCT detection in the 3 age groups, namely 0.582 versus 0.432, 0.553 versus 0.228, and 0.416 versus 0.332, respectively.In total, 2.5% (241/9765) of women had atypical squamous cells of undetermined significance or worse by TCT. High-risk (HR)-HPV infection was found in 70 triage participants. Neoplastic changes were confirmed in 95 patients  by biopsies. Among the different age groups, the positive rate of abnormal cytology was significantly different (The results of this study indicate that TCT testing is suitable as a cervical cancer screening method for women ≥35 years old in primary hospitals. Triage testing for women with HR-HPV has a high negative predictive value, reduces the rate of misdiagnosis, seems to be an excellent triage method for repeat atypical squamous cells of undetermined significance, and reduces the number of referral colposcopies preventing unnecessary overtreatment. The results of this study provide a crucial foundation for a unified guideline cervical cancer screening for primary health care institutions. In contrast to the declining trend of cervical cancer incidence in developed countries, the incidence of cervical cancer in China has significantly increased. Hence cervical cancer is still one of the main threats to women's health in China.Cervical cancer is the fourth most common cancer of women worldwide. The on-going policy for cervical screening is to perform cytology-based cervical tests for women every 3 years within primary care. However, in primary hospitals, the detection of high-risk human papillomavirus (HR-HPV) is not widely implemented. It is recommended that patients with abnormal cytology undergo a cervical biopsy. Cervical cancer screening work throughout China has not been evenly distributed, with various regions having different conditions. Nevertheless, HR-HPV screening has been implemented as the triage of equivocal cytological results in some places.Cervical screening has been implemented in some rural and urban areas in women aged 35 to 59 and 35 to 64 years since 2009.,5 However, the TCT has low predictive value in detecting high-grade cervical cancer precursors, as its accuracy depends on sample acquisition, as well as the skill and experience of the physician.With the improvement of screening technologies, cervical pap smear, and visual inspection with acetic acid and Lugol's iodine are currently only used in remote and economically deficient areas in China. Thin-prep cytologic Test (TCT) screening has become the primary screening method in China.,8 The persistence of HR-HPV infection in epithelial cells of the cervix is essential to progression towards high-grade cervical disease and ICC. Without treatment, the risk of ICC increases with an increasing degree of dysplasia by affecting about 50% of women with cervical intraepithelial neoplasia grade 3 (CIN3). Human papillomavirus (HPV) screening techniques have the advantages of good reproducibility, easy training, and high sensitivity. Recent findings have suggested that HPV DNA detection on self-collected cells has advantages. TCT, combined with HPV detection, significantly improves the screening sensitivity and specificity for cervical cancers. However, it may not be suitable for women in primary hospitals because it is expensive. Therefore, the value of TCT and/or HR-HPV testing for primary screening is mostly unknown. There is also a lack of evidence and reliable data on the differences in the efficacy of TCT and/or HR-HPV detection methods in women of different ages.Many studies have shown that most invasive cervical cancers (ICCs) are caused by HR-HPV infection.Therefore, it is of great importance to find a screening program that has specific sensitivity and specificity in primary hospitals. The objective of this study was to compare and evaluate the efficacy of TCT and HR-HPV screening methods for cervical cancer in adults of different ages to find an optimal screening method that can improve the sensitivity and specificity of detection, save medical costs, and reduce overdiagnosis and treatment for women in primary health care institutions.22.1This was a retrospective cohort study that targeted women who underwent cervical cancer and precancerous lesion screening at 3 primary hospitals from December 2017 to December 2018. The 3 primary hospitals included 2 community hospitals  and 1 township hospital . The Medical Ethics committee approved the survey of the First Affiliated Hospital of Nanjing Medical University. Informed consent was obtained from all individual participants. The inclusion criteria for participants were:1)patients with rural household or urban household registration;2)patients who were not pregnant or puerperal;3)patients who were not having a menstrual period; and4)patients voluntarily participated in the physical examination. Patients with acute genital inflammation, or severe other diseases of the genital tract, or had a history of cervical or total uterus resection were excluded. The cervical cancer screening examination included a gynecological examination, primary cytology screening, triage HPV testing, and colposcopy with cervical biopsy. The screening flowchart is shown in Figure 2.2 The grade categories of the results from low to high severity in order were:The participants were asked to refrain from vaginal lavage or sexual intercourse for 48 hours before the examination. Cervical TCT was performed after routine gynecological examinations. Patients adopted the bladder lithotomy position and exposed the cervix. Cervical secretions were wiped with a sterile cotton ball. Then, a special cervical brush was gently inserted into the inner cervix (1 cm), and the cervical brush was rotated 3 to 5 times clockwise to collect the cervical cells. Next, the brush was placed in the cell preservation solution. Then the specimens were prepared at the Inspection Agency, and tablets for TCT microscopic examination were read. The cytology results were reported using the Bethesda System terminology.1)no intraepithelial lesion or malignancy (NILM);2)atypical squamous cells of undetermined significance (ASC-US);3)ASC that cannot exclude a high-grade lesion (ASC-H);4)low-grade squamous intraepithelial lesions (LSIL);5)high-grade squamous intraepithelial lesions (HSIL); and6)squamous cell carcinoma or cervical adenocarcinoma. ASC-US and more severe grades were considered abnormal cytology results.2.3After the TCT, women with abnormal cytology results were advised to undergo triage HPV DNA testing as an adjunct to the cytology test. Some patients whose TCT results showed NILM but presented with clinically observed cervical contact bleeding, cervical ectropion, or other suspected cervical lesions were also advised to undergo HPV DNA testing Fig. . The cer2.4Women with a positive outcome of any of the 2 indices underwent colposcopy with biopsies and/or endocervical curettage (ECC). All cervical biopsies were performed under direct vision by colposcopy. Specimens were obtained from the area with the most severe dysplastic cervical lesions according to visual examination and colposcopy with VIA + VILI. Four-quadrant cervical biopsies were taken by colposcopy if the lesion area was not found. The specimens were routinely fixed in formaldehyde. The pathological results of cervical tissue were used as the gold standard for diagnosis. The diagnosis was divided into:1)normal/inflammation;2)cervical intraepithelial neoplasia grade 1, cervical intraepithelial neoplasia grade 2 (CIN2), CIN3; and3)invasive carcinoma. Histopathological examination results were used to identify neoplastic changes and other infections with a positive diagnosis referring to cervical intraepithelial neoplasia grade 1 and more severe lesions (CIN1+) and a negative diagnosis referring to normal/inflammation.2.5P < .05 was considered statistically significant.All the original data were carefully checked and recorded. Statistical analysis was performed using SPSS 20.0 . The association between the categorical variables was analyzed using the Chi-squared test. The pathological test results were used as the gold standard. The receiver operating characteristic curve and Youden index was used to evaluate the diagnostic value of TCT and HPV on positive screening results by age group. The area under the curve (AUC) = 0.5–0.7 was defined as low accuracy, AUC = 0.7–0.9 was defined as moderate accuracy, and AUC > 0.9 was defined as high accuracy. 3A total of 9765 women aged 35 to 64 years were included in this study Table . They we3.1P = .003). Between the groups of 35 to 44 and 45 to 54 years, the difference was statistically significant (P = .024). Similarly, between the groups of 45 to 54 and 55 to 64 years, the difference was statistically significant (P = .002). However, no statistically significant difference was detected between the 35 to 44 and 55 to 64-year age groups (P = .270)  had NILM, and 241 (2.5%) had abnormal cervical cytology  (Table P = .299)  and normal cytology but clinical suspicious cervical lesions (n = 196) were advised to undergo triage HPV DNA testing. Valid results for both cytology and HPV were obtained for 437 women (Table 41 and no3.3+ was 21.7% (95/437). The results showed that the 55- to 64-year age group had the highest rate of CIN1+ detection , followed by the 45- to 54-year age group , whereas the 35- to 44-year age group had the lowest detection rate . The difference between the various age groups was statistically significant (P = .018). With increasing age, the detection rate of cervical precancerous lesions increased  women, cervical intraepithelial neoplasia grade 1 in 61 (13.96%), CIN2 in 19 (4.35%), CIN3 in 13 (2.97%), and invasive cervical carcinoma in 2 (0.46%). The detection rate of CIN13.4+ was moderate .Primary hospitals have the main task of national free screening for cervical cancer in China. This study shows the value of detecting cervical cancer and precancerous lesions using primary TCT screening and triage HPV DNA testing in different age groups in the primary care setting. In the present study, we retrospectively analyzed women with abnormal cytological in an organized free cervical cancer screening program based on cytology screening strategy to perform triage HPV-DNA testing before referral to colposcopy to evaluate the value of HPV testing, with the goal of optimizing the strategy of current cervical cancer screening and providing some references. According to previous studies, the detection rate of cervical precancerous lesions increases with increasing age.P = .003). Cytology is the most frequently used primary screening test due to its better sensitivity. The stages of cervical carcinogenesis include the persistence of HPV infection rather than clearance of the virus, which is linked to the development of a high-grade precursor lesion or “precancer”. Some precancer stages, such as chronic cervicitis, wet squamous changes, and cervical lesions, can be reversed and/or treated if they are detected early enough, which can radically reduce cervical cancer incidence.,17 So, HPV is recommended as a triage testing or co-testing for women with cytology. However, in China, TCT and HPV co-testing has only been conducted in a small number of areas in the National Cervical Cancer Screening Program, which does not include Jiangsu Province. With limited financial support, more women can participate in screening, and the benefits may be more significant. The optimal strategy for cervical cancer screening is to expand screening rates to maximize the benefits of screening while minimizing potential hazards. In our study, women with ASC-US were recommended to undergo triage HPV-DNA testing as an adjunct to cytology. Of these, among 67 HPV positive women with abnormal cytology, 43 had ASC-US, 6had ASC-H, 2 had HSIL, and 11 had LSIL. Five of the women with LR-HPV infection had ASC-US. The proportion with HR-HPV did statistically significantly differed between women with abnormal cytology and normal cytology (P < .001), with increasing age, and the positive rate of HPV increased, but with no significant difference among the different age groups (P = .299). The infection rate of HPV different from the reported by You et al, which was high in the age groups of 26 to 30 and 51 to 55 years, accounting for 87.7% (71/81) and 79.7% (51/64), respectively, while it was lower in the >55 years group at 28.6% (14/54) in Shandong provinces of China; this difference was considered to be related to the age of the population and size of the sample. Due to its relatively high specificity  and positive predictive value  in detecting CIN1+ lesions in the present study, HR-HPV seems to be useful in the triage of repeat ASC-US or worse among different age groups, which reduces the rate of misdiagnosis. However, our results should be interpreted with caution because a selection bias cannot be ruled out, due to the fact that the use of HR-HPV test was not systematic in this setting.In the current study, there was a significant difference in abnormal cytology in different age groups (,19 In other words, the screening rate of cervical cancer and precancerous lesions should be increased as much as possible, and the rate of missed diagnosis should be reduced with minimum economic investment. Hence, the sensitivity of screening methods should be increased as much as possible. An excellent screening method should not miss any true-positive patients and also exclude all true-negative patients, limiting the economic, and psychological burden on women.For primary hospitals, screening methods should be economical and effective. reported that cumulative risks for cancer of persistent HPV infection, CIN2, and CIN3 were 56%, 2.8%, and 1.1% while follow-up 1 year, respectively; follow-up 2 years were 37%, 4.8%, and 2.0%.In this study, the sensitivity of TCT detection was higher than that of HPV DNA testing across all age groups. By contrast, the specificity, positive predictive value for triage HPV testing was significantly higher than that of TCT in all age groups. These results suggest that triage TCT testing is successfully able to diagnose patients with cervical lesions as the primary method of secondary prevention of cervical cancer and is suitable for women of all ages. HR-HPV has a better ability to screen people with virtually no cervical lesions as an auxiliary screening, which should reduce the number of referral colposcopies, thereby preventing unnecessary overtreatment. Katki et al Potential adverse effects of colposcopy procedures related to cervical cancer screening included pain, bleeding, discharge, and anxiety. In the present study, triage HPV testing of repeat abnormal cytology increased its specificity and positive predictive value and reduced the unnecessary injuries to patients.Furthermore, a meta-analysis based on the results of RCTs found a significantly lower rate of cervical cancer among women with negative HPV testing results.,26 However, the present study had some differences. On the 1 hand, the population of HPV testing was limited. The HPV as a triage testing was conducted based on people with abnormal cytology results before colposcopy referral to exclude more low-risk people, thus reducing the chances of invasive surgery and saving medical costs. On the other hand, CIN1+ was used as the endpoint of diagnosis to identify more high-risk populations. These factors can help in the follow-up care of high-risk groups, including repeat TCT test after 12 months. HPV testing alone or co-testing is the main difference between the European and American guidelines: the American guidelines recommend co-testing with HPV and cytology, while the European guidelines recommend HPV alone. China has a large population and regional economic distribution. The current cervical cancer screening program in China is implemented in a “3-Step” strategy, including TCT, HPV testing, and colposcopy, and is carried out in a diversified manner, adopting different screening strategies for different groups of people. Opportunistic screening, which aims to screen out abnormal cases at a minimum cost and select the optimal screening method according to local conditions, can bring the most significant benefit to the majority of women at limited medical cost. Therefore, primary hospitals should expand screening coverage of cervical cancer with more options for low-cost screening strategies.Some previous studies have shown that HPV-DNA detection is more sensitive but less specific compared with cytology for high-grade CIN. which is a public health service system run by the government of People's Republic of China. The group was organized by the government in primary medical institutions, with clear screening guidelines. The data provided an accurate indication of the cervical lesions and cancer prevalence in the basic population of the region. The limitation of the present study is relatively small of sample size, which may need further large-scale studies to confirm the conclusion.The strength of the present screening strategy was that it was based on an opportunistic screening based approach focusing on a community health care maternal child health system,5In conclusion, the results of this study indicate that TCT testing is suitable as the primary cervical cancer screening method for women of all ages in primary hospitals. High-risk HPV as triage testing has a high specificity, and positive predictive value, reducing the rate of misdiagnosis, seems to be an excellent triage method for repeat ASC-US, the numbers of referral colposcopies can be reduced to avoid unnecessary overtreatment. At present, there is no unified cervical cancer screening guideline. This study provides a crucial foundation for a unified cervical cancer screening guideline for primary health care institutions.The authors are thankful to Gilmour Tim for his help in preparing the manuscript.Z-YY: study design, literature search, data acquisition, manuscript preparation, and manuscript editing. X-XQ: study design, literature search, data acquisition, statistical analysis, manuscript preparation. ZD: literature search, data acquisition, statistical analysis, manuscript preparation. Z-HX: study design, the definition of intellectual content, and manuscript review. WJ: definition of intellectual content, manuscript review. Hongxiu Zhang orcid:0000-001-5995-6460Writing – review & editing: Hongxiu Zhang.


High-risk human papillomavirus (HR-HPV) is notoriously associated with tumor progression in a broad spectrum of malignancies. Detection of HR-HPV is clinically important in the management of HPV-related carcinomas, particularly in cervical cancer and oropharyngeal squamous cell carcinoma (OPSCC). Several methods for HPV detection are currently available including Polymerase chain reaction (PCR)-based techniques, DNA in situ hybridization (ISH), RNA ISH, and p16 immunohistochemistry (IHC). Currently, the guidelines for HPV detection in cervical carcinoma are available, while no clear consensus has not yet been reached on the gold standard for HPV testing in OPSCC. Multimodality testing could help to reliably identify patients with transcriptionally active high-risk HPV-positive.We propose a multiplex approach carrying out HPV RNA ISH and p16 IHC on the same slide to detect simultaneously HPV E6/E7 transcripts and p16INK4a overexpression. We tested this assay in two different series one of the cervical cancers with p16-positive, as control, and the other of oropharyngeal squamous cell carcinomas with blind p16 status.The multiplex HPV RNA ISH /p16 IHC results in the series both of the cervical cancers and the oral-oropharyngeal cancers were fully concordant with the previous results achieved through the classic p16 IHC and HPV RNA scope carried out on two different slides.Our results suggesting several advantages of this technical approach, namely an easy interpretation fully in the light field, the feasibility in formalin-fixed paraffin-embedded tissue sections, complete automation and a potential wide spreadable for routine testing in several clinical laboratories. Human papillomavirus (HPV) is non-enveloped icosahedral, circular, dsDNA virus that can infect cutaneous and mucosal epithelia. Approximately 200 different HPV genotypes have been identified responsible of a broad spectrum of the clinical profiles, from benign lesions to HPV-related carcinomas , 2. HighThe US Preventative Services Task Force (USPSTF) recommends that women aged 30 to 65 years should be screened with cytology and HPV testing (co-testing) every 5 years . InsteadIn the last decade, HR-HPV has revolutionized head and neck oncology, since the HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) represents a unique cancer type with distinct clinicopathologic features and favorable prognosis. Thus HPV-OPSCC is now a well-recognized tumoral entity in the field of head and neck oncology, with an increasing incidence , 10. Sevp16 IHC represents a method relatively inexpensive, which is highly sensitive for transcriptionally active HR HPV , 19, 20.In this context, multimodality testing could help an accurate detection of HPV. We developed a multiplex approach carrying out HPV RNA ISH and p16IHC on the same slide to detect simultaneously HPV E6/E7 transcripts and p16INK4a overexpression. We validated this approach in two different series, one of the cervical cancers and the other of OPSCC .A series of 17 cervical cancers with p16-positive IHC and a series of 38 oral-oropharyngeal cancers with blind p16 status were included in our study. All cases were collected in our records at the University of Campania “L. Vanvitelli”. The series included surgical samples or wide biopsies. Sections of 4 μm thickness from each block with a mean of 3 blocks per tumor) were stained with hematoxylin-eosin. All cases were reviewed according to the histopathological classification.FZM, AR and RF).p16 IHC was carried out with a proprietary kit CIN tec Histology; MTM laboratories AG) using the clone E6H4 on a Ventana Benchmark autostainer  for the detection of p16INK4a antigen. A tonsil squamous cell carcinoma with high p16 expression was used as a positive control. The primary antibody was omitted from negative controls. The new guidelines proposed by the College of American Pathologists (CAP), for HPV testing in head and neck carcinomas in routine clinical practice proposed the interpretation of p16 IHC as follows: tumors with lack of staining or < 70% of nuclear and cytoplasmic staining are classified as HPV negative, while tumors with ≥70% of nuclear and cytoplasmic staining as HPV positive . Accordiμ mof each case are used to perform HPV RNA ISH test. Detection of high-risk-HPV E6/E7 mRNA was performed using Ready-to-use reagents from RNAscope 2.5 LS Reagent Kit-BROWN and the HPV-HR18 probe cocktail (Advanced Cell Diagnostics) that were loaded onto the Leica Biosystems’ BOND RX Research Advanced Staining System according to the user manual (Doc. No. 322100-USM). The slides were independently evaluated by three separate observers . Ubiquitin C and dapB were used as positive and negative controls, respectively. A positive HPV ISH test result was defined as positive if any of the malignant cells showed brown punctate dot-like nuclear and/or cytoplasmatic positivity [Sections 4 All steps are performed on the Leica BOND RX, automated system . We tested different technical conditions.In particularly, we have tested different dilutions of the antibody for the detection of p16INK4a antigen; different protocols namely first RNA ISH and then p16IHC or the opposite sequence; different colorimetric approaches including detection for HPV mRNA in DAB and p16 staining in Fast Red or conversely.Finally, our results showed that the sequential staining first RNA ISH in DAB and then p16 IHC staining in Fast Red represents the best technical approach  chromogen of the Bond Polymer Refine kit to staining HPV E6/E7 mRNA, the Fast Red chromogen of the Bond Polymer Red Refine kit to staining p16 and hematoxylin to counterstain.Detection of high-risk-HPV E6/E7 mRNA was performed using ready-to-use reagents from RNAscope® 2.5 LS Reagent Kit-BROWN and the HPV-HR18 probe cocktail (Advanced Cell Diagnostics) that were loaded onto the Leica Biosystems’ BOND RX Research Advanced Staining System according to the user manual (Doc. No. 322100-USM). The target-specific probes for the E6 and E7 genes of 18 HR-HPV genotypes HPV . The Ubiquitin C a constitutively expressed endogenous gene was used as positive control to assess the presence adequate RNA quality and avoid a false-negative result. The dapB test was used as negative control to assess non-specific staining, for a comparison in the cases with negative or weakly stained HPV staining.In brief, 4 μm sections were baked and deparaffinized on the instrument, followed by epitope retrieval using Leica Epitope Retrieval Buffer 2 at 95 °C or at 88 °C for 15 min and protease treatment 15 min at 40 °C. Probe hybridization, signal amplification trough different AMP reagent AMP 1–6) and colorimetric detection were subsequently performed. Several washes were performed, subsequently the ready-to-use primary antibody clone E6H4 for the detection of p16INK4a antigen was incubated and colorimetric detection was performed. Finally, a hematoxylin staining was carried out.When the run is completed and the slide trays are removed, the covertiles are carefully lifted upward by the neck to remove. The slides are dehydrated through 2 changes each of 70, 95, and 100% alcohol and 2 changes of xylene, before coverslipping. A schematic of multiplex HPV RNA ISH/p16 IHC assay workflow is presented in Fig. FZM, AR and RF) evaluating simultaneously HPV mRNA and p16 expression.A positive HPV ISH test result was defined as positive if any of the malignant cells showed brown punctate dot-like nuclear and/or cytoplasmatic positivity. p16 IHC was positive if nuclear and cytoplasmic red staining was observed according to the above score. The slides were independently analyzed by three separate observers .Fisher’s exact test was used to assess the correlation between the results obtained from multiplex HPV RNA ISH /p16 IHC and the achieved through the classic p16 IHC and HPV RNA scope carried out on two different slides. Statistical significance was set at The cervical cancers selected from our archive on the basis of p16-positive IHC status were tested for RNAscope HPV-test. All 17 cervical cancers p16-positive showed RNAscope HPV-test positive results (100% concordance). The multiplex HPV RNA ISH/p16 IHC analysis showed a complete correspondence with the data obtained using the p16 IHC test and RNAscope HPV-test carried out separately on two different slides Fig. .Fig. 2RFirstly, the p16 IHC and the RNAscope HPV-test were carried out separately on two different slides for each case. The p16 immunohistochemical expression was found in 7 (18.4%) of 38 cases of oral-oropharyngeal cancers, particularly 3 cases p16-positive (7.9%) showed high expression, 2 cases (5.3%) moderate expression and 2 cases (5.3%) low expression. The HPV mRNA was found in 3 (7.9%) out of 38 cases, particularly 1 case (2.6%) showed high positivity and 2 cases (5.3%) low positivity, all other cases were negative.Overall, only two cases (5.3%) out of 38 showed concurrently p16 and HPV RNA expression, particularly one showing high expression and other low.Overall, 6 (15.8%) out of 38 showed no corresponding test results between IHC and ISH. The p16 IHC analysis yielded positive results for five cases where there was an absence of detectable HPV mRNA by ISH. One case HPV RNA ISH positive was p16 IHC negative.All 38 cases of oral-oropharyngeal cancers were tested with the multiplex HPV RNA ISH/p16 IHC and the results were compared with those obtained with the p16 IHC and HPV RNA test performed on two different samples.The multiplex HPV RNA ISH/p16 IHC results showed 100% concordance with the previous results achieved through the classic p16 IHC and HPV RNAscope carried out on two different slides Fig.  and 4. TDetection of HPV-DNA can be performed through PCR or ISH, while the detection of HPV E6/E7 mRNA is based on ISH assay, and finally, p16 IHC represents an indirect method to establish a possible infection with the virus integration. DNA ISH has limited sensibility, mainly for tumor samples with low HPV copy numbers resulting in false negative results . Moreovep16 overexpression has been described in approximately 20% of metastatic skin and lung HPV unrelated SCCs without a real association with HR-HPV . PreviouThe multiplex HPV RNA ISH/p16 IHC assay could be a promising assay to use in our routine considering its many advantages. This method is able to identify in a single assay both the p16 overexpression and the viral transcripts resulting in an increase in sensitivity and specificity to detect clinically relevant HPV-infections. The main advantage of this method is the easy interpretation of the results by a pathologist, since it is fully interpretable in light field. Furthermore, multiplex HPV RNA ISH/p16 IHC is technically feasible in FFPE tissue sections) and cytological samples, thus wide spreadable for routine testing in several clinical laboratories. The complete automation of the multiplex HPV RNA ISH/p16 IHC allows the reduction of time-consuming and the reproducibility of the technique, reducing any operator-dependent bias.The novel multiplex approach proposed in this study could have some limitations that may concern particularly the expertise in the interpretation of the results and the optimization of the technique on other automation systems. Unlike the immunohistochemistry which has wide diffusion in all laboratories and is easily interpretable, the ISH represents a not widespread technique and moreover not always easy to interpret by all observers. In this view, this multiplex approach may be difficult to interpret for the observers with little expertise in the analysis of the in situ hybridization. In terms of the automation, although we have optimized this method on plataform Bond Leica, not too many technical difficulties are conceivable in the translation of our multiplex HPV RNA ISH/p16 IHC on other automatic platforms.In the cervical carcinomas the HPV diagnosis is currently performed using the DNA-based molecular, rather than HPV RNA ISH . HPV RNAAccording to the current clinical practice, the multiplex HPV RNA ISH/p16 IHC approach could improve the identification of HPV-related SCCs. In head and neck cancer, HPV-SCC patients overall have significantly improved outcomes when compared to HPV-negative , 31. HPVThe 8th edition of the cancer staging manual defined HPV-related OPSCC as a distinct entity, with staging now completely dependent on testing for p16 as a surrogate of HPV status . in the Previous authors proposed HPV diagnostic algorithms for OPSCC using the p16-IHC as the first stepwise to define tumors HPV-unrelated based on exclusively p16-IHC negative. Conversely, p16-positive potentially HPV-driven OPSCC are addressed to second molecular assays, namely ISH or PCR, to confirm the presence of HR-HPV , 36–38. Moreover, we hypothesize a further application field of our technique on cytological samples, particularly in OPSCC patients at an advanced stage when the cytology specimens are the only sample available to establish HPV-status , 40.Our results suggesting that the multiplex HPV RNA ISH/p16 IHC have several advantages, namely the feasibility in FFPE samples, the complete automation and a potential wide spreadable for clinical practice in several Pathology Unit. This novel approach could help to completely eliminate the percentage of misdiagnosed HPV-related cases saving time, costs and biomaterials.


N = 293) dental student and forty-one (N = 41) post-graduate dental resident questionnaires were available for a total sample size of N = 334. In brief, although the majority of dental students and residents agreed that vaccines are safe and effective, less than half of dental students (37.5%) or dental residents (48.7%) had discussed the HPV vaccine with a physician or had received the vaccine themselves. In addition, a significant percentage of dental students and residents felt they did not have enough information regarding the HPV vaccine  or had significant concerns about the side effects (17.1%). Conclusions: The data suggest more specific information in dental school microbiology and immunology courses might be needed to increase awareness and knowledge of the safety and effectiveness of vaccines, including the HPV vaccine. This enhanced education might also serve as a curricular focal point to answer questions regarding vaccine-related side effects and provide a mechanism for answering important questions regarding this vaccine.Introduction: The recent development of a vaccine that is highly effective against the human papillomavirus (HPV) has been met with widespread clinical and public health professional acceptance. However, social and societal barriers to vaccination may hamper public health efforts to prevent HPV-mediated diseases. Although a few studies have evaluated knowledge or awareness of HPV vaccination among dentists or dental educators, few studies have evaluated the acceptance, knowledge and awareness of HPV vaccination among dental students and post-graduate dental residents. The primary goal of this study is to evaluate survey responses regarding acceptance, knowledge and awareness of HPV vaccination among dental students and post-graduate dental residents. Methods: This study was a retrospective analysis of a previously administered and collected questionnaire. The original protocol was reviewed by the UNLV Biomedical Institutional Research Board (IRB) and was deemed excluded from IRB review (OPRS#0811-2911). Results: Two hundred and ninety-three ( The recent development of a highly effective vaccine against the human papillomavirus (HPV) has been met with widespread clinical and public health interest and awareness based on scientific and lay literature reports of its effectiveness and resulting reductions in associated cancer risk ,2,3,4. SHowever, in spite of these positive reports of HPV vaccine effectiveness, social and societal barriers to vaccination may hamper public health efforts to prevent HPV-mediated diseases . For exaAlthough a few studies have evaluated knowledge or awareness of HPV vaccination among practicing dentists or dental educators, it is unclear whether this information receives sufficient emphasis in dental education to mold student views on vaccination that lead to their implementation by students when they enter dental practice. Few studies have evaluated the awareness, knowledge and acceptance of HPV vaccination among dental students and post-graduate residents ,16. The Based upon this information, the primary goal of this study is to evaluate dental student and post-graduate resident awareness, knowledge and acceptance of HPV vaccination during a curricular module devoted to immunization practices and HPV-specific vaccination.The original protocol was reviewed by the UNLV Biomedical Institutional Research Board (IRB) and was deemed excluded from IRB review (OPRS#0811-2911) November 12, 2008. Informed consent was waived pursuant to the exemption to human subjects research under the Basic HHS Policy for Protection of Human Research Subjects, (46.101) Subpart A (b) regarding IRB exemption for research involving the use of education tests  in which the subjects cannot be identified directly or through identifiers.n = 312) and post-graduate residents (n = 48) were asked to complete a voluntary questionnaire, based upon a previously validated HPV vaccine awareness, knowledge, perceptions and clinical practice survey discussed within the context of the required Microbiology and Immunology course session focused on HPV and HPV vaccination [In brief, this study was a retrospective analysis of previously administered and collected student responses. All enrolled dental students  and find evidence-based information to support their answers. Each student would take one of the four selected questions and find peer-reviewed articles to support (or refute) their answers. The students then answered questions on a separate survey regarding how and where they found this peer-reviewed or evidence-based information (information literacy skills). Neither the HPV questionnaire or information literacy survey had any identifying information regarding the respondents. At the completion of this exercise, students were allowed to voluntarily turn in either the HPV questionnaire, the information literacy survey or both. The information from the literacy survey will be reviewed and published separately.n = 17 total questions, divided between two sections. The first section consisted of eight non-specific vaccine related questions assessing general knowledge, awareness, perceptions and clinical practice guidelines. Four possible responses were available . These questions included:Vaccines are necessary to protect public healthThere are too many required vaccinesVaccines are generally safeVaccination can make you sickSome vaccines are dangerousVaccines are generally effectiveI follow the vaccine guidelines for myselfI adhere to the vaccine guidelines for my family The second section was comprised of nine HPV-specific questions, which included:I am aware of a vaccine for human papillomavirus (HPV)HPV vaccination is important for meHPV vaccination is important for (my) spouse/partnerHPV vaccination is important for (my) daughter(s)HPV vaccination is important for (my) son(s)I have discussed HPV vaccination with a doctorI do not have enough information about the HPV vaccineI am concerned about possible HPV vaccine side effectsI have already received the HPV vaccineThe full questionnaire consisted of Compliance with IRB and Informed Consent exemption required that no demographic or other personal information could be collected at the time of the questionnaire collection, therefore no summary statistics or analysis for these participants and the corresponding responses can be provided. All questionnaire responses were transcribed in an Excel spreadsheet and descriptive statistics for the percentage of responses from each question were reported. Differences in responses between dental (DMD-level) students and post-graduate residents were analyzed using Chi square analysis and on-line software from GraphPad .n = 312). Almost three hundred students completed this questionnaire, yielding an overall response rate of 93.9% (n = 293/312). The overall demographic breakdown of the overall student population was approximately 55% male and 45% female  and half non-White minorities (50.6%), including 39.1% Asians and 8.7% Hispanics. These percentages were also not significantly different from the DMD survey respondents, p = 0.5692.The voluntary questionnaire was administered to all enrolled dental (DMD) students . Forty-one residents completed this questionnaire, yielding an overall response rate of 85.4% (n = 41/48). The overall demographic breakdown of the overall post-graduate resident population was approximately 48% male and 52% female  and half non-White minorities (54.2%), exclusively Asian. These percentages were also not significantly different from the survey respondents, p = 0.7512.The voluntary questionnaire was also administered to all post-graduate dental  residents  was the same as the percentage that agreed vaccines are generally effective (97.5%), p = 1.000.To assess the knowledge and awareness of positive aspects of vaccinations, including vaccine safety and efficacy, two specific questions from the questionnaire were evaluated: 3. Vaccines are generally safe, and 6. Vaccination is generally effective . Althougp = 0.0001. Similarly, the percentage of PGDR that agreed vaccines can make you sick (34.1%) was higher and significantly different from the percentage who agreed that some vaccines are dangerous (9.8%), p = 0.0001. Although the percentage of DMD students and residents who agreed vaccines can make you sick was similar (30.0% versus 34.1%), a significantly higher proportion of DMD students responded that some vaccines were dangerous (19.1% versus 9.8%), p = 0.0001.To assess the knowledge and awareness of negative aspects of vaccinations, including vaccine-related illness and side effects, two specific questions from the questionnaire were evaluated: 4. Vaccines can make you sick, and 5. Some vaccines are dangerous . There wp = 0.8962. The percentage of PGDR that had discussed HPV vaccination with a doctor (48.7%) was significantly higher—and much higher than the percentage of DMD students—than the percentage that had received the HPV vaccination (41.4%), p = 0.0001.To assess specific aspects of HPV vaccination, two specific questions from the questionnaire were evaluated: 14. I have discussed HPV vaccination with a doctor, and 17. I have received the HPV vaccine . These dp = 0.396. In addition, the same percentage of both the DMD and PGDR responders indicated they had concerns about possible HPV vaccine side effects (17.1%).Finally, to assess the knowledge and awareness regarding HPV vaccinations, two additional questions from the questionnaire were evaluated: 15. I do not have enough information about this vaccine, and 16. I am concerned about possible HPV vaccine side effects . AnalysiThe few previous studies that evaluated the awareness, knowledge and acceptance of HPV vaccination among dental students and post-graduate residents found deficiencies in both knowledge and awareness ,16,17,18Moreover, the finding that nearly twice the percentage of dental students (approximately 20%) compared with dental residents (approximately 10%) agreed that some vaccines were dangerous may suggest that these concerns about vaccine safety, in general, may be more widespread and must be addressed within the dental school curriculum ,24. AlthThe findings that higher percentages of PGDR had discussed or received the HPV vaccination than DMD students may have several possible explanations. For example, the average age of a first year dental student is approximately 25 years old, while the average age for a post-graduate dental resident is closer to 30 years old—a difference of nearly five years that provides significant opportunities to interact with other healthcare providers and vaccination experts ,27. In aThis study also includes several limitations, which must also be considered. For example, this study is a retrospective analysis of previously collected data from an educational seminar and was therefore not designed specifically to collect demographic data and other variables that would allow for more in depth and comprehensive analysis of the respondents. In addition, more recent studies have developed and implemented psychometrically tested HPV surveys among college students, which may provide more accurate and useful information for comparison ,31,32.Despite these limitations, the data suggest more specific information in dental school microbiology and immunology courses might be needed to increase awareness and knowledge of the safety and effectiveness of vaccines, more specifically including the HPV vaccine and the relationship to oral cancer prevention. This enhanced education might also serve as a curricular focal point to answer questions regarding vaccine-related side effects and provide a mechanism for answering important questions regarding this vaccine.


Evaluate the effects of carboxy-methyl-beta-glucan on cervical epithelialization and on the vaginal microbiota in patients with HPV infection or low-grade cervical preneoplastic lesion (CIN 1).  Seven-hundred eighty-four women with positive HPV tests or diagnosed with CIN 1 were enrolled in a retrospective case-control study. All the recruited women performed, at baseline and after 6 months, Pap test, HPV test, evaluation of vaginal health according to the Amsel criteria, colposcopy, and punch biopsy. The study population was then divided into 2 groups in relation to the therapy performed during the follow-up period. Group A performed treatment with vaginal gel based on carboxy-methyl-beta-glucan (1 application/day for 20 days per month for 3 months). Group B was the control group.  The patients of group A had a significant improvement in the ectopia pattern and a greater number of cases with metaplasia in the maturation phase with a significant increase in Lugol uptake. In the experimental group, a significant improvement in the pH indices, a negative Swift test and a resolution of the leucorrhoea were observed. A negative result of the 37.1% Pap test and the 39.9% HPV test  were demonstrated in the treatment group with respect to the control group. A negativization of the colposcopic pictures was observed with a reduction in the amount of CIN 1 found higher in the treatment group.  Vaginal therapy based on carboxy-methyl-beta-glucan has been able to improve overall vaginal health; this effect seemed to positively impact the risk of persistence and progression of CIN. HPV (human papillomavirus) infection is transmitted mainly by sexual contact, and the cervix is the organ most sensitive to the oncogenic action of papillomavirus. The cervix is covered by two epithelia: the esocervical one and the paved type and the endocervical one and the cylindrical type. This transition zone represents, from a biological point of view, an area of instability because it allows easier access to the basal site of the target reserve cells of oncogenic agents such as HPV .The HPV infection is very common especially in young women (highest incidence peak between 20 and 30 years). There is a percentage of women at risk who are positive for the infection but in whom the virus did not cause cervical precancerous lesions (CIN). When the virus integrates into the cells of the cervical epithelium, it determines a process of cellular transformation and gives rise to the CIN lesions [Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii appear to dominate the vagina of most healthy women. The composition of vaginal microbiota is influenced by numerous factors: ethnicity, cyclical secretion of oestrogen and progesterone throughout the menstrual cycle, menstruation or menopausal state, the widespread use of synthetic hormones for contraceptive purposes, sexual intercourse, hygiene practices, and infection [Recent data show a significant correlation between immune status and persistence of the virus. The vaginal microbiota plays an important role in modulating the immune system of the female genital tract . Lactobanfection .There is much evidence to correlate the persistence of HPV with the altered presence of vaginal lactobacilli and an altered microbiota. A balanced situation of the vaginal microbiota ensures a better response against HPV –9. Even Current thinking suggests that the development of papillomavirus-associated disease requires the infection not just of an epithelial basal cell but more specifically an epithelial tissue stem cell at a pluristratified cutaneous or mucosal site. At the cervix, the transformation zone is maintained by a specialized type of cell known as the reserve cell. The cervical neoplasia develops primarily at the squamocolumnar junction because these cells fail to properly regulate viral gene expression, leading to a nonproductive or abortive infection rather than a productive infection. The idea that papillomaviruses generally reside in an epithelial stem cell following infection is compatible with our understanding of latency and reactivation from latency. During latency, the virus can be undetectable to the common diagnostic test , 10.Maintaining adequate or improving the state of vaginal health and acting positively on the cervical epithelium and the vaginal microbiota could be a new strategy to prevent both the acquisition and persistence of HPV infection and the progression of CIN lesions.This study is aimed at evaluating the effects of local therapy with vaginal gel based on carboxy-methyl-beta-glucan on cervical epithelialization and the vaginal microbiota in patients with HPV infection or low-grade cervical preneoplastic lesion.Seven-hundred eighty-four women with positive HPV tests or diagnosed with CIN 1, referred to the Colposcopy and Cervical Pathology Center of our Department, were enrolled in a retrospective case-control study.Age between 18 and 60 yearsCaucasian originPositivity to HPV testing and/or positivity to CIN 1 at punch biopsyAbsence of contraindications to the proposed therapiesNegative medical history for any type of pharmacological treatment that acts on the elements in the current or recent study (<3 months)Negative medical history due to systemic pathology which can influence virus natural history, in particular immune disorders, and diabetesAll patients meeting the following enrollment criteria were selected:Age under 18 or over 60 yearsPositivity to biopsy for CIN 2+ lesionSuspected or diagnosed hypersensitivity/allergy to the components of the vaginal gelA positive medical history for each type of treatment that acts on the current or recent study outcomes (<3 months)States of specific deficiency of the immune systemThe exclusion criteria were as follows:number protocol: 260/18).At the time of recruitment, each person was informed about the aims and methods of the study with the fulfillment of a written informed consent. The study was approved by the ethical committee of the Federico II University .A targeted punch biopsy during colposcopy was obtained in case of the presence of ZTA .Group A: 392 women with positive HPV test or CIN 1 diagnosis who performed the treatment with vaginal gel-based carboxy-methyl-beta-glucan (Colpofix®: 1 application/day for 20 days per month for 3 months);Group B: 392 untreated women, as the control groupThe study population was then divided into 2 groups concerning the therapy performed during the follow-up period:In Group A, the aspects of compliance, treatment security, and the onset of side effects or allergic sensitization were also evaluated.β = 80% the sample size must be of at least 356 patients per group. Considering a 10% dropout (for lack of data completeness), it was decided to increase this value to 392 patients.The number of the sample under examination was calculated starting from the assumption that, as reported in the literature , about 6p < 0.05.SPSS software (version 20.0) was used for statistical analysis. The significance was set at a value of t-test for data with parametric distribution  and with the χ2 test for ordinal variables . The differences in the number of CIN lesions and HPV test positivity between the two groups were evaluated with the χ2 test. The measurement of the OR (odds ratio) was used to assess the risk of persistence of infection or lesions.The demographic and clinical data of the two groups were compared with the Student p = NS).At the end of the data analysis, 725 patients were found to meet the inclusion criteria and completed the follow-up as required by the protocol: 358 belonging to group A, 367 belonging to group B. The demographic and anamnestic variables of the subjects under study are reported in As regards the vaginal and tissue health outcomes, it was observed that the patients who performed the therapy, concerning the control group, had a significant improvement in the ectopia pattern Figures  and 2: aIn the experimental group, a significant improvement in the pH indices, a negative Swift test, and a resolution of the leucorrhoea were observed .No adverse effects were reported by patients in therapy .The analysis of risk factors for the persistence of infection or lesions  showed tMany factors have been reported regarding the risk of persistence of HPV infection and the progression of a CIN lesion. In recent years, many studies have shown that the stability and composition of the vaginal microbiome can influence viral clearance and probably also the progression of cervical preneoplastic lesions , 4–9.The treatment used is based on carboxy-methyl-beta-glucan, hydrophilic polymers capable of forming on the vaginal mucosa a mucoadhesive film that protects from external microbial agents and assists in maintaining and controlling the physiological conditions of the processing areas of the cervicovaginal mucosa by hydration. Carboxy-methyl-beta-glucan contributes to the maintenance and/or restoration of the vaginal microbiota through a prebiotic effect.Our data have shown how in vivo carboxy-methyl-beta-glucan in polycarbophil vaginal therapy can improve cervical epithelialization. Indeed, an improvement in the ectopia pattern was observed with a reduction in severe ectropion (>2/3) in the therapy group compared to the control group, with a corresponding increase in the rate and extension of metaplasia and an improvement in the uptake of the Lugol test with a reduction in the proportion of patients with a noncaptive test.The observed metaplasia and Lugol's test uptake were expressions of the repair process of ectopia with the conversion of a delicate tissue such as the cylindrical one into tissue more resistant to vaginal insults such as the multilayered squamous tissue.This repair probably involves a reduction in the rates of tissue inflammation and predisposition to the development of infection. Indeed, the therapy has been shown to improve the vaginal health of patients who presented clinical signs of bacterial or fungal vulvovaginitis at the first visit. The therapy induced a resolution of the leucorrhoea, a normalization of the vaginal pH, and a negativization of the Swift test in a more significant percentage of cases compared to the group not in therapy.n = 85/358) than in the control group .Our data demonstrated a regression rate at 6 months from the histological diagnosis of CIN 1 significantly higher in the therapy group . The effect probably depended on the ability of the medical device to promote greater stability of the vaginal environment.The presence of bacterial vaginosis (VB) has been associated with a delay in HPV clearance in patients with CIN, suggesting that the presence of a poor lactobacilli microbiota (community state type-IV-CST IV) may play a role in this persistence. It was also shown that greater diversity of the vaginal microbiota (CST-IV) is associated with greater severity of CIN lesions .2O2 seems protective in preventing the progression of dysplasia and ultimately the carcinogenic process. A greater prevalence of L. jensenii and L. coleohominis, both producers of H2O2, has been demonstrated in women with LSIL compared to HSIL. Furthermore, regardless of lactic acid concentrations, Lactobacilli spp. is capable of being cytotoxic when cultured in vitro against cervical cancer cells, but not in normal cells, highlighting an even more complex interaction between cervical cells, the microbiota, and the vaginal environment [The presence of lactobacilli-producing Hironment .This study has shown that correcting the vaginal microbiota and favoring cervical epithelialization with a specific therapy favors CIN 1 lesion regression.Our data confirmed that the presence of leucorrhoea, a positive Swift test, and a basic vaginal pH correlate with an at least doubled risk of persistence of CIN lesion at the cervical level.Active and passive cigarette smoking is one of the major risk factors in the development of CIN in the presence of HPV infection . Min et Alcohol consumption is known as a potential risk factor in acquiring HPV infection, although the data on the risk of persistence of lesions, the presence of cervical cancer, and alcohol consumption are few and controversial –19. In oThe high number of patients, the execution of diagnostic tests , and the short follow-up times (6 months) represented the strengths of this study.The choice of enrolling only patients with reassessment time at 6 months from the first visit has allowed to minimize the variations of the vaginal environment induced by other environmental factors and to exclude that the observed improvement of colposcopic pictures and histological findings may be linked to the natural history of HPV infection. The choice of enrolling only Caucasian patients in the study erases the impact of ethnicity on the risk of CIN and cervical microbiota differences in other populations. Indeed, it has been demonstrated that the overall HPV prevalence rate of migrant women is four times higher than the overall prevalence observed among Italian women .The limitations of the study were its retrospective nature and the lack of randomization with a placebo group.In conclusion, vaginal therapy based on carboxy-methyl-beta-glucan turned out to improve overall vaginal health with a positive impact on the risk of persistence and progression of low-grade cervical lesions of the uterine cervix.


A growing proportion of head and neck cancers (HNC) result from HPV infection. Between HNC aetiological groups (HPV positive and HPV negative) clinical evidence demonstrates significantly better treatment response among HPV positive cancers. Cancer stem cells (CSCs) are identified in HNC tumour populations as agents of treatment resistance and a target for tumour control. This study examines dynamic responses in populations of a CSC phenotype in HNC cell lines following X-irradiation at therapeutic levels, and comparing between HPV statuses. Variations in CSC density between HPV groups showed no correlation with better clinical outcomes seen in the HPV positive status. CSC populations in HPV positive cell lines ranged from 1.9 to 4.8%, and 2.6 to 9.9% for HPV negative. Following 4 Gy X- irradiation however, HPV negative cell lines demonstrated more frequent and significantly greater escalation in CSC proportions, being 3-fold that of the HPV positive group at 72 hours post irradiation. CSC proportions of tumour populations are not fixed but subject to change in response to radiation at therapeutic dose levels. These findings imply a potential effect of aetiology on radio-responsiveness in CSCs, illustrating that clonogen treatment response may be more informative of therapy outcomes than inherent population density alone. Two principal aetiologies of HNSCC are those cancers associated with agents such as tobacco and alcohol and those resulting from the human papillomavirus (HPV) infection, which displays an anatomical preference for the base of tongue and tonsils, resulting in oropharyngeal squamous cell carcinoma (OPSCC)3. The most important distinction between these aetiologies is the significantly better treatment responses shown by HPV positive HNSCC, where the 5-year overall survival rate is around 80% compared to 50% for HPV negative HNSCC5. Moves toward more individualised treatment for HNSCC patients in terms of HPV status are hampered however, largely because the mechanisms and predictability of different treatment sensitivities according to HPV status remaining poorly understood8.Head and neck cancers are invasive carcinomas of the mucosal epithelium involving the complex anatomy of this area and frequently requiring radiotherapy as part of an approach aimed at tumour control whilst conserving normal tissue function. Globally, around 3.8% of cancers are head and neck cancers of which approximately 95% are squamous cell carcinomas (HNSCC)9. They demonstrate changes in divisional dynamics that can accelerate tumour re-population and are the numerical beneficiaries of tumour plasticity whereby de-differentiation of non-CSCs further increases the CSC population14. Consequently, therapy needs to account for CSC elimination to achieve tumour control16. CSCs have been identified in HNSCC tumour populations by several cellular markers tested by serial xenotransplantation of which CD44 and aldehyde dehydrogenase (ALDH) are found to be markers of stemness in both HPV statues21. Density of tumour CSC populations is recognised as a strong prognostic marker in a range of cancers22 but evidence of CSC density in HNSCC, by HPV status, is conflicting and fails to correlate with the better treatment outcomes of the HPV positive group25. These mixed results strongly imply additional factors are involved in CSC treatment response among HNSCC that result from HPV infection, and the prognostic interpretation of tumour CSC density may depend on HNSCC aetiology. Greater radiosensitivity and limited DNA repair following irradiation has been demonstrated in HPV positive tumours in pre-clinical studies28. Differences in the response to treatment according to HPV status in HNSCC has been widely researched, but data on the role played by CSCs, in terms of their HPV status, is scarce. What role CSCs play, as a result of HPV status, in response to treatment through radioresistance or the plasticity of tumour populations to replace them, remains unclear.Cancer stem cells (CSCs) are a small but critical subset within tumour cell populations, having unlimited capacity for self-renewal and generation of differentiated progeny. CSCs are mediators of metastases and recurrence following therapy and the most treatment resistant of tumour cellsin vitro study was on the quantitative evaluation of cell line dynamics during fractionated radiotherapy among HPV positive and negative aetiologies alike. Our previous work in this area, as a pilot study, examined only one cell line each HPV status, as such being insufficient to draw comparison between aetiologies, but suggestive of need for further investigation29. The current work comprises of data from 6 cell lines and provides valuable additional information, enabling not only qualitative but also quantitative comparison between HNSCC aetiologies by an examination of dynamic responses among CSCs to therapeutic levels of radiation in terms of their HPV status. While general understanding of HPV positive and negative responses are known, dynamic quantitative data for up to 6 cell lines has not been reported in literature so far30. This study therefore progresses current knowledge from generic toward specific where findings demonstrate significant differences in CSC population response to X-ray irradiation between the HPV groups, implying greater repopulating ability in HPV negative HNSCC.The aim of this study has been to investigate behavioural responses of CSCs to irradiation, in terms of their HPV status, measuring inherent CSC proportions of HNSCC cell lines and changes in CSC populations in response to irradiation as a function of HPV status. The key focus of this in vitro experimental study measured a) the inherent proportions of the CSC phenotype CD44+/ALDH + in HNSCC cell lines, b) changes in CSC proportions of population over time following 4 Gy X-ray dose, c) parallel assays to compare cell population response in T25 and 6 well plates .This 31. Cells were cultured in T25 flasks using Dulbecco’s Modified Eagle’s Medium with 4500 mg/L glucose,  supplemented with with 10 mM HEPES, 10% foetal bovine serum (FBS), 100 U/mL penicillin and with 0.1 mg/mL streptomycin . Cells were incubated in humidified atmosphere of 5% CO2 at 37 °C. Cell lines were tested for mycoplasma used MycoAlert detection kit  and were negative.Six HNSCC cell lines were selected, 3 of each HPV status. UM-SCC-1, UM-SCC-17a, UM-SCC-22a and UM-SCC-47 were purchased through Merck Millipore . UPCI-SCC-090 (ATCC CRL-3239TM) and UPCI-SCC-154 (ATCC CRL-3241TM), both HPV positive, were purchased from ATCC (Manassas VA). UM-SCC-1, UM-SCC-17a and UM-SCC-22a are HPV negative and UM-SCC-47, UPCI-SCC-090 and UPCI-SCC-154 are HPV positive  to achieve full scatter conditions. Flasks were filled with media (21 mm depth over the cell monolayer) to achieve electronic equilibrium at the cell layer. One T25 flask of each cell line, for each analysis time point  was irradiated with a 4 Gy dose. 4 Gy was chosen as the treatment dose as it is low enough to be at the upper end of what a single fractionated dose might be in therapy, and sufficiently high to produce cell killing and a clear response among survivors. Matching controls for each flask were sham irradiated. Media was changed post irradiation and flasks for assay were incubated with cells remaining in situ until analysis.Cell cultures were irradiated in T25 flasks using a 160 kV, 25 mA, RS2000 X-ray cabinet irradiator  calibrated using the AAPM TG61 protocolCSCs were putatively identified in the 6 HNSCC cell lines by 2 cellular markers: cluster of differentiation 44 (CD44), a cell surface protein and adhesion molecule, and aldehyde dehydrogenase (ALDH), a metabolic enzyme. CD44 expression was determined by anti-mouse/human CD44 antibody, clone IM7, fluorophore APC . ALDH expression was measured using Aldefluor . Gating for CD44 was based on the APC isotype control and diethylaminobenzaldehyde (DEAB) was used for gating of ALDH expression Fig. . Cells wBaseline proportions of CSC populations were established through concurrent flow cytometry of the 6 unirradiated cell lines for their CD44+/ALDH + population. Measurements of CSC proportions in unirradiated and irradiated cell line populations were conducted 7 times at 24, 48 and 72 hours following a 4 Gy X-ray dose. Time points at 1, 2 and 3 days were chosen as they reflect normal intervals between radiotherapy fractions during which CSC density may increase or potentially re-establish populations.in situ following irradiation until analysis, parallel assays were conducted to test for significance of any difference in results. For comparison, cells in T75 flasks, also under 21 mm of media and associated sham irradiated controls, were irradiated with 4 Gy then detached using TrypLE Select  centrifuged and re-plated in 6 well plates until concurrent analysis with cells from T25 flasks.To test for associative effect on CSC proportions as a result of cells being contained Flow cytometry data was analysed in Microsoft Excel and Prism v7.01 (GraphPad Software Inc. La Jolla CA). Data from repeated cell line analysis (n = 7) was reported as mean and SEM for each cell line. Multiple t-tests were used to determine significance in CSC population change following 4 Gy dose. Two way ANOVA and Sidak’s multiple comparisons were used to test significance for difference in ratio of response. Unpaired t-tests were used to compare CSC data for control cells of T25 versus 6 well plate methods and irradiated cells. Significance considered to be p < 0.05 .Proportions of putative CSCs in untreated cultures of 6 HNSCC lines were determined by staining for CD44 and ALDH. The proportions of CSC varied between 2% to 10% of the total cell population. Large variations in CSC density was observed among the cell lines, irrespective of HPV status  at 72 hours following irradiation  and another CSC, representing self-renewal. But in response to tumour volume loss from irradiation, cellular signalling may switch the CSC divisional process to a symmetrical one where both daughter cells are CSCs, rapidly increasing their proportion in the tumour population and thus their potential for proliferation37. Furthermore, plasticity among progenitor and differentiated cells, in response to irradiation, can increase CSC populations by dedifferentiation where these cells acquire the CSC phenotype and the capacity for self-renewal13. Observed increases in CSC proportions demonstrate that CSC numbers are not static but may be responsive to therapeutic levels of irradiation where a significantly greater effect was observed in the HPV negative group. This observed difference suggests a potential difference in radio-responsiveness among HNSCCs depending on aetiology, and warrants further investigation. The fact that recurrent primary HNSCC tumours are the most common cause of mortality points to the importance of our finding that HPV status seems to have differential effects on the relative proportions of putative CSCs among the entire cell population of HNSCCs after a single 4 Gy dose of X-irradiation and thus may also affect the relative repopulating ability of CSCs according to their HPV status38.To test for differences in the radio-responsiveness of the CD44+/ALDH + phenotype of HPV positive versus negative HNSCC cells, cell cultures were irradiated with a 4 Gy dose and analysed for changes in the proportion of this phenotype over intervals of 24, 48 and 72 hours, reflecting time intervals between fractionated doses in external radiotherapy. Resulting data is novel in that it provides a quantitative comparison of dynamic population responses among HSCC CSCs, by their HPV status, at therapeutically relevant dose and time points following irradiation. Among irradiated cell cultures, only 1 of the 6 cell lines (UM-SCC-22a) showed any increase in CSC density at 24 hours. Although CSC density increased among the HPV positive lines, UPCI-SCC-154 and UPCI-SCC-090, the HPV negative populations of HNSCC cells more often had significantly greater increases in the proportion of CSCs during the 3 days after irradiation. To compare how differences in HPV status might affect the overall extent to which CSC density responded at 24, 38, and 72 hours after irradiation, we calculated the ratio of the proportions of putative CSCs post-treatment to baseline (untreated) cell line populations, at each time point for either the HPV positive or the HPV negative HNSCC cell lines. We found that this ratio increased steadily during the 3 days after irradiation so that the proportional change in CSC density of the irradiated HPV negative HNSCC cells was significantly greater than that of the irradiated HPV positive cells by day 3. The increased proportions of CSCs over the 3 days may indicate that several different biological processes are at work including preferential survival, altered divisional dynamics and/or cellular plasticity by de-differentiation. The danger of clonogen-driven acceleration in tumour repopulation, in response to sublethal or delayed doses of radiation, has been earlier described by Withers, in situ in T25 flasks demonstrated less range of error than the 6 well plate method and was felt to introduce fewer confounding effect from cell handling.Comparison of protocols (T25 flask vs 6 well plate) for potential effects on CSC counts, found one significant difference in results from these methods among the 6 comparisons of means. This may be indicative of the extent to which methods can influence this data and needs to be considered in the interpretation of results. The measurement of CSCs from cells remaining inherent CSC densities among the investigated HNSCC cell line populations did not associate with HPV status;the study compared changes in CSC densities within the cell line population, by HPV status, in response to radiation;quantitative differences in CSC populations were shown using 6 cells lines, even amongst HPVpositive and negative sub-groups, based on their origin;the behavioural response of putative CSCs among the 6 cell lines investigated demonstrated elevations in CSC densities following a 4 Gy dose that were significantly greater in the HPV negative group, potentially indicating stronger repopulating ability;in vitro, in vivo and in silico (modelling studies) that require specific quantitative inputs.the dynamic quantitative data obtained for the 6 cell lines facillitates progress of knowledge from generic to specific, as only qualitative studies have been reported so far in the literature; this information is important for future There is very little quantitative data in the scientific literature on the cellular dynamics of cancer stem cells originating from HNSCC, in response to therapeutic levels of X-irradiation, which expressly compares CSCs by HPV status. This work provides additional insight using data from 6 cell lines (3 HPV positive and 3 HPV negative), thus enabling for the first time quantitative comparison between HNSCC aetiologies by analysis of dynamic responses among CSCs to therapeutic levels of radiation in terms of their HPV status. The main highlights of the current work are as follows:Data on responsiveness of HNSCC clonogens to therapeutic levels of radiation, comparing HPV statuses, is of vital interest in finding better clinical applications. Given the number of clinical trials investigating dose de-escalation for HPV positive HNSCC, quantification of intrinsic capacity for response to treatments by CSCs in both HPV statuses is of critical concern and value to informing approaches toward de-escalation.The differential response of CSC densities to therapeutic levels of X-ray dose, according to HPV status, warrants further studies. Apoptotic studies and FACS analysis, to measure preferential survival against proliferative of differential changes in phenotype proportions are required to measure their influence in resulting phenotype proportions over time following irradiation. Testing of intrinsic radiosensitivity by proliferative capacity following fractionated irradiation, using clonogenic assays of cell line populations, needs to be part of future work determine dynamic response to repeated dose. Any changes in cell survival, as a function of dose and HPV status, following repeated 4 Gy dose would provide data on changes to intrinsic radiosensitivity reflective of the therapeutic use of radiation.