Using lasso-regression, a 24-miRNA signature predicting HPV-status was built in a multicentre cohort and validated in a single-centre cohort. Its combination with p16/HPV DNA status improved clinically relevant risk stratification, allowed the identification of an HPV-associated patient subgroup with impaired overall survival, and might be considered for future clinical decision-making. miRNA-transcriptome integration identified HPV-specific signaling pathways.Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC), regarded as a distinct clinical entity, are characterized by a considerably favourable prognosis after radio(chemo)therapy and a not yet fully understood distinct molecular pathogenesis. We aimed to develop a miRNA-signature that identifies HPV-associated HNSCC according to their specific molecular pathogenesis, and to characterise the transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD−L1 expression/PD−1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG  High-risk human papillomavirus (HPV) driven head and neck squamous cell carcinoma (HNSCC), regarded as a distinct clinical entity, is characterized by a considerably favourable prognosis after radio(chemo)therapy and a not yet fully understood distinct molecular pathogenesis ,2,3,4. Tn = 229) of a multicentre and a single centre cohort, both comprising patients treated by adjuvant radio(chemo)therapy. We aimed to develop a miRNA signature that identifies HPV-positive HNSCCs according to their specific molecular pathogenesis, and to characterise the transcriptome compared to HPV-negative HNSCCs.Here, applying comprehensive miRNA profiling, we characterized HPV-positive and HPV-negative HNSCC specimens  and then = 101; ,18). AllThe DKTK-ROG cohort originally comprised 221 HNSCC patients who were treated at one of eight different DKTK partner locations . InclusiThe LMU-KKG cohort included all HNSCC patients treated with adjuvant radio(chemo)therapy at the Department of Radiation Oncology, LMU, Germany, between 06/2008 and 01/2013 with available tumour tissue specimens . A mediaClassification of Malignant Tumours, 7th edition.Tumour stage was assessed using the UICC TNM Median time from diagnosis to the first day of radiotherapy treatment was 66 days (IQR 52–80) for the DKTK-ROG and 68 days (IQR 55–83) for the LMU-KKG cohort. Median time from surgical tumour resection to the first day of radiotherapy treatment was 46 days (IQR 37–54) for the DKTK-ROG and 42 days (IQR 34–51) for the LMU-KKG cohort.Formalin-fixed paraffin-embedded tumour specimens were derived from treatment-naive surgically resected tumour tissues. Haematoxylin-eosin-stained tissue sections were histopathologically reviewed by a pathologist . The tumour area was defined and micro-dissected, whereby only samples with at least 60% tumour cells were included. Total RNA including the small RNA fraction was extracted using the Qiagen miRNeasy FFPE (DKTK-ROG) or the AllPrep DNA/RNA FFPE Kit (LMU-KKG) according to the manufacturer’s protocols .INK4a immunohistochemistry (IHC) in combination with HPV DNA detection as described previously [INK4a staining was performed using the CINtecTM Histology Kit  on a Ventana Benchmark LT automated immunostainer  according to the manufacturer’s protocol. As positive and negative controls, FFPE sections from embedded p16-positive (UPCI SCC154) and p16-negative HNSCC cell lines  were used. Tumour specimens with strong and diffuse nuclear and cytoplasmic staining in more than 70% of tumour cells were classified as p16-positive, while tissue with only weak diffuse or absent staining was considered p16-negative [INK4a-stained tissue sections was performed by two independent observers. Detection of mucosotropic HPV DNA was performed by quantitative real-time PCR (q-PCR) in combination with SYBR green chemistry was subjected to q-PCR reactions (10 µL) on a ViiA 7 q-PCR system  using GP5+/6+ primers detecting the L1 gene   [p16/HPV DNA status of LMU-KKG specimens was determined by p16eviously . p16INK4negative . Evaluat 142 bp) . The b-g 142 bp) . Two HPVINK4a IHC and HPV DNA status assessed by GP 5+/6+ q-PCR  [A tumour specimen was finally classified as HPV-positive if it was positive for both p16., 2007) .Whole RNA sequencing on LMU-KKG tumour specimens was performed as described previously  and in tINK4a IHC  and HPV DNA status assessed using the LCD-Array HPV 3.5 kit , as described previously [p16 and HPV DNA status for DKTK-ROG specimens was provided by the DKTK-ROG consortium [HPV16 E6/E7 RNA status for DKTK-ROG specimens was provided by the DKTK-ROG consortium ,28. HPV1miRNA-microarray profiling using SurePrint G3 8x60K Human miRNA Microarrays  was performed as described previously . ScannedDifferentially expressed miRNA between HPV-positive and HPV-negative tumours (DKTK-ROG and LMU-KKG combined) were determined using the Limma approach . Signifip-value < 0.05 in univariate association testing (Mann-Whitney test) were used as priors in feature selection by penalized lasso regression. Overfitting was addressed by 100 repeated 8-fold cross-validation using ROC-AUC as the summary metric for model optimization, while lambda was varied per iteration between 0.001 and 0.3 in steps of 0.001. The miRNA expression matrix of the DKTK-ROG cohort (n = 128) was used as a training data set with p16/HPV DNA status as ground truth. The prediction performance of the model was tested in the LMU-KKG miRNA-expression data set (n = 101). To assign patients of the discovery and validation sets to the HPV-negative and HPV-positive groups, the logit function was applied on the scalar product of signature-miRNA expressions and the appropriate model coefficients for the calculation of the prediction probability score per patient. Tumours were predicted as HPV-positive if the prediction probability score was >0.5, and as HPV-negative if ≤0.5. For the assessment of model performance, prediction accuracy, sensitivity, specificity, and ROC-AUC were used. All machine learning and performance assessment steps were performed using the caret R-package.We built a generalized-linear model with HPV status (ground truth: p16/HPV DNA status) as the response variable, and a signature of miRNAs as explanatory variables. miRNAs with a Overall survival was calculated from the start date of radiotherapy treatment to the date of death from any cause, locoregional control from the date of radiotherapy treatment start to the date of locoregional recurrence, and freedom from recurrence from the date of radiotherapy treatment start to the date of locoregional or distant recurrence. In the absence of an event, patients were censored at the date of the last follow-up visit (or the date of death).p-values < 0.05 were considered statistically significant. Hazard ratios (HR) with 95% confidence interval (CI) were reported.Ground truth (p16/HPV DNA) and predicted HPV-status were tested for prognostic significance in the combined DKTK-ROG and LMU-KKG cohort for clinical endpoints overall survival, locoregional control and freedom from recurrence. A univariable Cox proportional-hazard model was fitted using the survival function as the response variable and the binary HPV-status as the explanatory variable. Log-rank test Whole RNA sequencing on LMU-KKG tumour specimens was performed as described previously : RNA intn = 30 LMU-KKG tumour specimens were included into the analyses: (1) n = 15 p16/HPV DNA-positive and 24-miRNA signature positive cases with available RNA-seq data and the top 15 highest prediction values; (2) n = 15 p16/HPV DNA-negative and 24-miRNA signature negative cases with available RNA-seq data and the top 15 lowest prediction values : Differentially expressed genes between HPV-positive and HPV-negative tumours were determined by the approach as implemented in the DESeq2 R package while significance was accepted for |log2-fold change > 0.5| and FDR < 0.1 .p-values < 0.05 [The activity of 14 cancer-associated signaling pathways was inferred from the transcriptome data using the Bioconductor R package Progeny . The z-ss < 0.05 .To characterize the functional impact of the identified miRNA-signature in HPV-positive and HPV-negative tumours, we integrated the signature miRNA-expression with the mRNA-expression data of the appropriate groups.http://www.gsea-msigdb.org, accessed on 17 January 2022) using the enrichKEGG function of the clusterProfiler R package. Enrichments were considered statistically significant if resulting false-discovery controlled p-values were <0.05.Possible signature-miRNA-mRNA interactions revealed by seed sequence-based prediction were downloaded from DIANA, Miranda, PicTar and TargetScan using miRNAtap R function, as implemented in the SpidermiRdownload_miRNAprediction function of the SpidermiR R package (v1.26.0) . The obtn = 128; n = 38 HPV-positive, n = 90 HPV-negative) and 5.2 years (IQR 4.0–6.3) for the single-centre LMU-KKG  cohorts. All patients were treated by adjuvant radio(chemo)therapy. HPV-positive cases were mainly oropharyngeal tumours  in HPV-positive (n = 61) versus HPV-negative tumours (n = 168), with 37 down-regulated and 18 up-regulated miRNAs , locoregional control, and freedom from recurrence , a trend towards poorer OS  was seen for cases (n = 10) predicted HPV-negative by the 24-miRNA signature compared to HPV-positive predicted cases . On the RNA expression level, 6/10 were E6/E7 HPV positive , and n = 3 HPV16 E6/E7 negative DKTK-ROG cases might have involved another HPV subtype other than HPV16 , 24-miRNA-positive  did not differ regarding OS , with 452 down-regulated and 206 up-regulated genes in 24-miRNA-/HPV-positive versus 24-miRNA-/HPV-negative tumours (MAP kinase, Estrogen, EGFR, TGFbeta and WNT signaling activity was significantly decreased in HPV-positive compared to HPV-negative tumours (To obtain insights into the biological regulatory function of the signature miRNAs, RNA-seq analysis was performed, including ectively . n = 658 tumours A. MAP ki tumours B.In a next step, miRNA-mRNA interactions calculated separately for 24-miRNA-/HPV-positive and 24-miRNA-/HPV-negative tumours. were subjected to KEGG pathway enrichment analysis. Most significantly enriched pathways were overlapping in the HPV-positive and HPV-negative groups. PD−L1 expression and PD−1 checkpoint pathway in cancer, autophagy was significantly enriched exclusively in HPV-positive and TNF signaling pathway, cellular senescence, focal adhesion, EGFR tyrosine kinase inhibitor resistance exclusively in HPV-negative tumours .MAP kinase, Estrogen, EGFR, TGFbeta and WNT signaling pathway; In addition, genes likely to be regulated by the signature miRNAs and belonging to the signaling pathways that showed differential activity between HPV-positive and HPV-negative cases (INK4A and HPV DNA-positive were considered HPV-positive [Here, we report a 24-miRNA signature that predicts HPV-status in HNSCC. The signature was identified in a multicentre miRNA dataset and validated in an independent single-centre dataset. In a simulation study, this strategy was shown to minimize the prediction error, leading to an increased chance for generalizability of the prediction model . Both HNpositive . We delipositive .In the clinic, HPV-positive tumours are considered as a distinct entity, which is also reflected at different molecular levels including the miRNA level ,2. PreviIn HPV-negative HNSCC, we did not detect differences in overall survival between subgroups defined by the 24-miRNA signature in this study, as was the case in a previous study of HPV-negative HNSCC in which we identified a 5-miRNA signature predictive of recurrence and survival and identified HPV-negative HNSCC subgroups with different prognosis .TGFbeta signaling activity has been shown to be decreased in HPV-positive HNSCC as a result of HPV infection, increasing sensitivity to radiation and chemotherapy by homologous recombination repair deficiency [WNT signaling activation was suggested for HPV-negative patients consuming tobacco [EGFR tyrosine kinase inhibitor resistance. The clinical benefit of the approved EGFR targeting monoclonal antibody Cetuximab is limited; with advanced disease status, resistance development and pleiotropic cellular functions of the EGFR pathway playing a role [EGFR signaling pathway activity observed in 24-miRNA-/HPV-positive patients in our study might be a contributing factor to the unexpected trial results. The EGFR signaling pathway is a complex network that largely overlaps with other signaling pathways. For instance, EGFR can activate the MAPK signaling pathway, for which we have also demonstrated higher activity in 24-miRNA signature-/HPV-negative HNSCC. miRNA-mRNA interactions identified a number of commonly enriched pathways, as well as pathways exclusively associated with 24-miRNA signature-/HPV-negative HNSCC: the TNF signaling pathway, for which a promoting effect on lymph angiogenesis in HNSCC was suggested [PD-L1 expression and PD-1 checkpoint pathway in cancer. Increased PD-L1 expression and an immunologically active tumour microenvironment has been demonstrated for HPV-positive HNSCC, suggesting immunotherapy as a promising treatment option [miRNAs act as important post-transcriptional integrative regulators and interfere, due to the high promiscuity of a single miRNA to bind multiple mRNAs, with multiple signaling pathways, making it difficult to infer clear biological functions. However, using the transcriptome data for the analysis of differential signaling activity and integration with miRNA data in HPV-positive and HPV-negative tumours, we gained insights into the biological regulatory function of the 24-miRNA signature. Our findings are further strengthened by the fact that the functional assignment of the signature miRNAs suggests an important post-transcriptional integrative role in signaling pathways frequently deregulated in HNSCC, which have also been associated with the differential molecular biology of HPV-positive and HPV-negative tumours. As such, ficiency . Stronge tobacco , also sug a role . Furtherg a role . The siguggested ; cellulauggested ,42; and uggested . In addit option ,45. Whilt option ,47. OverIn conclusion, the 24-miRNA signature accurately predicts HPV-status and, within HPV-positive HNSCC, even better reflects prognosis and molecular biology compared to HPV-typing using p16/HPV DNA status alone. Future clinical decision-making on treatment de-escalation in HPV-positive HNSCC might consider 24-miRNA signature classification combined with p16/HPV DNA status. Specifically, deregulated signaling pathways in HPV-positive or HPV-negative HNSCC identified by 24-miRNA signature-mRNA integration enable a deeper molecular understanding of both tumour entities. Moreover, the functional assignment of miRNAs suggests an important post-transcriptional integrative role in key deregulated pathways in HNSCC.


Exciplexes possessing thermally activated delayed fluorescence (TADF) characteristics have received much attention in the fields of organic light-emitting materials and devices over the past decade. In general, an exciplex is a physical mixture between a donor (D) with hole transport properties and an acceptor (A) with electron transport characteristics, and the energy difference between the lowest excited singlet state and the lowest excited triplet state is usually fairly small in terms of the long-range charge-transfer process from D to A. In the processes of photoluminescence and electroluminescence, triplet excitons can be converted to singlet excitons through reverse intersystem crossing and then radiate photons to achieve TADF. As a consequence, triplet excitons can be effectively harvested, and the exciton utilization can be significantly enhanced. Up to now, a large number of exciplexes have been developed and applied to organic light-emitting devices. Notably most of them showed green or red emission, while blue exciplexes are relatively few owing to the spectrum characteristics of the large red-shift and broadened emission. In this study, the latest progress of blue exciplex–based organic light-emitting materials and devices is briefly reviewed, and future research is prospected. Among tof <10 V , phosphoof <10 V , tripletof <10 V , traditiof <10 V , hyperflof <10 V , singletof <10 V , excipleof <10 V , aggregaof <10 V , and mulof <10 V  have beeof <10 V . Neverthof <10 V  or highloll-offs .EST) between the lowest excited singlet state (S1) and the lowest excited triplet state (T1) by means of the long-range CT process from D to A –excited state between the highest occupied molecular orbital of an electron donor (D) and the lowest unoccupied molecular orbital of an electron acceptor (A), leading to a fairly small energy difference (Δm D to A . In the m D to A . Up to nm D to A . This ism D to A . In thism-MTDATA):tris-[3-(3-pyridyl)mesityl]borane (3TPYMB) exciplex system, which showed a relatively high maximum external quantum efficiency (EQEmax) of 5.4% in view of a rather low photoluminescence quantum efficiency (PLQE) of 26%, exceeding the corresponding limit of fluorescence-based OLEDs (max of 10.0% and a maximum power efficiency (PEmax) of 47.0 lm W−1 were achieved by changing the exciplex system from m-MTDATA:3TPYMB to m-MTDATA:2,8-bis(diphenylphosphoryl)dibenzo thiophene (PPT)  .λEL = 471 nm) with an EQEmax of 8.0% based on 1,3-bis(N-carbazolyl)benzene (mCP):2,4,6-tris[3-(1H-pyrazol-1-yl)phenyl]-1,3,5-triazine (PO-T2T) (N-carbazolyl)triphenylamine (TCTA):4,7-di-phenyl-1,10-phenanthroline (Bphen), which displayed a low EQEmax of 2.65%. H-benzimidazol-2-yl)benzene (TPBi), while a blue-emitting device containing TPAPB:TPBi exhibited a low Von of 3.2 V, a high EQEmax of 7.0 ± 0.4%, and CIE coordinates of . Liu and coworkers reported an efficient blue exciplex emitter consisting of 4,4′-bis(9-carbazolyl)-2,2′-dimethylbiphenyl (CDBP):PO-T2T, which showed obvious TADF emission and intrinsically high T1, making itself an excellent candidate as a blue emitter or a host for green and red phosphors (H-carbazole (mCBP) and PO-T2T as the electron donor and acceptor, respectively, and a high EQEmax of 7.66% with CIE coordinates of  were realized. max of 7.8% with blue emission peaked at 488 nm. Based on the acceptors xTPOTZ , which are triphenyltrazine derivatives substituted with diphenylphosphine oxide groups at ortho-, meta-, and para-positions, respectively . Thus, in virtue of the strongest electron-withdrawing effect and appropriate steric hindrance, an efficient sky-blue OLED based on mCP:pTPOTZ realized an ultralow Von of 2.5 eV, a high CEmax up to 26.2 cd A−1, a high PEmax of 32.4 lm W−1, and an EQEmax of 11.1%. H-carbazol-3-yl)silane (dCzPSi) and 3,3′-sulfonylbis(9-phenyl-9H-carbazole) (dCzPSO2). The electron-transporting acceptor, PO-T2T, was introduced to give two exciplex-forming systems, dCzPSi:PO-T2T and dCzPSO2:PO-T2T. The dCzPSi:PO-T2T–based device revealed a Von as low as 2.4 V and a high EQEmax of 8.6% with the CIE coordinates of , significantly higher than that of the device based on dCzPSO2:PO-T2T (EQEmax = 1.8%), which is due to the fact that dCzPSO2:PO-T2T possessing a large ΔEST is unfavorable in forming a CT complex.Since then, exciplex-based emitters have aroused widespread attention by virtue of their fascinating optoelectronic properties, and a number of efficient blue exciplex–based OLEDs have been realized . In 2014(PO-T2T) . In 2015hosphors . Meanwhiectively , Duan etmax of 6.1%, 16.0%, and 6.5%, respectively. N,N′-bis(3-methylphenyl)-N,N′-diphenylbenzidine (TPD):Bphen, which exhibited a relatively low EQEmax of 0.46%, possibly resulting from the low PLQE of the exciplex. max of 9.1% and deep-blue emission with λEL = 433 nm. max of 10.2% along with a low roll-off.In 2019, Chapran and coworkers investigated the exciplex properties by selecting PO-T2T as an electron acceptor with different electron donors, and the emissions of these exciplexes spanned from blue to orange-red regions . The bluvia a rigid linker, the electronic coupling between the donor and acceptor units is sufficient to allow for efficient direct absorption by the CT state. In contrast to more flexible or less strongly coupled samples, the three rigid sky-blue exciplex emitters, 1′-[4-phenyl]-10-phenyl-10H-spiro (DM-B), 1′-[3-phenyl]-10-phenyl-10H-spiro (DM-Bm) and 1′--10-phenyl-10H-spiro (DM-G) possess very high PLQEs of >90% when incorporated in a solid matrix. The sky-blue OLEDs based on DM-B achieved a fairly high EQEmax of 27.4% along with a small efficiency roll-off. H-spiro (2tDMG) and 2,7-di-tert-butyl-10-[4-(tert-butyl)phenyl]-1′-[4-phenyl]-10H-spiro (3tDMG). 2tDMG and 3tDMG achieved extremely high EQEmax of 30.8% (evaporation-process) and 20.2% (solution-process), respectively. These excellent results opened new avenues for the study of spatial electronic interactions in organic light-emitting materials. max of 18.9%. Wang and coworkers reported the design of π-stacked dendrimers consisting of cofacially aligned D and A for highly efficient OLEDs, and the dendritic structure and orthogonal configuration led to the TSCT emission  are an attractive class of emitters with spatial D/A architecture and TADF characteristics . For intemission . Thus, tVon and high efficiencies  using a blue exciplex cohost of mCP:bis-4,6--2-methylpyrimi-dine (B3PYMPM) and a phosphorescent emitter of iridium (III)bispicolinate (FIrpic), and meanwhile, the OLED exhibited a low Von of 3 V and low-efficiency roll-off. In 2015, Ban and coworkers designed and synthesized a novel electron-transporting molecule bis(diphenylphosphine oxide) (POPH), and the solution-processed blue PhOLED incorporating a blue exciplex–based cohost TCTA:POPH displayed an extremely low Von of 2.7 V, a high PEmax of 22.5 lm W−1, and a very low-efficiency roll-off even the luminance was up to 10,000 cd m−1  ,which is lower than the T1 levels of the consisting molecules of mCP (2.94 eV) and PO-T2T (2.99 eV), and higher than that of FIrpic (2.63 eV), so that the exciplex system well confines the excitons in the exciplex state, followed by energy transfer to a blue dopant of FIrpic. In 2016, Ban et al. realized a highly efficient blue PhOLED based on a blue exciplex cohost system of TCTA:1,3,5-tris(diphenylphosphoryl)benzene (TPO) with the CEmax of 23.8 cd A−1 and PEmax of 15.8 lm W−1 (H-benzo [d]imidazol-2-yl)benzene (TPOB) to form an exciplex-type cohost with TCTA, and the solution-processed blue PhOLED achieved an extremely low Von of 2.8 V and a high PEmax of 22 lm W−1 along with a low-efficiency roll-off . Yun and coworkers designed n-type molecules with isomeric molecular structure, while the corresponding exciplex cohosts formed by mCBP:3-phenyl]-1,3,5-triazin-2-yl)benzonitrile (mSiTrz-mCN) showed blue emission . Afterward, Yun et al. developed a bipolar n-type host material, 9-phenyl]-1,3,5-triazin-2-yl)-9H-carbazole-3-carbonitrile (mSiTrzCzCN), and the blue-emitting mCBP:mSiTrzCzCN exciplex system showed a high T1 energy close to 3.0 eV (max of 21.8% and a lifetime elongation of more than double relative to the conventional n-type host-based device. Kim et al. developed three n-type hosts to form blue exciplex with mCBP (1 of 2.95 eV and the fabricated blue PhOLED showed a rather high EQEmax over 23%.In 2014, 0 cd m−1 . Lee et 8 lm W−1 . Afterwaroll-off . Lim et emission . The deeo 3.0 eV . The mCBith mCBP . Among tIn summary, this study provided an overview of blue exciplex–based organic light-emitting materials and devices. The research background and luminescence mechanism were briefly introduced. Benefiting from the intriguing merits of exciplex-based OLEDs including low-driving voltages and low-efficiency roll-offs, simultaneously, as well as simple device structures, exciplexes have drawn significant attention on account of the potentials for efficient electroluminescence or for the use as high-performance cohost materials. Manipulating blue exciplex emissions by adjusting molecular structures gives an ideal strategy to fully utilize all exciton energies for high-performance OLEDs. We believe that our work will be conductive to the future development of blue exciplex–based OLEDs with high efficiencies and simplified device structures. Meanwhile, we expect that further systematic investigations of the excited-state dynamics and the structure-property relationships will be of benefit for the development of more efficient exciplex–based emitters and cohost materials oriented for WOLEDs and solution-processed OLEDs.


N = 1628) were recruited from 13 clinics, community agencies, and social media sites in Los Angeles, California, and New Orleans, Louisiana, that specialized in serving sexual and gender minority youths (SGMY), especially males at risk for HIV. A cross-sectional comparison of sociodemographic and risk histories of HPV vaccinated/unvaccinated youths was conducted using both univariate and multivariate regressions. Results: About half (51.9%) of youth were vaccinated, with similar percentages across states and across genders. Sexual and gender minority youths  and their heterosexual peers (15%) were equally likely to be vaccinated (54%), even though their risk for HPV-related cancers is very different. Vaccinations were higher among younger youth, those not using condoms, youth with greater education, that possessed a primary health care provider, and youth diagnosed with HIV. Vaccinations were lower among youth that were out-of-home due to mental health inpatient hospitalization, drug treatment, homelessness, or incarceration. Conclusions: Special programs are required to target youth experiencing multiple life stressors, especially out-of-home experiences, those with less education, and without the safety net of health insurance or a provider.Background: Human papillomavirus (HPV) is epidemic among young people, especially those at highest risk of acquiring HPV-related cancers. Methods: Youth aged 14–24 years old ( Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) worldwide and is associated with cervical cancer and genital warts ,2,3. TheBecause HPV vaccinations were initiated five years later for men than for women , vaccinaAll SGMY in the U.S. are at risk for HIV , especiaVaccination rates may also vary by risk behaviors and protective factors, and we examine a range of these factors. This study’s sample was drawn from agencies serving youth with histories of mental health hospitalizations, substance abuse, homelessness, and incarceration, as well as those marginalized for being SGMY. Histories of sexual risk acts, those placing the youth at high risk for HIV and HPV, were particularly common among these youth, as sex was often a means of economic survival. SGMY also lack knowledge about the acquisition and availability of HPV vaccines . There aClinicalTrials.gov NCT03109431). All youths were recruited with voluntary informed consent. Youths aged 14–24 years old present at 13 adolescent clinics and community agencies specializing in services to SGMY and young people who were homeless, subsisted on low income, and lived in neighborhoods with high seroprevalence rates for HIV in New Orleans, Louisiana, and Los Angeles, California (N = 1628) were recruited from 2017–2020 [All study procedures followed the guidelines of the Declaration of Helsinki and were approved by the Institutional Review Board of the UCLA  or not (0). A variable was created, “out-of-home”, reflecting youth who had experienced any one of these four experiences: homelessness, mental health hospitalization, drug treatment, or incarceration. HPV vaccination status was assessed using the following question: “There is a virus called Human Papillomavirus which can be sexually transmitted. There is a vaccine that can prevent you from getting diseases from this virus. Have you completed the two-shot or three-shot HPV vaccine?” Possible responses included “yes”, “no”, and “I don’t know”, or the option not to answer the question. This question was gated so that youth answering “yes” were queried regarding the number of doses received. HIV status was determined by rapid-HIV testing with CLIA-waived Alere  Determine HIV-1/2Ag/Ab Combo finger stick. Those who reported a recent high-risk HIV exposure or had flu-like symptoms received the Cepheid  Xpert HIV-1 Qual Assay to test for acute HIV infection that would not be detected by rapid antibody tests. In addition, participants reported on the number of times that they had been diagnosed with an STI  and their history of sexual protection via either abstinence or consistent condom use with all sexual partners. 2) tests to examine possible differences between those vaccinated and not. Multivariate logistic regression model with a binary outcome for HPV vaccination status evaluated factors associated with vaccination [We stratified the sample on self-reported HPV vaccination status and considered those who did not know if they received/completed the vaccination and those who refused to answer the question as missing. We performed bivariate analyses using chi-square (χcination . For theAs noted in N = 1317) responded to the interviewer’s question regarding HPV vaccination (excluding those who indicated that they did not know if they had received the vaccine and those who did not answer the question) with 51.9% reporting that they had received the vaccine. Participants who responded to the HPV vaccination question differed from non-respondents in that they were more likely to be assigned male sex at birth, white, and unemployed.Of the 1628 participants, almost 80% (N = 1099). After adjusting for the other predictors in the model, older youth , those who have been out-of-home , and those who are sexually active and not using condoms consistently  have significantly lower odds of receiving the HPV vaccination. Additionally, those who have a healthcare provider , and those who are living with HIV  had higher odds of being vaccinated for HPV. Moreover, education level was associated with higher odds of being vaccinated. Notably, recruitment site, gender, and being an SGMY are unrelated to HPV vaccination in the multivariate analyses.This study examined factors influencing HPV vaccination among youth who are at increased risk for or living with HIV. These youth have significant histories of risk behaviors associated with HIV , as well as out-of-home placements . In contrast to recent studies of HPV vaccinations among young people , our samOverall, cisgender women and participants with an “other”  sexual or gender identity have the highest rates of vaccination by gender and sexual identity when examining these factors in the univariate analyses. However, these results do not appear as significant in the multivariate analyses. Given the higher rates of HPV-related cancers among gay, bisexual, and transgender persons, much higher rates of vaccination would be important among these young people. There are now very conflicting data on the uptake of HPV vaccinations among SGMY. Similar to our study, SGMY in a much smaller sample found an HPV vaccination rate of 10% . Yet, otMost previous research found a need for increased targeting of young men for HPV  and thatStudies have also shown that HPV vaccination increases for young adult women when healthcare providers recommend the vaccination series . A systeThere is a significant association between HPV vaccination and level of education, which is consistent with a previous study’s findings . Most prThis relatively large sample of SGMY and the sample’s focus on young people with histories of multiple concurrent life challenges  is a key benefit of this study. Since the data are based on self-reports , these rResults suggest that despite national efforts to increase the uptake of the vaccine, many youths at high risk for HPV have not received it. SGMY are at the highest risk of acquiring HPV cancers ,8, but d


Objectives: Italy was the first European country to introduce universal vaccination of adolescents, for both males and females, against Human Papilloma Virus (HPV) starting in 2017 with the NIP 2017–2019′s release. However, vaccine coverage rates (VCRs) among adolescents have shown a precarious take-off since the NIP’s release, and this situation worsened due to the impact of the COVID-19 pandemic in 2020. The aim of this work is to estimate the epidemiological and economic impact of drops in VCRs due to the pandemic on those generations that missed the vaccination appointment and to discuss alternative scenarios in light of the national data. Methods: Through an analysis of the official ministerial HPV vaccination reports, a model was developed to estimate the number of 12-year-old males and females who were not vaccinated against HPV during the period 2017–2021. Based on previously published models that estimate the incidence and the economic impact of HPV-related diseases in Italy, a new model was developed to estimate the impact of the aggregated HPV VCRs achieved in Italy between 2017 and 2021. Results: Overall, in 2021, 723,375 girls and 1,011,906 boys born between 2005 and 2009 were not vaccinated against HPV in Italy . As compared with the 95% target provided by the Italian NIP, between 505,000 and 634,000 girls will not be protected against a large number of HPV-related diseases. For boys, the number of the unvaccinated population compared to the applicable target is over 615,000 in the ‘best case scenario’ and over 749,000 in the ‘worst case scenario’. Overall, between 1.1 and 1.3 million young adolescents born between 2005 and 2009 will not be protected against HPV-related diseases over their lifetime with expected lifetime costs of non-vaccination that will be over EUR 905 million. If the 95% optimal VCRs were achieved, the model estimates a cost reduction equal to EUR 529 million, the net of the costs incurred to implement the vaccination program. Conclusion: Suboptimal vaccination coverage represents a missed opportunity, not only because of the increased burden of HPV-related diseases, but also in terms of economic loss. Thus, reaching national HPV immunization goals is a public health priority. In 2008, Prof. H. Zur Hausen received the Nobel Prize for discovering the cancerogenic role of HPV, which he initially discovered in 1976. Following that, in 1989, Prof. Ian Frazer discovered the HPV vaccine that later in 2006 became available globally ,4, was iAfter many studies showed both the cost effectiveness and the efficiency of HPV vaccination in women ,6, the BThe achievement of high coverage in the population, as well as for other vaccinations, guarantees indirect protection (herd immunity) and, consequently, represents an important public health goal. In Italy, the data on vaccination coverage are provided by the regions to the Ministry of Health and are published annually. The ministerial reports, albeit with some data gaps, represent the official source of monitoring the coverage objectives envisaged by the 2017–2019 NIP currently in force.To a generalized picture of precarious take-off of this vaccination, especially in males, in 2020, the impact of the COVID-19 pandemic was added, which lasted into 2021, as a result of the commitment at the forefront of the vaccination centers in the influenza and COVID-19 vaccination campaigns. In July 2020, the Ministry of Health summarized the results of an investigation conducted on the vaccination centers in the aftermath of the outbreak of the pandemic. The survey revealed that 97 local health authorities (LHAs) witnessed a decrease in vaccinations; in 68% of cases, it was adolescents’ vaccinations that had suffered the greatest delay due to the pandemic [This study, in light of the above, aims to estimate the epidemiological and economic impact of the drop in vaccine coverage rates (VCRs) due to the pandemic on those generations that missed the vaccination , and, inTwo previously published models in Italy estimated the impact of HPV-related diseases on the Italian population regarding health conditions and death risk ,12. ThesAchieved VCRs: in this scenario, we considered the actual vaccination coverage rate recorded in Italy for cohorts 2005–2008 reported from the official ministerial HPV vaccination reports, and we made assumptions of VCRs achieved in the 2009 cohort, whose VCRs are not publicly available at the moment of this publication .Target coverage rate: in this second scenario, the optimal vaccine target defined by the Italian NIP 2017–2019 was considered  Table 1Table 1.Vaccine coverage rates’ (VCRs) scenarios definition: by extracting the VCRs recorded in the official ministerial HPV vaccination reports for the cohorts of girls and boys born between 2005 and 2008 (eligible on their 12th year of age and therefore vaccinated between 2017 and 2020), two scenarios were developed:Model adaptation for gender, coverage rate and efficacy: the previously developed model, considered the starting point for this analysis ,14, inclModel simulation and economic effects: the model was performed considering the assumed scenarios and absolute differences were calculated for HPV-related events, direct and indirect costs.The analysis develops according to three different steps:Every year, the Italian Ministry of Health publishes national and regional coverage data for vaccination against HPV in the female and male populations . The mod‘Worst case scenario’: Coverage rates for males and females vaccinated in the years 2020 and 2021 (birth cohorts 2008 and 2009) remain the same as for those registered in 2020  . No catc‘Best case scenario’: in this scenario, the model assumes a national coverage rate for the two cohorts equal to the maximum level registered in the Italian regions in 2020  .Given that the last ministerial report refers to VCRs performed in the 2008 cohort during the pandemic year (2020) and that this cohort might have undergone a catch-up in 2021, the model will make some assumptions regarding the VCRs in these two cohorts . For the purpose of the simulation and to account for two different scenarios of COVID-19′s impact on VCRs on the most affected cohorts, the model defines a ‘worst case scenario’ and a ‘best case scenario’ as follows:All hospital admissions were identified through administrative archives, according to the International Classification of Diseases (ICD-9 CM). Information related to the hospital discharges of all accredited public and private hospitals, both for ordinary and day care regimes, was taken into account.We included hospital admissions related to resident patients presenting with one of the ICD-9-CM codes as a primary or secondary diagnosis for: genital warts (GW): ‘condyloma acuminatum’ (078.11); ‘cervical intraepithelial neoplasia (CIN)’ ; ‘anal cancers’ (AC) (154.2–154.8); ‘oropharyngeal cancers (OC): ‘oropharyngeal cancer’ 146.0–146.9) and ‘head, face and neck cancers’ (171.0); and genital cancers (GC): ‘penis cancer’ (187.1–187.9) and ‘cervical cancer’ (180.0–180.9) ,14. Data6.0–146.9A deterministic analysis was performed considering the estimated age-relative risk of event, the estimated efficacy and the direct and indirect costs applied to the estimated live population of the cohort at the same age. Real-world data were applied to the unvaccinated cohorts, which considers the overall hospitalization rates for each disease, as already done in the Marcellusi, Mennini et al. study . For theThe uncertainty associated with the model’s outcomes was estimated through a deterministic sensitivity analysis (DSA). In the DSA, each sensible parameter of the model was subject to a variation derived from the literature (CI of vaccine efficacy data) or from an arbitrary variation , as repoIn 2021, 723,375 girls and 1,011,906 boys born between 2005 and 2009 were not vaccinated against HPV in Italy . As compared with the 95% target provided by the Italian NIP, between 505,000 and 634,000 girls will not be protected against a large number of HPV-related diseases. For boys, the number of the unvaccinated population compared to the applicable target is over 615,000 in the ‘best case scenario’ and over 749,000 in the ‘worst case scenario’ .Overall, between 1.1 and 1.3 million young adolescents born between 2005 and 2009 will not be protected against HPV-related diseases during their lifetime, and the expected lifetime costs of non-vaccination will be over EUR 905 million. If the 95% optimal VCRs were achieved, the model estimates a cost reduction equal to EUR 529 million, the net of the costs incurred to implement the vaccination program.The model also estimated the lifetime cost associated with the five simulated cohorts by considering three different scenarios. Without the HPV immunization program (scenario (a), no vaccination), the expected lifetime costs would be over EUR 905 million . Assuming the VCRs ‘worst scenario’ due to the pandemic were true, the sub-optimal coverage rates performed between 2017 and 2021 would lead to a model estimate of the cost reduction equal to EUR 260 million (−28% vs. no vaccination scenario), with a significant economic burden for the NHS of the EUR 644,618,178 of HPV-related diseases. Finally, if VCRs scored up to the optimal NIP targets for the selected cohorts , an additional EUR 269 million could be saved (−58% vs. no vaccination scenario). To synthesize, protecting adolescents born between 2005 and 2009 by reaching the NIP’s optimal HPV immunization rates would potentially avoid up to EUR 529,6 million in the lifetime horizon as compared to ‘no vaccination’. The COVID-19 pandemic is likely to have compromised the VCRs of the 2008 and 2009 cohorts as modelled in our ‘worst case scenario’. In this scenario, which also account for sub-optimal VCRs in the 2005–2007 cohorts, the total economic and social burden of HPV-related conditions will remain significantly high .This analysis shows, in an intuitive and accurate way, the epidemiological and economic impact of the ‘lost generations’ to anti-HPV vaccination due to sub-optimal historic VCRs and to the likely impact of the pandemic on the 2020 and 2021 immunization campaigns [In light of these results, it becomes important to stress HPV immunization as a public health priority and to identify organizational solutions capable of promoting adolescent-friendly prevention, vaccine confidence and a full recovery of coverage after the pandemic emergency ,22,23.The PNPV 2017–2019, which is still in force as it was extended for the COVID-19 pandemic emergency, defines a flowchart addressed to achieve high coverage through a repeated and carefully monitored ‘invite and remind’ system and presents the operating procedures to promote the active and free offer of vaccinations . Since HA consensus conference held in 2019 issued a series of recommendations for implementing HPV vaccination that were recognized to be highly recommended both by the international literature and by the opinions of Italian experts, based on the most effective recognized strategies .The included recommendations were:(i) Sending reminders to parents of 11–12-year-olds a few days before the agreed upon vaccination appointment following the active call from the vaccination program and in the case of a no-show at an agreed appointment;(ii) The implementation of reminder strategies aimed at health professionals to remind them to propose vaccination during a visit to a citizen who has not been previously vaccinated, but for whom vaccination is planned. In particular, it is recommended to activate reminder strategies aimed at the pediatrician of free choice (PLS), the heads of the vaccination clinic and general practitioners (GPs);(iii) The provision of informational/educational strategies aimed at vaccination targets and/or their parents are weakly recommended since informational/educational actions for the population are generally ineffective in increasing compliance with vaccination and may encounter economic sustainability problems;(iv) The implementation of reminders/informational activities should, on the other hand, be based mainly on the direct trust relationship between the health worker and the vaccination target/parents of the target or be implemented at the school level.The consensus identified, while acknowledging the difficult implementation of the strategy, that secondary school is the ideal venue for information, education and vaccination opportunities.The implementation of strategies that provide, among other interventions, the carrying out of vaccination in the school setting, have proved effective in various contexts, despite encountering organizational difficulties at the local level.Given the situation of poor coverage and the real risk of missing the HPV immunization for more than half of the boys and girls born after 2004, it seems speculative to stop at the evidence of the literature and their transferability to the Italian context: evaluating alternative vaccination and information settings become an imperative in this circumstance . CountriThe COVID-19 pandemic may leave an indelible mark on the reproductive health of millions of adolescents; however, the response to this same emergency has shown us rare examples of resilient organizational systems, and it is hoped that this moment will be also an opportunity to promote real organizational innovation in organizational models for the promotion of vaccine prevention ,33,34.Looking closely at the results of this analysis, it emerges strongly that, overall, between 1.1 and 1.3 million of young adolescents born between 2005 and 2009 will not be protected against HPV-related diseases during their lifetime, with an expected lifetime cost of non-vaccination that will be over EUR 905 million. If the 95% optimal VCRs were achieved, the model estimates a cost reduction equal to EUR 529 million, the net of the costs incurred to implement the vaccination program.The results of recent studies ,36 highlConsequently, this analysis highlights and demonstrates how suboptimal vaccination coverage represents a missed opportunity, not only because of the increased burden of HPV-related diseases, but also in terms of economic loss. Thus, reaching national HPV immunization goals is a public health priority.The study presents some limitations and some strengths. The lifetime projection model has a high level of uncertainty, though the sensitivity analysis showed important advantages. Again, the model is based on assumptions for 2020–2021 coverage, and yet a scenario analysis was conducted. The study, in light of the highlighted results and in terms of both the economic and epidemiological benefits, is a valuable tool for decision makers to be able to make an informed decision regarding vaccination policies in Italy.


In the recent years, the prevalence of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) has increased significantly. Currently, nearly 80-90% of all oropharynx tumors are HPV-positive. In addition, it is now recognized that HPV-positive tumor status is associated with good prognosis and improved response to chemoradiation. However, within this setting, there are still patients with HPV-positive OPSCC who will experience recurrence. With the increasing incidence of HPV-mediated OPSCC, recurrent HPV disease is also becoming more prevalent and there is an increasing need to understand the unique presentation and treatment of recurrent HPV-mediated disease. In this review, we will discuss epidemiology of recurrent HPV-positive OPSCC, role of surgical salvage, re-irradiation, and the role of upcoming novel treatments and immunotherapy. Historically, recurrent oropharyngeal disease has been associated with poor prognosis and high morbidity. However, recent advances have transformed the landscape for salvage treatment of HPV-mediated OPSCC. Liquid biomarkers offer potential for early detection of recurrence, robotic techniques may reduce morbidity of surgical salvage, improvements in re-irradiation approaches reduce toxicities, and novel immune based therapies on the horizon are offering promising results. These advances combined with the improved prognosis of HPV-positive disease offer to transform our approach to recurrent disease of the oropharynx. Despite the improved prognosis conferred by human papillomavirus (HPV)-positive tumor status, recurrences and distant failures still occur in this population with progression free survival of 72-74% at 3-years , 2. RecuPatterns of recurrence with regard to local, regional and distant sites do not differ significantly by HPV status , 4, 6. Wth edition stage was not independently associated with progression free survival in either p16-positive and p16-negative OPSCC patients  as a biomarker of treatment response and recurrent disease. Early studies established the relationship between detectable serum or saliva HPV DNA with increased risk for recurrent disease . In a prAfter recurrence, HPV-positive patients still demonstrate improved outcomes compared to their HPV-negative counterparts , 42, 56.In cases of recurrent OPSCC, surgical salvage with curative intent should be offered when feasible. Historically, recurrent oropharyngeal disease has been associated with poor prognosis with lower rates of survival and higher rates of surgical complication compared to other head and neck subsites , 61, 62.TORS for recurrent oropharyngeal disease has been associated with decreased post-operative complications, including lower long-term tracheostomy and feeding tube dependence , 70. WhiA patient’s burden of recurrent disease and functional status play an important role in selecting patients most likely to benefit from surgical salvage. As previously discussed, selection for surgical salvage should foremost consider ability to achieve negative margins , 67–69. Given that patients with advanced primary disease are more likely to recur, 90-95% of patients who recur will have received prior radiation either in the primary or adjuvant setting , 59. In Isolated neck recurrence may provide greater chance at complete resection of recurrent disease, however nodal recurrence is associated with high rates of further recurrence and metastasis , 73. PriFor patients with resectable recurrence after prior radiation treatment, there is strong evidence that adjuvant therapy following salvage surgery reduces progression free survival and is recommended by NCCN guidelines , 82. OPSFor patients with unresectable disease or who are unable to undergo surgical resection, re-irradiation with or without chemotherapy is an option that has historically demonstrated limited benefit for non-nasopharyngeal sites, and carries high rates of toxicity , 61. TriWhen distant metastasis occurs, generally systemic treatment options are favored. However, one recent study demonstrated increased survival associated with surgical salvage for distant metastases from OPSCC, with an increase of median survival from 12.5 to 35 months . For patFor patients who do not have surgical or re-irradiation options, systemic chemotherapy regimens are the mainstay for recurrent metastatic head and neck cancer. The EXTREME regimen including cetuximab, platinum and 5-fluorouracil (5-FU) had been the standard treatment regimen for the past decade , 86. Thevia PD1 blockade and antibody dependent cellular cytotoxicity of IgG1 antibodies, Sacco et al. combined pembrolizumab and cetuximab in recurrent/metastatic HNSCC patients who were refractory to or poor candidates for platinum  for a given HPV antigen and HLA type has introduced the possibility of engineering T cells to express the relevant TCR. These strategies have been tested in clinical trials using HPV-16 E6 and E7 TCRs. Interestingly, a clinical trial of an HPV-16 E7 TCR recently demonstrated a 50% response rate in refractory tumors . While tIn this evolving field, novel combinations are under study that attempt to leverage the benefits demonstrated with anti-PD1 blockade including adding agents that target TIGIT, CD47, and LAG3 . FurtherAlthough HPV-mediated OPSCC is associated with improved prognosis and decreased rates of recurrence, recurrent disease still occurs. With the increasing incidence of HPV-mediated OPSCC, recurrent HPV disease is also becoming more prevalent and there is an increasing need to understand the unique presentation and treatment of recurrent HPV disease. Recurrences in HPV-positive patients may occur later, and patients may have improved outcomes after recurrence compared to HPV-negative counterparts. Emerging data demonstrates that detection of recurrences may be aided by evaluation of circulating tumor HPV DNA. Surgical salvage is the preferred treatment when feasible, and robotic approaches can decrease morbidity. New advances in re-irradiation and immune based therapies are offering promising results for this patient population.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All authors  provided substantial contributions to the conception of this work, drafting and writing as well as approval of publication.TG is supported by 1KL2TR001444 from NIH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To verify the association of high-risk human papillomavirus (hrHPV) viral load reflected by cycle threshold (Ct) values from HPV testing on Cobas4800 assay with the histologic grades of cervical lesions via analysis on the combined data from two cervical cancer screening trials and to explore the referability of Ct values in management of the abnormalities from cervical cancer primary screening.We analyzed the data from Chinese Multi-Center Screening Trial (CHMUST) and BUJI Cervical Cancer Screening Study Project (BUJI Study). All data to be analyzed in this paper were related to provider-collected samples. One-way ANOVA was performed to compare the Ct values among different histological groups, and Kendall’s tau-b correlation was applied to examine the association between Ct values and cervical lesion grades. The stepwise incidence of CIN2+ and CIN3+ in every 100 HPV positive individuals were calculated according to the descending of the genotype specific Ct values. The highest Ct values related to CIN3+ incidence rate 4% (or 25%) were used as the cutoffs to distinguish low-Ct value cases from the high-Ct value ones.P < 0.05). No significant difference was found among different grades of lesions in term of correlated CtV of HPV18-plus (positive of HPV18 only or HPV18 plus pooled 12-HPV) (P > 0.05). The CIN2+ or CIN3+ incidence per 100 positives significantly increased corresponding to the descending of the CtV of hrHPV, HPV16-plus and pooled 12-HPV. Compared with high-CtV groups , the relevant risks (RRs) of CIN2+ for hrHPV and pooled 12-HPV positive groups with low-CtV  were 3.2 (95%CI 2.18–4.80) and 2.3 (95%CI 1.50–3.45). Similarly, the RRs of CIN3+ for hrHPV and pooled 12-HPV positive groups with low-CtV were 6.5 (95%CI 2.83–14.80) and 2.7 (95%CI 1.15–6.39), respectively. The RRs of CIN2+ for medium- (30.3 < CtV ≤ 37.4) and low- (≤ 30.3) CtV HPV16-plus positives were 5.1 (95%CI 0.68–38.38) and 20.6 (95%CI 2.96–143.92) related to high-CtV (> 37.4) groups, and the CIN3+ incidence in low-CtV value group was nine-fold higher of that in medium-CtV ones . In comparing with the algorithms of “HPV16-plus/18-plus + cytology ≥ ASCUS for pooled 12-HPV”, triage algorithm “HPV16-plus/18-plus + Ct value ≤ 33.2 for pooled 12-HPV” could achieve a comparable sensitivity of 93.2%.A total of 1376 women in CHUMUST and BUJI Study who were aged 30–59 and positive of hrHPV for provider-collected samples on Cobas4800 with complete data in terms of the relevant Ct values (CtV) and the histological diagnosis were included for analysis. Our data showed significant difference among different histological grades of cervical lesions in the CtV of hrHPV, HPV16-plus (positive of HPV16 only or HPV16 plus 18 and/or pooled 12-HPV), and pooled 12-HPV (HPV viral loads reflected by Ct values for hrHPV, HPV16-plus and pooled 12-HPV from Cobas4800 HPV testing were directly associated with the severity of cervical lesions. A lower HPV genotype-specific Ct value prompted a significantly high CIN3+ risk of 4% or higher in women positive of hrHPV, HPV16-plus or pooled 12-HPV, indicating that HPV viral load reflected by Ct values on Cobas4800 may be a promising risk indicator in management of abnormalities from primary cervical cancer screening.The online version contains supplementary material available at 10.1186/s13027-022-00440-4. Cervical cancer is the fourth leading cause of cancer morbidity and mortality among women worldwide, and 88% of the new cases are from less developed countries . HrHPV iCervical cancer is preventable via early diagnose and proper management . HPV tesSeveral HPV testing assays can report indicators reflecting viral loads in addition to indicating hrHPV infections, such as HC-2  , 14, HBRSeveral clinical studies have verified the sensitivity, specificity, and reproducibility of Cobas 4800 for detecting hrHPV genotypes –25. BesiIn this study, we reanalyzed the data from two clinical studies completed by our team, Chinese Multi-Center Screening Trial (CHMUST) and BUJI Cervical Cancer Screening Study Project (BUJI Study), to further confirm the potential correlation between hrHPV viral loads and the grades of cervical lesions in women positive of hrHPV via age-grouping, projecting to provide evidences for the referring HPV genotype-specific Ct values from Cobas 4800 to manage the abnormalities from HPV primary screening.This study was designed to analyze the data of the hrHPV positives for provider-collected sample on Cobas4800 with recorded Ct values from CHIMUST and BUJI study.CHIMUST  is a multicenter population-based cross-sectional cervical cancer screening study led by our team, in which 10,885 women were screened at 6 sites located in 6 regions nationwide in China from Aug 2016 to Jan 2018. Women enrolled were those who were aged from 30 to 59 years, sexual exposed, and non-pregnant, had not been screened for at least 3 years based on participant report, had no history of hysterectomy and pelvic radiation, and signed the consent form. The trial was approved by the Ethics Committee of the Peking University Shenzhen Hospital (IRB:PUSH2016001) and the Institutional Review Board of Cleveland Clinic (USA) (IRB:15–1549).Both self-collected vaginal sample and provider-collected endocervical sample were taken from each participant, which were split for HPV testing on Cobas 4800  and SeqHPV . The provider-collected samples were additionally prepared for cytology testing . Cytology slides were analyzed by 2 senior cyto-pathologists from Peking University Shenzhen Hospital (PUSH) and reported according to the Bethesda classification , as negaParticipants who were positive of hrHPV from Cobas 4800 assay and/or SeqHPV for both or either self- and/or provider-collected samples were referred to colposcopy. Cytology results were not referred for triage in this study . ColposcBUJI study  were conducted in Buji Community, Shenzhen, China, from August 2016 to September 2017. Totally ten thousand  women who were 19 to 80 years of age, sexually exposed and non-pregnant, had no prior hysterectomy and pelvic radiation, and consented for participation in writing were enrolled for cervical cancer screening with co-test of HPV testing on Cobas4800 and cytology. The study was approved by the Ethics Committee of Peking University Shenzhen Hospital (No. PUSHGYN2015005).The provider-collected endocervical samples were prepared for hrHPV testing on Cobas 4800  and cytology testing using AutoCyte® thin-layer liquid-based technology . Participants were called back for colposcopy if they were (1) positive of HPV16 and/or HPV18, or (2) positive of pooled 12-HPV plus abnormal of cytology (≥ ASC-US); or (3) negative of hrHPV but Cytology ≥ LSIL. Cytology and histology slides were analyzed by PUSH pathologists, and the interpretations were classified following the same criteria as followed in CHIMUST. Ct values of the 763 women who were positive of HPV from Cobas 4800 testing from the first 7000 women in BUJI study were recorded, of them 705 were at the age matching with CHIMUST (from 30 to 59).Cobas 4800 HPV assay is a multi-PCR based HPV assay, which simultaneously detects a total of 14 hrHPV types in three channels: HPV16, HPV18, and pooled 12-HPV , in addition to a separate channel of β-globin as reference. Cobas 4800 records CtV of each positive HPV channel as well as the reference channel. CtV-cutoff for all the HPV channels were determined by the manufacturers to define as positive . When CtV equals to or lower than the cutoff, we get a positive result and the corresponding CtV is recorded. CtV of negative channels is beyond the default CtV-cutoff and not reported.In this analysis, hrHPV positive results were categorized according to 3 hierarchies: HPV16-plus, HPV18-plus, and pooled 12-HPV. If not specially indicated, HPV16-plus refers to a result that is positive of HPV16 only or positive of HPV16 plus 18 and/or pooled 12-HPV; HPV18-plus refers to a result that is positive of HPV18 only or positive of HPV18 plus the pooled 12-HPV; while pooled 12-HPV refers to a result that is positive of the pooled 12-HPV only.Kendall’s tau-b correlation was used to examine the association between Ct values and cervical lesion grades.Statistical analysis was based on the data of the women who were enrolled in both the two projects, aged of 30–59, positive of hrHPV for provider-collected sample on Cobas 4800 testing with complete data regarding to the relevant Ct values and the histological diagnosis . The main outcome of this analysis was the association of genotype-specific Ct values and the histological grading of the lesions. HPV and histological examination results of the provider-collected samples from CHIMUST and BUJI Study were jointly analyzed. Ct values were described by mean ± standard deviation, and the differences of CtV among histological lesion grades were compared by one-way ANOVA. P-values of diagnostic accuracy between algorithms were obtained by McNemar’s test.The stepwise incidence of CIN2+ and CIN3+ in every 100 HPV-positives were respectively calculated along with the descending of genotype-specific Ct values. According to the 2019 ASCCP risk-based consensus guidelines for abnormal cervical cancer screening tests and cancer precursors (2019 ASCCP guidelines) , the higP < 0.05.SPSS v.26.0 software  was used for all data analysis in this study with the significance level Totally, there were 2051 women in CHUMUST and BUJI Study who were aged 30–59 and positive of hrHPV for provider-collected samples on Cobas4800 with Ct-value reported, including 1346 from CHIMUST and 705 from BUJI Study. After deleting 225 cases from CHIMUST cohort for unreturning for colposcopy and 419 cases from BUJI Study cohort for having no pathology diagnosis because of negative cytology and 31 for unreturning for colposcopy, 1376 women were included in the analytic group, including 1121 from CHIMUST and 255 from BUJI.Among the positives in the analytic group, the mean age was 44.3 (± 7.64) years. 14.03% (193/1376) were positive of single HPV16, 5.81% (80/1376) of single HPV18, and 71.95% (990/1376) of pooled 12-HPV; 20.49% (282/1376) of them were categorize as HPV16-plus, 7.56% (104/1376) as HPV18-plus (Table P > 0.05) , 32 (± 5) and 31.1 (± 5.4), respectively. As no significant difference was observed when compared the CtV between any two HPV genotypes and between HPV-16 or -18 single-type infection and multi-type co-infection (P < 0.001). The CtV of hrHPV and HPV16-plus showed gradually descending along with the upgrading of the histological severities. Detailed grouping analysis showed that (1) Ct values of hrHPV and HPV16-plus correlated to non-CIN and CIN1 were both respectively significant higher than those correlated to CIN2+ and CIN3+ (P < 0.05); (2) CtV of pooled 12-HPV correlated to non-CIN was higher than that correlated to CIN1 and CIN2+ (P < 0.05); (3) CtV of pooled 12-HPV correlated to CIN1, CIN2+ and CIN3+ showed virtually identical (28.9–29.1); and (4) no significant difference was found among different grades of lesion in terms of correlated CtV of HPV18-plus (P > 0.05) ; and  no signiAnalysis also showed that the CIN2+ or CIN3+ incidence per 100 positives significantly increased corresponding to the descending of the Ct values of hrHPV, HPV16-plus and pooled 12-HPV , indicating that the higher the lesion is the more consistent the low-CtV is with the lesion. We then tried to construct algorithms for positive triage base on CtV and compared them with multiple recommended algorithms, and demonstrated that, in comparing with the algorithms of “HPV16-plus/18-plus+ cytology ≥ ASCUS for pooled 12-HPV”, making “HrHPV CtV ≤ 33.2” as the triage algorithm  could achieve a lower sensitivity of 91.9% and lower specificity of 37.9% for CIN3+ , while making “HPV16-plus/18-plus+ CtV ≤ 33.2 for pooled 12-HPV” as triage algorithm  could achieve a comparable sensitivity of 93.2% and lower specificity of 28.4% for CIN3+ , with 72.8% of colpo referral rate  and specific histology grades were showed in Table Our results showed that the Ct values of hrHPV, HPV16-plus and pooled 12-HPV genotypes were inversely associated with the severity of cervical intraepithelial neoplasia and the risk of CIN2+ or CIN3+. The viral load of HPV18-plus reflected by CtV had no such correlation with the degree of cervical lesions or the risk of CIN2+/CIN3+. This result is consistent with the prior analysis based on BUJI study , showed . reported that coinfection with lower hierarchy HPV genotypes added little risk for CIN2+ or CIN3+, [Linear descending with grading-up of the histological cervical lesions were shown in the CtV for HPV16-plus but not in that for pooled 12-HPV. The explanation for the nonlinear correlation between CtV for pooled 12-HPV and cervical lesion grades may possibly lay on the fact that Cobas4800 assay makes the 12 hrHPV genotypes  in pool which may result in different pathogenic activity to a specific precancer lesion and therefore impacts the correlation of the CtV to the cervical lesions, and it is unable to distinguish the Ct-value for each single-genotype infection from that for multi-genotype infection. Early studies had confirmed the differences in the pathogenicity, either viral load related or not, of different hrHPV genotypes. One study compared the positive predictive value (PPV) of 13 hrHPV genotypes for CIN2+ or CIN3+ and found that the cervical precancerous lesion risks were significantly different among these hrHPV genotypes; the alpha-9 species  are phylogenetically similar to HPV16 and closely associated with precancerous lesions; and the top three genotypes (HPV16/33/31) with the highest CIN2+ /CIN3+ risk were all within the alpha-9 species . Anotherr CIN3+,  indicatiOur work showed no correlative descending of HPV18-plus Ct values corresponding to the cervical lesions upgrading. The CtV for CIN3+ related HPV18-plus was not significantly different than that for CIN2+ related HPV18-plus, although little lower was observed. The small number of cases with HPV18-plus related CIN2+ (7 patients) may bias the finding in this study. In addition, several previous studies demonstrated the controversial correlation of the HPV18 viral loads and the cervical lesion grading. Some authors reported viral load increasing along with upgrading of the HPV18-postivie cervical lesions  or compaIn view of the correlation of HPV Ct values to the cervical lesion grades and its predictive value on CIN2+ or CIN3+ lesions, the viral loads reflected by Cobas4800 CtV may potentially be a referable indicator for CIN risk in management of abnormalities from primary cervical cancer screening. However, how to refer HPV Ct values to achieve the favorable management is still a great challenge. Previous studies mainly focused on the triage value of non-16/18 hrHPV viral load, and mostly based on the quartile CtV of the tested population , 18 or tOur work showed a strong correlation between CtV of HPV16 and the risk of cervical lesions. Since 2019 ASCCP guidelines recommends management of cervical cancer screening abnormalities based on the CIN3+ risk yielding from any combination of history and current test results , and ourOur study also demonstrated that triage based on HPV genotyping together with HPV genotype-specific CtV was more effective in triage than using CtV as the only indicator for triage, although there was no significant difference among the average CtV of each genotype of HPV Table . When maThis study showed the predictive effect of HPV16-plus Ct values for CIN3+ risk, which may be socioeconomically high valued in hierarchical management of HPV16-plus positives. When patients with 4% or higher CIN3+ risk are referred for colposcopy, 14.5% 41/282) of HPV16 positives wouldn’t need immediate colposcopy and no CIN3+ lesion would be missed. More importantly, the low-CtV (≤ 30.3) of HPV16-plus indicates a very high CIN3+ risk (≥ 25%) where expedited treatment or colposcopy is acceptable according to 2019 ASCCP guidelines. No correlation was observed between CtV for HPV18-plus and cervical lesions in this study. Considering the relatively low infection rate of HPV18, its high correlation with adenocarcinomas  and infe/282 of HOur study reviewed the data from two large clinical studies involving 1376 HPV positives from six diverse provinces of China, which rendered the results applicable to the general population in various settings. However, there are several limits in our study. Firstly, the viral load in the sample may be influenced by the quality of the sample. In order to reduce the bias caused by the sample cellularity difference in each sample, we obtained cervical exfoliated cell samples by a standardized sampling procedure of removing cervical secretions and rotating brush 3 times. By using this method, several studies published by different teams have all confirmed the clinical value of HPV viral load in triaging of HPV primary screening positives , 43, 44,In conclusion, HPV viral loads reflected by Ct values of hrHPV, HPV16-plus and pooled 12-HPV from Cobas4800 HPV testing were associated with the severity of cervical lesions. The Ct values of hrHPV and HPV16-plus, but not pooled 12-HPV, showed linear descendance with the histological upgrading of the cervical lesions. Ct values of HPV18-plus showed no correlation with the grades of cervical lesions. A lower HPV genotype-specific Ct value prompted a significantly high CIN3+ risk of 4% or higher in women positive of hrHPV, HPV16-plus or pooled 12-HPV, this CIN3+ risk can even get up to 25% or higher in HPV16-plus positives with low-CtV, indicating that HPV viral load reflected by Ct values on Cobas4800 may be a promising risk indicator in management of abnormalities from primary cervical cancer screening.Additional file 1: Table S1. Comparison of the Ct values of specific HPV genotype among different age groups.