E6 and E7 into the host cell, leading to the inactivation of several tumor suppressor genes, such as Rb. HPV status can affect prognosis in OPSCC, but its role as a predictive biomarker remains to be elucidated. Given the favorable prognosis associated with HPV-positive disease, the concept of de-escalation treatment strategies has been developed with the primary intent being the reduction of treatment-related long-term toxicities. In this review, we aim to depict current data regarding treatment de-escalation in HPV-associated OPSCC and discuss ongoing clinical trials.HPV-related head and neck squamous cell carcinoma (HNSCC) has emerged as a diverse clinical and biological disease entity, mainly in young patients with oropharyngeal tumors who are nonsmokers and nondrinkers. Indeed, during the past few years, the pendulum has shifted towards a new epidemiological reality, the “HPV pandemic”, where the majority of oropharyngeal squamous cell carcinomas (OPSCCs) are attributed to HPV. The oncogenic potential of the virus is associated to its capacity of integrating oncogenes  TP53, EGFR and CDNK1A genes [Recent epidemiological evidence suggests a substantial rise of Human Papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC), which accounts for more than 60% of OPSCC cases in the contemporary area ,2,3. Com1A genes  and diff1A genes .Most importantly, HPV+ OPSCC has been traditionally associated with significantly better outcomes ,9,10,11.Given the remarkably better survival rates observed in HPV+ OPSCC, juxtaposed with the substantial morbidity caused by the standard of care treatment of cisplatin and RT combination in locally advanced disease, the concept of treatment de-escalation has been proposed as a new treatment paradigm. In this review, we seek to summarize current data on deintensification approaches in HPV+ OPSCC and discuss the goals of future de-escalation studies in this regard.The retrospective analysis of the RTOG 0129 trial published by Ang et al. identified a low-risk group of HPV+ OPSCC patients who exhibit high survival rates (90–95% at 3 years), implying a high chance of cure . AlthougThe choice of suitable candidates for treatment de-escalation trials has become a matter of paramount importance. Two groups of patients should probably be excluded from participation. The first group (a) consists of patients with a smoking history (more than 10 pack years) due to a substantially worse prognosis, with a 3-year OS of approximately 70% . Of noteImportantly, the recognition that the American Joint Committee on Cancer (AJCC) 7th edition staging system does not adequately reflect the prognosis of HPV+ OPSCC, given the paradoxically favorable survival associated with the advanced stage, led to the development of the modified 8th edition clinical stage paradigm for HPV+ disease that has been more recently adapted . HoweverAlternative methods for risk stratification that are currently under investigation include the incorporation of genomic signatures , expressAlthough there are many ongoing de-escalation strategies, the most commonly assessed in clinical trials are reduction of RT dose, field, or schedule when given either as adjuvant or definitive therapy and omission, dose reduction, or replacement of concurrent chemotherapy. Some of these clinical trials are presented below and in The cumulative dose of delivered RT has been substantially correlated with long-term functional deficits, which are lessened with doses below 60 Gy . The ratDefinitive chemoradiotherapy. In the setting of definitive chemoradiation, the phase II NRG-HN002 (NCT02254278) trial evaluated the efficacy of a reduced-dose RT (60 Gy) in patients with low-risk disease based on stratification by Ang’s criteria, namely patients with 7th edition stage T1-2N1-N2bM0 or T3N0-N2bM0 OPSCC with a ≤10 pack-year history of tobacco consumption [2 or to IMRT alone over 5 weeks. This study showed that despite the satisfactory efficacy of concurrent CRT arm (2-year PFS of 90.5%), the RT alone arm did not meet acceptability criteria (2-year PFS 87.6%). Of note, 2-year OS was similar between the two groups.sumption . Patient2 weekly) [In the same context, Chera et al. reported the results of a phase II trial that evaluated the combination of reduced dose IMRT (60 Gy) with cisplatin at a lower dose (30 mg/m weekly) .In a similar context, EVADER is a single arm phase II study that seeks to evaluate the effectiveness of reduced RT volume to selected lymph node regions in patients with early stage (T1-3 N0-1M0 AJCC 8th edition) HPV+ OPSCC . On the Deintensification of definitive RT after induction chemotherapy. Response to induction chemotherapy (IC) has been used as a biomarker for selection of appropriate candidates for deintensified RT in phase II trials. All trials demonstrate improved outcomes compared to standard controls, warranting the design of phase 3 trials.The Quarterback trial was a phase II study that enrolled patients with HPV+ disease and a <20 pack year smoking history who received induction TPF. Following IC, clinical responders (20 out of 23 patients) were randomized to receive a standard dose (70 Gy) or reduced dose (54 Gy) RT with weekly carboplatin . In a meE1308 was a larger phase II trial with 90 participants, all stage III-IV (AJCC 7th edition) HPV+ OPSCC patients who were assessed for response at primary site and lymph nodes after IC with cisplatin, paclitaxel, and cetuximab. Patients with complete response (CR) were treated with RT at a reduced dose of 54 Gy, whereas patients with less than CR received standard dose RT of 69.3 Gy; both RT schedules were given in combination with weekly cetuximab . ImportaOn the other hand, the OPTIMA phase II trial included patients with low risk  and high risk (defined as T4 or ≥N2c or ≥10 pack-year smoking history) HPV+ OPSCC, who, depending on their response at IC with a carboplatin/nab-paclitaxel combination regimen, were treated with reduced dose RT (low-risk with good response) or CRT (low-risk with medium response or high-risk with good response) or standard dose CRT (patients with any other response) . Two-yeaFinally, the RAVD trial similarly sought to assess the efficacy of a de-escalated approach that incorporated a reduced RT volume areas based on response to IC in patients with locally advanced HNSCC . ConsequAdjuvant therapy. Adjuvant CRT is currently indicated in patients whose tumor pathology includes positive surgical margins or extracapsular lymph node extension. On the other hand, adjuvant RT is considered in tumors with positive lymph nodes and deep invasion .p = 0.011). Based on this study, TORS plus adjuvant reduced dose RT should be evaluated in a phase III trial in comparison to standard radical CRT in patients with HPV+ OPSCC.The ECOG-ACRIN 3311 trial included patients with resectable p16+ OPSCC and cT1-T2 stage III/IV 7th edition AJCC without matted nodes. After transoral resection (TORS), patients were stratified depending on their pathological features to low risk , no extracapsular extension (ENE)) who had no additional treatment (Arm A), intermediate risk  who were randomly assigned to either postoperative reduced dose RT 50 Gy (Arm B) or RT 60 Gy (Arm C), and high risk  who were managed with adjuvant cisplatin-based CRT (Arm D) . For intMC1273 was a phase II trial that sought to assess the efficacy of a remarkably reduced RT dose administered as adjuvant treatment following surgical management of stage III-IV (AJCC 7th edition) HPV+ OPSCC . EligiblOn the other hand, the AVOID trial, a phase II trial with distinct design, focused on patients with LN involvement after surgical treatment with TORS . Sixty pPATHOS is an ongoing trial which transitioned into phase III after successful completion of the phase II cohort. Patients with stage T1–T3 N0–N2b HPV+ OPSCC are stratified to pathological subgroups following TORS and LN dissection. Patients in the low-risk group receive no further treatment, patients in the intermediate-risk group are randomized to standard or reduced dose RT, and patients in the high-risk group are randomized to CRT or RT ,42. The p = 0.001) and 2-year relapse rate .Given the significant toxicity correlated with cisplatin-based CRT and the promising efficacy of cetuximab-based bioradiotherapy in patients with OPSCC demonstrated in the IMCL 9815 trial , the ideRTOG 1016 was a noninferiority phase III trial published in same year as De-ESCALAaTE-HPV, which randomized patients with HPV+ OPSCC to either two cycles of cisplatin in combination with RT or cetuximab plus RT . It was 2 and RT; primary endpoint was symptom severity [p = 0.015).In addition to these two trials, TROG 12.0, a phase III trial conducted in Australia was recently reported at ASCO 2021 . In thisseverity . SimilarAs we discuss in detail a variety of heterogeneous deintensification trials in HPV-related OPSCC, we understand that it is currently not the proper time for a broad application of de-escalation therapy in HPV+ patients outside of clinical trials. However, any patient with low-risk disease and no/limited history of smoking should be considered a candidate for participation in ongoing trials. A very tempting approach is the incorporation of immunotherapy in de-escalation strategies, since HPV+ OPSCC has an immunogenic profile and has been found to produce high response rates with immunotherapy . Two triNevertheless, in addition to including a more stringent definition of HPV positive status and accurate information of the OSCC subsite, the identification and evaluation of novel biomarkers is a most important step in de-escalation trials and patient selection. The diagnosis of an HPV+ tumor can be improved in future clinical trials by adding the analysis of HPV DNA or HPV RNA to p16 immunohistochemistry, since around 10–15% of all p16+ oropharyngeal cancer is not HPV DNA+ . ClinicaOngoing de-escalation clinical trials in HPV+ OPSCC are shown in


Human Papillomavirus (HPV) is a small, non-enveloped, icosahedral and double-stranded DNA virus with a genome of 8 kb, belonging to the papillomaviridae family. HPV has been associated with 99.7% cases of cervical squamous cell carcinoma worldwide. The HPV E6 protein is known as a potent oncogene and is closely allied with the events that result in the malignant transformation of virally infected cells. The present study aims to target plant derived anticancer molecules for HPV driven cancer using a computational approach. Methods: In this study, E6 oncoprotein was targeted by 101 plant-derived nutraceuticals using the molecular docking method. The multiple sequence analysis and phylogenetic analysis of low risk and high risk 28 HPV E6 proteins were performed. Withanolide D, Ginkgetin, Theaflavin, Hesperidin, and Quercetin-3-gluconide were identified as the potential inhibitors of HPV 16 E6 protein. The zinc finger domain was identified on all variants of HPV E6 oncoprotein while high-risk HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV58, HPV68 and HPV73: probable risk HPV53 and low-risk HPV43 and HPV70 contain PDZ domain. The current study using bioinformatics analysis approaches reveals a promising platform for developing anti-cancerous competitive inhibitors targeting HPV. Cancer is a multifactorial disease, which involves the degradation of several cell signalling pathways  recent study identified various metabolites for onset of cancer . There are various risk factors responsible for developing cancer, including oncogenic viruses. Human papillomavirus (HPV) is a well-known oncogenic virus and the incidence of HPV-related cancer is higher across the world.. HPV has been associated with 90% of cervical squamous cell carcinoma cases worldwide, as well as some other cancers related to the vagina, vulva, penis, anus, head and neck, oral cavity and oropharynx . Human papillomavirus (HPV) is a small, non-enveloped, icosahedral and double-stranded DNA virus with a genome of 8 kb, belonging to the Papillomaviridae family. More than 10% of human cancers are developed by the infection of HPV . On the basis of potential oncogenic HPVs can be categorized into two subsets: low-risk type and high-risk type . HPV genConsequently, HPV 16 and 18 are included in high-risk HPVs. Out of the eight types of proteins expressed in HPV, E6 and E7 proteins are reported as cooperative viral oncoprotein due to their expression in all HPV types. Naturally occurring chimeric proteins have a tendency to cause cancer hence, they are known as oncoprotein . The HPV E6 protein is known as a potent oncogene and is closely allied with the events that result in the malignant transformation of virally infected cells . The HPV E6 and the E7 proteins modulate cellular proteins that control the cell cycle. The E6 oncoprotein binds to the p53 tumour suppressor protein and targets it for accelerated ubiquitin-mediated degradation and telomerase activity . E6 oncoprotein disturb transcriptional pathways, disrupt cell adhesion and architecture, inhibit apoptosis, abrogate DNA damage responses, induce genome instability and immortalize cells .Based on their association with cervical cancer and precancerous lesions, HPVs can also be grouped into high-risk and low-risk HPV types. Low-risk HPVs are types: 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81. These types can cause genital lesions but are considered non-carcinogenic types as they are not associated with cancerous lesions and are very rarely associated with precancerous lesions. HPV 6 and 11 are the most common cause of genital condylomas . High-risk HPVs are types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 73, and 82. These high-risk HPVs are associated with cervical as well as other anogenital cancers, and they are referred to as the carcinogenic or oncogenic HPV types. HPV 16 and 18 are the most common risk factors in cervical cancer; however, infections with HPV 16 or 18 do not always result in cancer .HPV is the well-known risk factor for the development of cervical cancer. Globally, 99.7% prevalence of HPV has been reported in cervical cancer. The rate of cervical cancer is higher in South-Eastern Asia with excessive burden especially in India, Latin America and Saharan Africa. . In Western asia specifically the prevalence of HPV infection in Iranian women is 29.3% (871/2969) where the frequency of high risk HPV infection is 584(19.7%) and low risk HPV infection is 453(15.3%) .Prevalence of HPV infection varies among different parts of India. Frequency of HPV infection is 51.3% in Eastern India, 15.5% in Western India, 48.6% in South India and 35.2% in North India  Especially in the western part of india, the frequency of the cervical cancer is high, 31 (59.6%) patients were infected with HPV 16 and HPV 18. Of these 31 HPV-positive cervical cancer patients, 28 (90.3%) were infected with HPV 16 and 3 (9.7%) were infected with HPV 18 . HPV 16 and HPV 18 positivity was observed in 56% and 15% cases, respectively, whereas 6% of the cases showed co-infection with both these types .Natural product is a chemical compound or substance produced by a living organism that is, found in nature. It has been a rich source for novels explored in drug discovery . These compounds show superior structural diversity, bioactivity and more complexity as compared to the compounds present in synthetic drug libraries. They have the capacity to constrain some targets which are considered “undruggable”, such as protein-protein interactions; all the natural products are secondary metabolites. Recent studies have clearly indicated the role of vitamins exhibits the antiviral property . Natural products and synthetic drugs limit the overlapping. Drug repurposing study based on artificial nural network has shown higher affinity towards post viral prognosis  These characteristics not only indicate the potential for new targets for therapy but also can help reduce the cost of the development of new treatments since these molecules already exist in nature, and these compounds offer additional options for combination therapiesIn this study, we are focusing on a computational approach and target oncoprotein with plant-derived natural compounds. Computational approaches for drug discovery and development are valuable and significant tools. Several computational methodologies are appropriate in the identification and investigation of new drug candidates . We also performed the evolutionary analysis of different variants of E6 oncoprotein.Protein and ligand preparationThe high resolution three dimensional X-ray crystal structures of the E6 oncoprotein of HPV-16 retrieved from protein data bank (PDB) (http://www.rcsb.org/) using their accession IDs 2FK4. For plant-derived natural anticancer compounds, NPACT database is crucial; 101 natural compounds have been selected from this database (Table S1), which were further assembled into data-sets and used for the docking purpose. The protein and natural compounds were prepared for molecular docking using AutoDockTools 1.5.6. and OpenBabel 3.1.1 consequently.Molecular docking studyThe binding affinity of natural compounds with the E6 oncoprotein of HPV 16 targets was determined by molecular docking method. The molecular docking was performed using blind docking method in Autodock Vina 1.5.6 .The grid box is outsized enough to protein structure to encounter any probable protein-ligand interactions. The binding poses were clustered and ranked in the order of their binding affinities. The molecular interactions (hydrogen bonds and hydrophobic interactions) between the target proteins and compounds were studied using LigPlot + version 1.4.5 .Multiple sequence alignment and phylogenetic analysisThe multiple sequence analysis and phylogenetic analysis of low-risk and high-risk HPV E6 protein were performed. The protein sequence of 28 E6 oncoprotein was retrieved from the UniprotKB database (https://www.uniprot.org/) . MultiplCurrently, there are various conventional therapies for HPV infection, but they all of them have side-effects. More than 50% of all the drug forms used in the clinical fields around the world have originated from the compounds extracted from plants. Secondary metabolites of plants are known as splendid sources of medicinal compounds. Plants based compounds play a significant role with no side effects. In this bioinformatics study, the virtual screening of ligands against E6 oncoprotein achieved and Withnolide D, Ginkgetin, Theaflavin, Hesperidin, Quercetin-3-gluconide, Silymarin, Epigallocatechin gallate (EGCG), Flavonol 3-O-glycoside, Ginkgolides B and Curcumin were identified as potential inhibitors of HPV 16 E6 protein See . Among aAnother promising compound Epigallocatechin gallate (EGCG), exposed impartial interaction in perspective to 10 H-bonds visualized in Multiple sequence alignment commonly used algorithms for the set of biological sequences . The aim of a MSA method is to align the sequences in a way that will either reflect their evolutionary, functional or structural relationship . In multiple sequence alignment, zinc finger domain and PDZ binding motif were identified in HPV E6 . The CXXPDZ domain (X-T/S-X-V) recognition motifs encoded by pathogenic viruses, including high-risk HPVs, target several cellular proteins via protein-protein interactions and those PDZ-containing proteins for proteasome-mediated degradation. In this study high-risk HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV58, HPV68 and HPV73:probable risk HPV53 and low risk HPV43 and HPV70 contains PDZ domain; these HPV-PDZ interactions are in part associated with virally induced cancer progression .Phylogenetic analysis offers a detailed understanding of species evolution through genetic changes, analyses the path that joins an organism with its ancestral origin, as well as can predict the genetic variance that may occur in the future. In this study phylogenetic analysis clearly revealed that the high and low-risk strains of HPV E6 proteins have diverse branching . The phyRetreatment with platinum-based combination therapy offers some improvement in survival as compared to no therapy. A randomized trial of cisplatin versus the combination of cisplatin and paclitaxel demonstrated improved response rates for the combination but no impact on survival, which remained in the 8–9 month range. Recent trials in this patient population include the addition of targeted therapies such as cetuximab (GOG207), taxanes (GOG240) or immunotherapies (GOG265). HPV-induced progressive diseases are associated in absence of strong HPV-specific CD4+ and CD8+ T cell response and rather, are infested with immunosuppressive cells . In some circumstances, the balance of positive and negative immune factors may be changed, leading to clearance of lesions. This is an area where therapeutic vaccines are used against HPV.Viruses behaving as oncogenes through a variety of molecular processes end up with neoplasia. HPV oncoprotein, E6, forms complexes with cellular proteins and triggers modulation, such as mitigating telomere shortening, immortalization, host cell differentiation, controlling cellular pathways, regulating growth factors, tumour suppressor degradation and inactivation, disruption with DNA repair efficiency, and apoptosis and facilitate cell transformation, and increment of the hTERT gene . HPV is the key factor for developing cervical cancer in women. One of the most serious and lethal malignancies in women is caused by HPV. Although, surgery, radiation treatment, hormone therapy, chemotherapy combination therapies, and immunotherapy are all possibilities for treating early-stage cervical cancer, there is lacuna for effective prognosis thus no effective cure for a persistent HPV infection. One of the therapeutic options for cervical cancer is herbal extracts; numerous researchers have explored the influence of plant metabolites on cancer treatment. Plant derived herbal metabolites may be one of the solutions for targeting drugs for the HPV driven cancers. Various plant-derived substances have conventionally been investigated separately as promising resources against HPV-caused malignancy. The discovery of new inhibitors from herbal remedies against malignancies such cervical cancer might be possible using benefits from advances in computational approach. Computer-aided drug discovery (CADD) is a valuable tool for analysing the binding interactions between a ligand and its target protein, and it has merged as a dependable, cost-effective, time-saving, and fast method for pharmaceutical research . The existing virtual screening strongly addresses promising top 10 natural compounds Withanolide D, Ginkgetin, Theaflavin, Hesperidin, Quercetin-3-glucoside, Silymarin, Epigallocatechin gallate (EGCG), Flavonol 3-O-glycoside, Ginkgolides B and Curcumin against E6 oncoprotein. Withanolide D is a flavonoid present in Withania somnifera; it’s anticancer potential is also utilized in breast cancer, head and neck cancer, colon cancer, leukaemia, prostate cancer, thyroid cancer and cervical cancer . EGCG is a polyphenol present in Camellia sinensis. It has the potential against various cancers including colorectal cancer. To enhance the apoptosis . Ginkgetin is a type of bioflavonoid present in the leaves of Ginkgo biloba. Several studies have reported ginkgetin as an anti-cancer drug, anti-tumor potential and anti-viral drug . The flavin is a type of bioflavonoids present in Camellia sinensis. It has an ability to induce apoptosis and inhibit angiogenesis . Hesperidin is a flavanone glycoside found in citrus fruits. It is an economical by-product of citrus production and one of the most essential bioflavonoids in sweet orange and lemon. Hesperidin has the potential to fight against various cancers . Quercetin-3-glucoside is plant flavonoids present in Salicornia herbacea . Flavonol 3-O-glycoside found in Camellia sinensis plants. And it shows anti-inflammatory, anti-cancer and anti-oxidative potential . Ginkgolides B is a biologically active terpene lactone present in the Ginkgo biloba plant. Curcumin polyphenol present in the Curcuma longa plant. Curcumin has been described as a potent antioxidant and anti-inflammatory agent. Evidence has also been presented to suggest that Curcumin can suppress tumour initiation, promotion and metastasis . Quercetin-3-glucoside shows inhibitory effects against various cancers such as breast cancer, head and neck cancer, colon cancer, pancreatic cancer, liver cancer, blood cancer and cervical cancer . Silymarin is a bioflavonoid present in Silybum marianum. Silymarin also has a variety of anti-cancer as well as antiviral properties . Few recent in-silico analysis on HPV show the importance of computational approach in drug designing. Curcumin and EGCG also reported against E6 oncoprotein of HPV. Curcumin, Epigallocatechin-3-gallate (EGCG), Jaceosidin, Resveratrol, Indole-3- carbinol, Withaferin A, Artemisinin, Ursolic acid, Ferulic acid, Berberine, Gingerol, and Silymarin are possible effective sources of cancer treatment . Colchicine, Curcumin, Daphnoretin, Ellipticine, Epigallocatechin-3-gallate have potential against E6 and E7 oncoproteins of HPV 16 and HPV 18 . Withaferin A, Silymarin, Ferulic acid EGCG , Indole-3- carbinol, Artemisinin, Jaceosidin, Resveratrol, Ursolic acid, Berberine, 5 Gingerol - Curcumin are inhibitor against E6 oncoprotein of HPV 18 . Artemisinin, Withaferin A, Ursolic acid, Ferulic acid, EGCG, Indole-3-carbinol, Silymarin, Indole-3-carbinol and Gingerol have the capability of inhibiting the E6 oncoprotein of HPV 16 and HPV 18 .The E6 oncoprotein is the prime hotspot region involved in carcinogenesis. The PDZ domain-binding motif appears to be particularly crucial for neoplastic transformation in cultured cells, transformation of primary human keratinocytes, and hyperplasia and carcinogenesis in E6-transgenic mice . C-terminal PDZ-binding motif is particularly conserved among E6 proteins of high-risk HPVs . However, the multiple sequence alignment has shown the presence of PDZ-binding motif in all high-risk types along with low-risk HPV 43, HPV70 and probable risk HPV 53. The study of evolutionary links among molecules, traits, and species is known as phylogenetic analysis. The physicochemical characteristics of nucleic acids or amino acids are critical factors that impact their structures or activities, therefore it may give function prediction in the case of infections and pathogenicity. Relationship study of some isolates, as seen in the phylogenetic tree, may yield different results when other genes or proteins are employed as points of reference. The current study reveals the existence of a PDZ binding motif in low-risk HPV 43, HPV 70, and HPV 53, which makes them plausible disease-causing candidates, leading to evolutionary proximity to high-risk HPV. The determination of related phylogenetic categorization or the naming of HPV types is based on one of the most conserved L1 genes . One more study shows the relation of polymorphism in HPV and infection. The phylogenetic tree results for HPV E6 variant proteins revealed a distinctive pattern association, In this study HPV 72, HPV 81, HPV 56 and HPV 53 exhibited inconsistency for which branch it belongs to and revealed that HPV E6 variants have various biological and biochemical consequences , which have changed carcinogenic potential. Computational characterization of human papillomavirus variations plays a crucial role in the HPV driven cancer prognosis, development of vaccines, and other treatment approaches to address virus-induced disorders. The present study limits the pharmacogenomics approach to determine the specific mechanism and downstream pathways.In conclusion, within the limitations of the present study, Withnolide D, Ginkgetin, Theaflavin, Hesperidin, Quercetin-3-glucoside, Silymarin, Epigallocatechin gallate (EGCG), Flavonol 3-O-glycoside, Ginkgolides B and Curcumin were identified as potential inhibitors of HPV 16 E6 protein. The inhibitory effect of natural compounds against E6 protein has been studied , thus, advancements in computational science and bioinformatics are beneficial for the study of novel inhibitors from natural sources. The E6 protein of HPV 16 inactivates p53; therefore, the process of gene regulation is disturbed, which is a fundamental cause of cervical cancer. Thus, E6 protein of HPV-16 is of considerable interest for discovery and designing of novel molecules to overcome the challenges. This computational analysis reveals that it is a promising platform for developing anti-cancerous competitive inhibitors targeting HPV. In-silico analysis of E6 protein reveals that all HPV E6 proteins contain PDZ domain and zinc finger domain. The phylogenetic analysis demonstrates that HPV E6 low risk protein and high risk protein are distinct from each other except HPV 73, HPV 72 and HPV 81.In future, this computational characterization will have significant impact on wet-lab studies.The conception and design of study by SS & MP,acquisition of data, analysis and manuscript drafting by TJ, interpretation of data by TJ & MP and manuscript editing by SS.


Transportation barriers limit access to cancer care services and contribute to suboptimal clinical outcomes. Our objectives were to describe the frequency of Veterans reporting and the factors associated with transportation barriers to or from colorectal cancer (CRC) care visits.Between November 2015 and September 2016, Veterans with incident stage I, II, or III CRC completed a mailed survey to assess perceived barriers to recommended care. Participants who reported difficulty with transportation to or from CRC care appointments were categorized as experiencing transportation barriers. We assessed pairwise correlations between transportation barriers, transportation-related factors , and chaotic lifestyle , and used logistic regression to examine the association between the reporting of transportation difficulties, distance traveled to the nearest Veterans Affairs (VA) facility, and life chaos.p = 0.19), but chaotic lifestyle was both positively and significantly correlated with experiencing transportation barriers . Results from the logistic regression model modestly supported the findings from the pairwise correlations, but were not statistically significant.Of the 115 Veterans included in this analysis, 18% reported experiencing transportation barriers. Distance to the VA was not strongly correlated with the reporting of transportation barriers  and with support services . Transportation barriers, including availability and cost of transportation and distance and time traveled to care , limit aTransportation barriers and subsequent health consequences disproportionately affect older adults –11, patiVeterans Health Administration (VHA) research has supported findings related to the demographic  , 18, geoTo address these gaps in knowledge, the objectives of this study were to determine how widespread transportation barriers were among Veterans with CRC and to assess the patient-level and transportation-related factors associated with the reporting of transportation difficulties.We used the Veterans Affairs (VA) Cancer Cube, which contains diagnostic, clinical, and treatment information about suspected cancer cases , 22, to The Colorectal Cancer Patient Adherence to Survivorship Treatment (COAST) survey assessed perceived barriers to, and adherence with, recommended care . BetweenThis study was approved by the Durham VA Health Care System Institutional Review Board. All participants included in this analysis provided implied consent by returning a completed survey and completed a Health Insurance Portability and Accountability Act (HIPAA) authorization form.n = 5).The primary outcome was reporting difficulty with transportation to or from CRC treatment or a related appointment. Veterans who responded “Sometimes”, “Often”, or “Always” were categorized as having experienced transportation barriers, while those that selected “Never” were classified as having no transportation barriers . Consistn = 38), we classified race as white or persons of color . In addition, Veterans who reported full- or part-time employment were categorized as working. Veterans’ marital status was classified as married (married or living together), divorced or separated, widowed, or single (single or never married).Self-reported demographics included age, sex, race, education, employment, and marital status. Veterans were categorized as either 40 to 65 years of age or 66 years or older at time of survey completion based on their year of birth and date of survey completion. Due to a limited number of racial and ethnic minority respondents  encompasses individuals’ ability to organize and anticipate future events, as well as the consistency and predictability of their daily schedules , 29. ChaWe conducted statistical analysis in SAS version 9.4 . We examined differences in demographic, lifestyle, and transportation-related characteristics between Veterans who did and did not report transportation barriers using chi-square or Fisher’s exact tests. We also assessed pairwise correlations between transportation barriers, transportation-related factors, and chaotic lifestyle. Lastly, we used logistic regression to evaluate the association between the reporting of transportation barriers, distance traveled to the VA, and chaotic lifestyle.Of the 115 survey respondents that were included in this analysis  of Veterans reported experiencing transportation barriers to or from CRC care appointments. Demographics, transportation-related factors, and lifestyle characteristics were similar between Veterans who did and did not report transportation barriers, although Veterans experiencing difficulties with transportation were more likely to be younger  and less likely to be married  than those without barriers.Approximately 18% (p = 0.187), but mode of travel was significantly correlated with reporting transportation barriers   and distance to the nearest VA .Distance to the VA was not strongly correlated with reporting transportation barriers to or from CRC care visits   and lifestyle characteristics  associated with reporting transportation barriers among Veterans with CRC. Findings indicated that mode of travel was significantly correlated with transportation barriers to or from CRC care appointments, suggesting that Veterans who rely on family, friends, or public transportation may not have consistent access to or use of reliable transportation. Treatment side-effects  , 32, 33,Although transportation services to or from cancer-related appointments  have eased the burden of both patients and their caregivers , 37, nonWe also found that distance to the nearest VA facility was not significantly correlated with the reporting of transportation barriers to or from CRC care appointments. This finding suggests that Veterans may have accessed community services located closer to their homes or travel distance may not be a substantial deterrent to the care and support received at VA hospitals or clinics. Some Veterans have cited the importance of the patient-provider relationship , 40 as aIn some cases, Veterans live more than 100 miles from the nearest VA facility and distance may become a more pronounced barrier to or from care with each CRC treatment or surveillance appointment , 18. DueFinally, we noted that a chaotic lifestyle was significantly correlated with the reporting of transportation barriers to or from CRC care appointments. A chaotic lifestyle may affect Veterans’ ability to identify, schedule, and manage access to reliable transportation; however, transportation instability  may disrupt Veterans’ lives and yield a higher degree of life chaos. Future research is needed to explore this potentially endogenous relationship, as well as to discern which facet of a chaotic lifestyle is the most challenging for Veterans experiencing transportation difficulties and which transportation-related factor is driving life chaos . In turnThis study had several limitations. First, a small sample size and inability to adjust for potential measured confounders  contributes to lack of statistical significance and limits the interpretation and validity of our findings. Future studies should survey a broader population of Veterans with CRC to enable a robust examination of the factors associated with the reporting of transportation barriers. Second, our study focused on a regional sample of Veterans with non-metastatic CRC who had a valid home mailing address, which further limits the generalizability of our findings. Future research is necessary to determine if the frequency of and factors associated with reporting transportation barriers differs among female Veterans and those with advanced disease. Third, based on the definition of the primary outcome, our study did not determine how the association between life chaos and transportation-related factors may have varied across the degrees  of experiencing transportation barriers. Fourth, we measured distance to and convenience of respondents’ nearest VA location; however, we were not able to discern if this facility was the primary provider of CRC services. Fifth, similar to all surveys, our study may be subject to potential recall and response biases; however, the use of validated and previously tested measures may have minimized the effect of these biases . Sixth, Our findings suggest that transportation is an important barrier to or from CRC care visits, especially among Veterans who experience greater life chaos. Future research is needed to determine the association between perceived life chaos and receipt of CRC care . However, screening for life chaos should be integrated into routine clinical practice. Identification of Veterans who experience chaotic lifestyles would allow for timely intervention – organizational skills training, patient navigation, and/or telehealth services – which in turn, could lead to the potential modification of observed risk factors and support the continuity of care across the cancer care continuum , 46, 47.


Human papillomavirus (HPV) infection can cause various cancers and can be prevented through vaccination. The American Cancer Society (ACS) has set an HPV vaccination completion target in 13-year-old children to 80% by 2026. While HPV vaccine coverage (proportion ever vaccinated) estimates are available, annual uptakes (proportion initiating vaccine in a year) in the United States (U.S.) are not well-known.We analyzed MarketScan® claims database to assess HPV vaccination uptakes in the U.S. among the 9- to 26-year-olds in 2006–2016. The annual uptake was the ratio of the number of enrollees who had a first record of an HPV vaccine during the year, and the number of enrollees of similar age and sex that year.Uptake was below 1% among children turning 9 and 10 years old during the year. Since 2009 among female and since 2013 among males, the annual uptake has been the highest in those turning 13 years old . Catch-up vaccination among older adolescents and young adults increased after Advisory Committee for Immunization Practices (ACIP) recommendations, but eventually slowed down as more younger persons were vaccinated. Most young adolescents were vaccinated by pediatricians, whereas young adult women were predominantly vaccinated by obstetricians/gynecologists and young adult males by family physicians. While only about half of the adolescents had well-check visits, the majority of those who initiated HPV vaccination had one the same year.Continued increase in uptake is needed to reach the ACS 2026 goals.The online version contains supplementary material available at 10.1186/s12889-021-11664-1. Recent data indicate that approximately 14 million persons are newly infected with human papillomavirus (HPV) each year in the United States (U.S.), and approximately 79 million persons are currently infected . In the To date, three HPV vaccines have been approved in the U.S. for the prevention of HPV-related diseases: a quadrivalent (4vHPV), a bivalent (2vHPV), and nonavalent (9vHPV) vaccine –7. The 4The U.S. Advisory Committee on Immunization Practices (ACIP) recommended routine HPV vaccination of girls at the ages of 11 or 12 in 2006 . VaccinaIn this study, we assess HPV vaccination uptake since the first HPV vaccine was introduced in 2006 until 2016, within the 9- to 26-year-old population. We also examine who administered the vaccine, and whether vaccines are initiated during a well-check visit.This retrospective analysis was based on the IBM® MarketScan® claim database which comprises prescription data in addition to inpatient and outpatient medical utilization for millions of commercially insured individuals across the U.S. In 2016, the database covered approximately 10% of the total U.S. population aged 9 to 26 years old. It was selected because of its size, geographic representativeness, and diversity of plan types. IBM provided the de-identified individual-level data which are fully compliant with U.S. privacy laws and regulations, i.e. the Health Insurance Portability and Accountability Act (HIPAA). As a result, this study was IRB exempt, and no IRB approval was necessary.The annual HPV vaccination uptake was defined as the proportion of unique enrollees who had a first claim for a dose of the HPV vaccine (Current Procedure Terminology (CPT) code: 90649 (4vHPV), 90651 (9vHPV), and 90650 (2vHPV) during the year. Age of the subject was determined by difference between calendar year and year of birth so that the 1997 newborns were the 9-year-olds in 2006 (the database only provides the year of birth to protect anonymity). Since the HPV vaccine is indicated at 9 years old or above, we could assume, in 2006 for example, that everyone from birth cohort 1997 who was vaccinated was 9 years old. However, in 2006, subjects from birth cohort 1996 could be vaccinated either at 9 or 10 years old depending upon whether they had the vaccine before or after their 10th birthday, and therefore were referred to as the 9–10-year-olds. Each year, females and males turning 9 to 26 years old were included, for example,, in 2006, enrollees who were born between 1980 (turning 26 years old) and 1997 (turning 9 years old). Women with an International Classification of Disease (ICD 9/10) code for pregnancy  or CPT procedure codes listed in Supplementary material Table  were ideIn the Marketscan® claims database, there were 118 million unique enrollees who turned 9 to 26 years old from 2006 to 2016. Slightly less than half (49.4%) were females. Figure The percent of subjects with a documented well-check visit by age and sex for 2016 is reported in Table Figure We assessed HPV vaccine uptake in the U.S. within a large claims database covering several million 9 to 26 year-old children and young adults between 2006 and 2016. The study includes all the licensed HPV vaccines , and theOur results seem consistent with another claim-based study by the Blue Cross Blue Shield Association , that reThe study provides insight into how a new ACIP recommendation for a vaccine is implemented in a real-world setting. When the HPV vaccine was approved for women in 2006, a broad slice of the population across different age groups got vaccinated. There was catchup vaccination uptake in older age group, with 10.6% of those turning 20 years old and 4.5% of those turning 26 years old receiving a dose of the vaccine. Over time, more and more teenagers were vaccinated at younger ages which means less were available for vaccination as older adolescents and young adults. This translated into higher uptake for young adolescents  and lower uptake at older ages (1.1% of those turning 26 years old) in 2016. This pattern can be observed through the heat map. As vaccination is initiated, the hue is light green across a broader age range, including older adults. Moving to the right the cells start turning dark green for young adolescents indicating steadily increasing vaccination uptake for this group, whereas it gets lighter for older adolescent and young adults  program. We also could not identify vaccines not submitted for insurance reimbursement nor those that were received out-of-network as for example, enrollees were not all continuously in the database since 2006. Only non-missing values were considered, and no imputation of missing information was carried out. Lastly, only birth year was available so vaccine coverage by exact year of age could not be determined.We found that the ACIP vaccine recommendations directly impact HPV vaccination uptake in the population. When the HPV vaccination program started in 2006 for females and 2009 for males, there was catch-up in a broader age-group, followed by an increase in vaccine uptake among targeted young cohorts. While females had a head start (with early inclusion in the label for HPV vaccination) vaccination uptake in males is fast catching up. While uptake in young adolescents has been increasing, coverage is far below the ACS 80% goal, and catch-up vaccination in older cohorts is likely to continue into the near future as many children remain unvaccinated as they get older. Pediatricians, followed by family physicians remain the primary vaccine administrators for adolescents. Obstetrician/gynecologists and family physicians commonly vaccinate young adult women and young adult men, respectively. Thus, interventions to improve uptake should consider focusing on pediatricians and family physicians for children, and obstetrician/gynecologists and family physicians for young adults. Interventions to improve access to well-check visits and HPV vaccination within well-check visits should also be considered to improve HPV uptake. Our study provides an analysis of how many children and young adults get vaccinated, when, by whom, and in what context. This can guide the development of appropriate interventions and policies to help improve vaccination uptake and public health.Additional file 1: Table S1. ICD-9/10 codes used to identify pregnancy-related service claims. Table S2. ICD9/10 and CPT codes used to define well-check visits.


HPV is one of the diseases of civilization that causes cervical cancer, among other diseases. For this reason, a vaccination program has been introduced worldwide for preadolescent, sexually inactive seronegative girls. However, the decision to vaccinate young girls must be made by the parents. In Poland, vaccinations are recommended but not financed by the government, which affects their choices, and there is insufficient knowledge of the diseases caused by genital HPV types. In addition, there are cultural, social, and even religious factors to be considered. Therefore, the aim of the study was to analyze the state of knowledge about HPV and HPV vaccines among parents. Two hundred and eighty-eight parents participated in the study, but only 180 of them declared that they had ever heard of HPV (62.5%). Therefore, only these parents completed the entire questionnaire consisting of 34 questions. The parents’ answers were analyzed with the Fisher’s and chi-squared tests. The study showed that parents’ knowledge of HPV and HPV vaccination in Poland is low (49.4% of correct answers). Parents’ attitudes were only influenced by knowledge and education and not by other parameters such as age, gender, place of residence, and the number of children. This study indicates that parents need to be educated about the threats of HPV and the possibilities of prophylactic vaccination. Papillomaviridae and are non-enveloped icosahedral, circular, dsDNA viruses [Human papillomaviruses (HPVs) belong to the family  viruses . HPVs in viruses . There a viruses , most of viruses ,4. Among viruses . In the  viruses .In 2012, there were 530,000 cases of cervical cancer related to HPV, of which 370,000 were caused by HVP16/18 (71%) .In the European Union, about 34,000 new cases of cervical cancer related to HPV are diagnosed every year. The highest prevalence of cervical cancer associated with HPV infection are observed in Latvia , Bosnia and Herzegovina 23.9/100,000), and Estonia . On the other hand, the lowest prevalence is found in Malta , Switzerland , and Finland . In Poland, the prevalence is 9.4/100,000 women [/100,000,Currently, three vaccines are used in the world: 1. Cervarix (GlaxoSmithKline), a bivalent (2-V) vaccine targeting HPV16 and HPV18, the two most carcinogenic types; 2. Gardasil , a quadrivalent (4-V) vaccine targeting HPV16/18 and the low risk types, HPV6 and HPV11, that cause genital warts; and 3. Gardasil 9 (Merck Inc.), a nonavalent  vaccine targeting HPV6/11/16/18 and the next five most carcinogenic types (HPV31/33/45/52/58). Most recommendations, including from the WHO, recommend routine HPV vaccination in girls aged 9–14 years before becoming sexually active. The secondary target group is girls over the age of 15 years and young women. HPV vaccination of males is currently not recommended as a priority ,11. HoweHPV vaccination was first introduced in 2007 in Australia and, since then, many countries have joined the vaccination program. In 2020, HPV vaccination has been introduced in 107 (55%) of the 194 WHO Member States. In the Americas and Europe, 85% and 77% of the countries have already introduced HPV vaccination. A lower number of countries with a HPV vaccination programme was observed in Oceania (56%), Asia (40%), and Africa (31%) .The implementation of an HPV vaccine programme has created many controversies. The two most frequently cited sources of negative knowledge about HPV vaccination are the Canadian article “Guinea pigs” and the Danish paradocumentary “De Vaccinerede Piger” (The Vaccinated Girls—Sick and Abandoned). The article describes the vaccination programme as “the biggest Canadian science experiment in decades”. The paradocumentary tells the story of some girls who allegedly had a reaction to a HPV vaccination and suffered from POTS—postural orthostatic tachycardia syndrome. The paradocumentary presents the HPV vaccine in a very negative light, although no studies confirmed the described symptoms.The vaccination coverage level depends on at what age the first dose of vaccination is being administered (higher coverage level in younger groups). However, in general, a high level of vaccination coverage was observed in Australia (78.6%) and in the United Kingdom (81%), in contrast with countries with low vaccination coverage level such as Georgia (36.2%), Lithuania (29%) , and PolLack of knowledge about HPV infections and vaccines.a.lack of recommendations from the National Health Fund (NHF) and doctors,b.“bad attitude” towards vaccination in anti-vaccine environments, c.lack of support and conversations with parents about sexuality.Motivational obstacles for vaccination, including:a.availability of vaccination,b.the price of the vaccine,c.the need to repeat vaccination (compliance).Logistical barriers, including:a.sexual promiscuity,b.negative information about the vaccine in the media .Myths about the vaccine—mistaken beliefs, including:Until now, Poland was one of the EU countries where vaccination against HPV was recommended but not government-funded . There aOf these many barriers, the lack of support and conversations with parents about sexuality deserves special attention. This issue is critical because prophylactic vaccination of girls against HPV should be carried out in the period preceding sexual initiation. Therefore, the decision to vaccinate girls is the responsibility of parents who, by giving their consent, give them the chance to avoid a disease that is dangerous to their health and even their life when they become adults. The attitudes of parents and adolescents, using many research models ,16,17, hCervical cancer remains high in incidence and mortality rankings in Poland. Despite this, HPV vaccines are not covered by the government. Many local governments in Poland organize free HPV vaccination programs, but HPV immunization remains low.This study, the first of its kind to be undertaken in Poland, undertook an analysis to determine the barriers to vaccination acceptance. This study aimed to assess parents’ knowledge about HPV infections, assess parents’ knowledge about HPV vaccines, and assess the impact of parents’ knowledge about HPV infection and HPV vaccines on their attitude to primary prevention, i.e., vaccinations.An observational cross-sectional descriptive study was undertaken.All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Medical University of Warsaw (AKBE/123/16).The survey was conducted in 2018 in Warsaw, Poland, and included parents of the children admitted to the Department of Pediatric Dermatology at a multidisciplinary regional hospital.Data was collected using questionnaires presented on electronic devices  during the admission of the patients to the Department of Pediatric Dermatology. A researcher who monitored the study and helped with technical problems was present to ensure that only one parent was involved in the study.Four parents refused to fill in the questionnaire, answering “no time” as the reason. The study was completed when questionnaires from a minimum of 200 parents were obtained. None of the children of the surveyed parents were diagnosed with HPV-related lesions as a reason for admission to the department.A goal of the study was to include as many participants as possible. At the initial stage, no formal calculation of sample size was carried out. The final number of 288 parents ensured precision  of 4 percentage points for assessing a trait whose true prevalence was 50% .The analysis was conducted mainly with the use of descriptive statistics. The results are presented in the form of frequency tables and cross tables. Statistical inference was performed using the chi-squared test or, in the case of low frequencies of the analyzed features, Fisher’s exact test.p = 0.05.Fisher’s exact test is used in the case of samples that are too small—when the observed values calculated with the chi-squared test are below 5. The calculations were performed using the statistics program R 3.5.1. All tests were performed at a significance level The parents’ responses regarding their knowledge about HPV were presented depending on their age, sex, their place of residence, and education. The parents’ knowledge about vaccination was also analyzed. The parents’ responses to the questions regarding their knowledge about vaccinations were classified as correct or incorrect. Total test result and results regarding the parents’ knowledge about HPV and HPV vaccine was expressed as a percentage of available points.The questionnaire survey  for pareThe survey consisted of both single-choice and multiple-choice questions. The age question was an open-ended question. The rest of the questions were closed questions. Seven questions related to their knowledge about the HPV virus, and seven questions related to their knowledge about the HPV vaccine. The rest of the questions were about the parents’ attitudes to vaccination and demographic data. One hundred and eighty of the surveyed parents declared that they had heard about the HPV virus and filled in the entire questionnaire. The rest of the respondents answered “no” or “I do not know” and were asked to provide their age, gender, place of residence, and education.The study included the parents of the children admitted to the Department of Pediatric Dermatology in Warsaw, Poland. Two hundred and eighty-eight parents participated in the study. Most of the respondents were women (78.8%). Parents between the ages of 30 and 40 years accounted for 61% (n = 155) of the respondents, parents between the ages of 20 and 30 years accounted for 24% (n = 61), and parents between the ages of 40 and 70 years accounted for 15% (n = 38).Furthermore, 38 parents (13.2%) lived in the countryside, 26 (9%) lived in a city with up to 20,000 inhabitants, 33 (11.5%) lived in a city with 20,000–100,000 inhabitants, 36 (12.5%) lived in a city with 100,000–500,000 inhabitants, and 155 (53.8%) lived in a city with more than 500,000 inhabitants.Most of the respondents had higher education . Sixty-eight people had secondary education (23.6%), 13 people had basic vocational education (4.5%), and 2 people had primary education (0.7%). A summary of these data is presented in Among those surveyed, 180 declared that they had heard of the HPV virus and filled in the entire questionnaire. The characteristics differentiating these two groups are presented in From an analysis of the second part of the table, 2.8% of the parents achieved a score of up to 30% of correct answers, 47.8% scored in the range of 31–60 (n = 86), and 49.4% scored in the range of 61–100 (n = 89). Therefore, as many as 50.6% of parents would not pass the test if the pass mark for this test was a score above 61% of correct answers.From an analysis of the second part of the table, 2.2% of parents achieved a score of up to 30% of correct answers, 32.8% scored in the range of 31–60, and 65.0% scored in the range 61–100. Therefore, 35% of parents would not pass the test if the pass mark for this test was a score above 61% of correct answers.From an analysis of the second division, 6.7% of parents achieved a score of up to 30% of correct answers, 56.7% scored in the range of 31–60 (n = 102), and 36.7% scored in the range 61–100 (n = 66). Therefore, 63.4% of parents would not pass the test if the pass mark for this test was a score above 61% of correct answers.There were no significant statistical differences between the groups identified by the place of residence and the number of children. Considering the remaining characteristics , parents with a higher education and women had more correct answers, which was statistically significant. Parents with a higher education had an overall higher test score and statistically more correct answers to questions about vaccine funding and vaccine availability in Poland and identified girls over the age of nine years as the target group for the vaccine. The number of correct answers decreased with the age of the parents.Women scored a statistically significant higher percentage of correct answers regarding the availability of the vaccine in Poland and a statistically significant higher percentage of correct answers regarding vaccine funding and identified young girls before sexual initiation as the target group for the vaccine.There were more statistically significant differences for incorrect vaccine responses between the education groups. People with a higher education had overall higher test scores and statistically answered more questions correctly about preventing or reducing the possibility of an HPV infection and about identifying the routes of transmission of the virus . Women correctly identified most of the cervical cancer risk factors. The number of correct answers decreased with the age of the parents.The only factors differentiating the attitude towards vaccinations were knowledge and education. Parents with a test score in the third quartile indicated that the high effectiveness of the vaccine might influence their decision to vaccinate their children with the recommended vaccine. The remaining characteristics of the parents  did not significantly affect the attitude to vaccination. It was interesting that 55% of the parents would have vaccinated their children if the vaccination was covered by the government.This research is the first in Poland to assess the knowledge and attitudes of parents towards HPV vaccination. HPV vaccination may prevent diseases related to papillomaviruses, including cervical cancer. Research in this field has been conducted worldwide for many years ,31,32,33It should be emphasized, however, that the attitude of parents may be influenced by many factors, and one of the key influencing factors in Poland is the lack of recommendations and financing by the National Health Fund. According to a study by Gerend et al., people recommended by the NHF to be vaccinated against HPV were forty times more likely to get vaccinated . This isThe lack of recommendation and funding may have a negative impact on parents’ attitudes, especially when the price of the vaccine is high. In these studies, over one-third of the respondents indicated the high price of the HPV vaccine as a possible reason for refusing to vaccinate their children.However, it should be emphasized that in countries with recommendations and a reimbursement scheme, the problem of HPV vaccination is much more complex. As many as 28% of parents refuse to vaccinate in the USA, and 8% delay vaccination. The motivation of both groups for this type of behavior differs significantly. Parents who refused vaccinations gave reasons such as fear of promiscuity, lower vaccine efficacy expected, and higher expected side effects. Parents who delay vaccination do not rule out later vaccination after learning more about the vaccine . In the The state of parental knowledge has a crucial influence on the immunization of children. The study by Fishman et al. proved that mothers with more knowledge about HPV were not willing to vaccinate themselves or their daughters . The preLack of support from parents due to their fear of encouraging girls to engage in risky sexual behavior is also emphasized in the literature, reducing the motivation to vaccinate. Factors increasing parental acceptance of vaccination include HIV testing in the past, having an older daughter, having had more sexual partners, and having a family member with cancer . The mosAn interesting aspect is a conversation with their children about HPV vaccinations as an introduction to sexuality. In the study by Marlow et al., mothers of the girls stated the age of their daughters at which they would like to start discussions with them about vaccinations, sex, cervical cancer, sexually transmitted diseases, HPV, and HPV vaccination . The stuThe price of the vaccine is often discussed in the aspect of barriers that reduce vaccination. In the analysis, the low price of the vaccine was not a determinant for vaccinating children (0 answers). Most parents decided to vaccinate their child after a doctor recommended the vaccine or because the vaccine was very effective. However, in the case of HPV vaccination, more than one-third of the respondents indicated the high price of the HPV vaccine as a possible reason for not taking up vaccination (over two-thirds indicated possible side effects).In the literature, concerns about the cost of the vaccine include costs to be borne by the parents and by healthcare professionals, and costs of the vaccine to be borne by the healthcare insurer . TherefoThis research is the first attempt in Poland to identify the most important barriers to the effective implementation of an HPV vaccination program, and thus to the prevention of diseases connected with this virus. The presented analysis may be of assistance in the implementation of HPV vaccination programs in Poland.Parents’ knowledge of the HPV virus is insufficient and depends on sex, age, and education and is independent of place of residence and the number of children. The parents’ knowledge about the HPV vaccine is low and independent of the place of residence, the number of children, and depends on age, sex, and education. Further studies need to be carried out to provide information regarding pro-vaccine motivation tools.


Escherichia coli, is closely related to the safety and quality of the meat. Some diarrheagenic E. coli can cause serious foodborne diseases, and pose a significant threat to human life and health. In order to ascertain the current status of E. coli and diarrheagenic E. coli contamination during the pig slaughter process in China, we conducted thorough monitoring of large-sized slaughterhouses, as well as small- or medium-sized slaughterhouses, in different provinces of China from 2019 to 2020. The overall positive rate of E. coli on the pork surface after slaughter was very high (97.07%). Both the amount of E. coli contamination and the positive ratio of diarrheagenic E. coli in large-sized slaughterhouses  were lower than those in small- or medium-sized slaughterhouses . Combined with the current status of sanitary control in slaughterhouses, we determined that pre-cooling treatment significantly reduced E. coli and diarrheagenic E. coli in pork after slaughter, while microbiological testing reduced E. coli. Based on our monitoring data, China urgently needs to establish relevant standards to better control microbial contamination during pig slaughtering progress. This study provided a theoretical basis for the hygiene quality management of the pig slaughter industry in China.The microbial contamination of pork during the slaughter process, especially that of the hygiene indicator bacteria,  In recent years, foodborne pathogenic infections have occurred frequently, which has a significant impact on human health and economy, and has become a major public health problem worldwide . FoodborSalmonella, Shigella, diarrheagenic E. coli, Campylobacter, Yersinia enterocolitica, Clostridium perfringens, Vibrio and Listeria monocytogenes, etc , Shiga toxin-producing E. coli/enterohemorrhagic E.coli (STEC/EHEC), Shigella/enteroinvasive E.coli (EIEC), enteroaggregative E.coli (EAEC), and enterotoxigenic E.coli (ETEC), can cause diarrhea, with food transmission as the main route of infection  in 2019 and eight provinces  in 2020; four of the provinces was sampled in both years]. There were 33 large slaughterhouses, and 38 small- or medium-sized slaughterhouses. The configuration of the slaughter cold chain and the development of microbiological testing in the slaughterhouses are shown in The period of May to October was selected for centralized sampling. According to the current situation of large slaughterhouses in China, the slaughtered pork is placed at 4°C for more than 6 h for precooling. In our study, at least 20 carcass surfaces swabbed before precooling, and 25 carcass surfaces swabbed after precooling, were collected from large slaughterhouses. At least 20 carcass surface swabbed samples were collected from small- or medium-sized slaughterhouses without precooling. A total of 1,074 carcass surfaces swabs were collected from large slaughterhouses, and 939 carcass surfaces swabs were collected from small- or medium-sized slaughterhouses. The total number of samples was 2013.2 of cotton swab samples were applied to each sampling site. Environmental samples comprised the surface swabs of facilities and equipment, workers' hands, utensils, and the ground that directly or indirectly came into contact with the pig carcass during the slaughter process. The collected cotton swab samples were loaded into transport medium containing 10 mL phosphate-buffered saline, stored at low temperature, and transported to the laboratory for testing within 24–48 h.Cotton swab  samples (involving pre-cooling) were collected from the surface of carcasses after slaughtering. Three sampling sites  were selected on each sample, and 100 cmEscherichia coli counts were detected using commercial kits according to the industry standard SN/T 4547-2017, specifically using the Petrifilm E. coli test sheet , with reference to the instructions and the methods specified in the standard for detection. In short, 1 mL sample with appropriate dilution was added to the center of the Petrifilm plate, then slowly covering the upper film of the Petrifilm plate. After the sample was evenly covered the Petrifilm plate, place it horizontally at 36 ± 1°C and incubate for 24 ± 2 h. Record the number of blue colonies with gas on the Petrifilm plate and the corresponding dilution factor, expressed in colony forming units (CFU).E. coli. Diarrheagenic E. coli was identified using the multiplex polymerase chain reaction detection kit developed by our laboratory referring the study of Toma et al. . The contamination rate of pathogenic microorganisms was analyzed by the Pert distribution fitting in @RISK7 software , and the amount of E. coli contamination was analyzed by random distribution fitting. The function is obtained by random fitting. Monte Carlo simulation was performed using the Latin hypercube sampling method. Probability distributions were obtained from each time point in simulations of 10,000 iterations.The microbial contamination rate was analyzed by Excel software, and the amount of E. coli were detected and counted in pork after slaughter. Overall, there was a very high positive detection rate of E. coli in pork after slaughter in China, with an overall rate of 97.07% . Due to the fact that 55.26%  and small- or medium-sized slaughterhouses (96.81%).As a hygiene indicator, % 21/38,  small- oE. coli detected in large-sized, and small- or medium-sized slaughterhouses. As shown in E. coli contamination of pork in large-scale slaughterhouses was generally lower than that in small- or medium-sized slaughterhouses. The highest frequency of contamination detected in small- or medium-sized slaughterhouses was 74.99 colony-forming units (CFU)/cm2-133.35 CFU/cm2, while the most frequently detected contamination in large-sized slaughterhouses was 7.50 CFU/cm2-13.33 CFU/cm2, which is exactly an order of magnitude difference. These results suggested that although the detection rate of E. coli in small- or medium-sized slaughterhouses was significantly different from that of large-sized slaughterhouses, the amount of E. coli contamination was still higher than that of large-sized slaughterhouses, indicating that overall hygiene control was poorer than that of large-sized slaughterhouses.We separately compared the data distribution of E. coli in slaughtered pork was randomly distributed and fitted by @Risk RISK assessment software to determine the optimal function, and an exposure assessment was then carried out by Monte Carlo sampling . Meanwhile, the overall contamination rate of diarrheagenic E. coli in pork samples produced by large-sized slaughterhouses was 3.44% (32/931), and that of small- or medium-sized slaughterhouses was 5.71% (25/438)  . These rE. coli count analysis.From a hygiene perspective, after pre-cooling cold fresh meat, the temperature and humidity changes of the carcass surface can deactivate the pathogenic microorganisms contaminated on the surface of the pork during the slaughter process, which better guarantees the quality and safety of the meat. Therefore, in this study, cotton swabbed samples from pork surfaces before and after pre-cooling were collected in some slaughterhouses with pre-cooling treatment for E. coli contamination on the pork surface before and after pre-cooling (E. coli contamination before pre-cooling was 0.95–6.19 log (CFU/100 cm2) or 0.09 CFU/cm2-15,488.17 CFU/cm2; while the average was 3.76 log (CFU/100 cm2) or 57.54 CFU/cm2. After pre-cooling, the 90% probability of E. coli contamination was −0.25–5.19 log (CFU/100 cm2) or 0.005 CFU/cm2-1,548.82 CFU/cm2, and the average was 2.40 log (CFU/100 cm2) or 2.45 CFU/cm2  before pre-cooling and 3.56% (18/506) after pre-cooling  or 1.55 CFU/cm2-15, 4881.66 CFU/cm2; while the average was 4.92 log (CFU/100 cm2) or 831.76 CFU/cm2. In contrast, the 90% probability of the contamination of E. coli with microbiological detection was 0.61–6.23 log (CFU/100cm2) or 0.04 CFU/cm2-16982.43 CFU/cm2; while the average was 3.60 log (CFU/100 cm2) or 39.82 CFU/cm2 , which is similar to the 95.81% positive detection rate before evisceration in the United States  and the European Union in 2017 (4.4%)  for the monitoring of d States . Howevero 11.78% . In Chino 11.78% . Howevero 11.78% ; therefo in 2011 . In addi7 (4.4%) , 24.E. coli contamination was 57.54 CFU/cm2 before precooling and 2.45 CFU/cm2 after precooling. Similarly, the contamination rate of diarrheagenic E. coli in pork before pre-cooling was 4.52%, which was reduced to 3.56% after pre-cooling. Thus, chilled pork will become a meat consumption trend in the future. GB/T 20551-2006 “Evaluating specification on the HACCP certification in the slaughter of livestock and poultry” , establishment of a hygiene evaluation method for pig slaughter, and formulation of an evaluation index system, in order to improve slaughter hygiene, and the quality and safety of pork products.Research by H. Holly Wang et al. has shown that chilled meat and hot meat are the main pork types among Chinese consumers in the cities surveyed, accounting for 45 and 46%, respectively . The prepoultry”  clearly E. coli in large-sized slaughterhouses are comparable to those in developed countries, and the microorganisms in the slaughter process can also be effectively controlled by measures such as pre-cooling, the control standards of E. coli in the pig slaughtering process must be urgently addressed. In fact, China is also taking a series of measures to improve the hygienic control of pig slaughtering process in recent years. For example, firstly, we promotes the standardization of pig slaughterhouses, which requires microbiological monitoring; secondly, we promotes the quality and safety risk monitoring of the slaughter process, which also stipulates the monitoring of common pathogens such as E. coli and Salmonella; thirdly, the overall bio-safety prevention and control measures of the slaughterhouse under the influence of African Swine fever (ASF) have been strengthened. It is believed that the development of standards for the detection of E. coli at the slaughtering stage can directly ensure the health and safety of the pork entering the market, and is also conducive to international standards.Developed countries such as Europe and the United States place great importance on the monitoring, assessment, and prevention and control of pathogenic microbial contamination in livestock and poultry products during the slaughter process, and have successively established a strict scientific regulatory and standards system , 30. HowE. coli in large-sized slaughterhouses is better than that in small or medium-sized slaughterhouses in China, and E. coli contamination in the pig slaughtering chain in large-sized slaughterhouses after pre-cooling is comparable to that in developed countries. Therefore, corresponding standards are urgently needed to better control microbial pollution in pork during the slaughtering process.In conclusion, the control of pork The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This work was funded by the National Key Research and Development Program of China (2018YFD0500505).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.


This is a research article on oropharyngeal cancer (OPC). The aim of the study was to assess and compare basic immune parameters and ratios in patients with Human Papilloma Virus (HPV)+ and HPV− OPC, before and after radiotherapy (RT) or chemoradiotherapy (CRT), and to investigate their impact on overall survival (OS) and disease-free survival (DFS). The higher neutrophil-lymphocyte ratio (NLR) and systemic immune inflammation (SII) are significant adverse prognostic factors for HPV+ OPC patients, because they are significantly associated with both inferior OS and DFS in this group, whereas the higher platelet cells (PLT) count is significant adverse prognostic factor for HPV− OPC patients, because it is significantly associated with inferior OS and DFS in this group. This study confirmed that determination of HPV etiology as well as analysis of various hematological and immune parameters should be a standard management in OPC patients in order to properly treat them for improved prognosis.3), NLR (>2.13), SII (>448.60) significantly correlated with reduced OS, whereas high NLR (>2.29), SII (>462.58) significantly correlated with reduced DFS. A higher pre-treatment NLR and SII were significant poor prognostic factors for both OS and DFS in the HPV+ group. These associations were not apparent in HPV− patients. There are different pre-treatment and post-treatment immune and hematological prognostic factors for OS and DFS in HPV+ and HPV− patients. The immune ratios could be considered valuable biomarkers for risk stratification and differentiation for HPV− and HPV+ OPC patients.Several immune and hematological parameters are associated with survival in patients with oropharyngeal cancer (OPC). The aim of the study was to analyze selected immune and hematological parameters of patients with HPV-related (HPV+) and HPV-unrelated (HPV−) OPC, before and after radiotherapy/chemoradiotherapy (RT/CRT) and to assess the impact of these parameters on survival. One hundred twenty seven patients with HPV+ and HPV− OPC, treated with RT alone or concurrent chemoradiotherapy (CRT), were included. Patients were divided according to HPV status. Confirmation of HPV etiology was obtained from FFPE  tissue samples and/or extracellular circulating HPV DNA was determined. The pre-treatment and post-treatment laboratory blood parameters were compared in both groups. The neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), monocyte/lymphocyte ratio (MLR), and systemic immune inflammation (SII) index were calculated. The impact of these parameters on overall (OS) and disease-free (DFS) survival was analyzed. In HPV+ patients, a high pre-treatment white blood cells (WBC) count (>8.33 /mm It has been proven that significant alterations in the immunological system (IS) are observed in cancer patients. The body’s specific immune response to a cancer leads to various changes in levels of basic immune parameters such as number of white blood cells (WBCs), circulating lymphocytes (CLCs), circulating neutrophils (CNCs), circulating monocytes (CMCs) and platelet cells (PLTs) ,2,3. TheOPC can be associated with a typical risk factor such as smoking and alcohol abuse or with Human Papillomavirus (HPV) infection. HPV-related (HPV+) OPC is fairly responsive to radiotherapy (RT) or chemoradiotherapy (CRT) and has a better prognosis than HPV− ,5.AJCC Staging Manual reflected HPV infection status in determining the clinical stage of OPC [Because of the different nature of HPV+ OPC, the current 8th edition of the American Joint Committee on Cancer e of OPC ,5. It ise of OPC .The aim of the study was to assess and compare basic immune parameters and ratios in patients with HPV+ and HPV− OPC, before and after RT or CRT, and to investigate their impact on overall survival (OS) and disease-free survival (DFS).The analysis included 127 adults with OPC treated at I Radiation and Clinical Oncology Department of Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice Branch, Poland. There were 87 (68.5%) men and 40 (31.5%) women of the mean age of 60.62 ± 8.54 years (range: 30–80 years) in the studied group. The inclusion criteria were as follows: primary OPC , age > 18 years, radical RT or CRT as sole and definitive treatment. Exclusion criteria included: cancer recurrence, initial surgery, incomplete demographic and/or clinical data.The detailed analysis of clinicopathological parameters  in OPC patients is presented in This study was supported by a grant from the National Centre of Research and Development, Poland (grant TANGO2/340829/NCBR/2017). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee  and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.The blood was obtained under standard conditions, the patients in fasting state, between 7:00 and 9:00, by means of a vacuum Becton Dickinson  system, in sample tubes with anticoagulant ethylenediaminetetraacetic acid (EDTA). The full blood count was determined using the Sysmex XN-2000 analyzer .The pre-treatment (0) and post-treatment (1) basic laboratory blood parameters, including hemoglobin (Hb) and ret-hemoglobin (RetHb) levels, red blood cell count (RBC), reticulocyte count (Ret), white blood cell count (WBC), circulating lymphocyte count (CLC), circulating neutrophil count (CNC), and circulating monocyte count (CMC) were compared in both groups and the impact of immune and hematological parameters on survival was analyzed. The neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and monocyte/lymphocyte ratio (MLR), and systemic immune inflammation index (SII) were calculated and correlated with survival in both groups. The SII index was calculated according to the following formula: SII = platelet counts × neutrophil counts/lymphocyte counts . PatientAdditionally, the patients were divided into two subgroups according to the cut-off value of the mean NLR, MLR, PLR, and SII. The mean values of the NLR, MLR, and PLR among the entire study population, as well as HPV− and HPV+ patients, were set as the border value to divide high and low NLR, MLR, and PLR subgroups in order to perform statistical comparisons of clinicopathological findings between these subgroups. Clinicopathological factors were compared between these low and high immune ratio subgroups.Confirmation of the HPV etiology was obtained from tissue material and/or extracellular circulating HPV DNA.Formalin-fixed paraffin-embedded tumor samples were examined for high-risk HPV (HR-HPV) infection using a double-check algorithm including immunohistochemical assessment of P16(INK4A) protein expression followed by detection of HR-HPV DNA in tumor tissue using real-time PCR. Only cases with both p16(INK4A) expression and HR-HPV DNA amplification were classified as truly HR-HPV-positive .g and 1000× g, both at 4 °C for 10 min. DNA was extracted (according to the manufacturer’s instructions) from 1 mL of plasma by the Genomic Mini AX Body Fluids kit . Each measurement consisted of a standard curve of three dilutions of plasmid construct containing HPV16 genome, negative control and a samples. For HPV16 detection, reaction was performed using primers and probe set for the HPV16 genome. PCR reactions were performed using the Bio-Rad CFX96 qPCR instrument . If HPV16 was found, its presence would be confirmed with a second independent DNA isolation.Peripheral blood (12 mL) was collected into K3EDTA tubes . Plasma was separated within an hour by double centrifugation at 300× The stage of OPC was classified according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM classification system ,5.The median follow-up was 74.58 (0.1–165.58) months. Overall survival (OS) and disease-free survival (DFS) were analyzed in both groups.The flowchart diagram of our study is presented in U test was used to compare HPV+ and HPV− groups. The Wilcoxon test was used to compare pre- and post-treatment parameters in all patients and both HPV groups separately. Prevalence and frequency were expressed as number and percentage. Cox proportional-hazards models were used to estimate hazard ratios (HRs) for OS and DFS. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off values for prognostic factors related to DFS and OS. Youden’s index was selected as the approximate cut-off value for each parameter. The Kaplan–Meier curves were constructed for comparison of OS and DFS between the two groups.The categorical variables were presented as numbers and percentages. Continuous variables with normal distribution were expressed as the means and standard deviations. The Shapiro–Wilk test was used to determine statistical distribution in the analyzed patients. The Mann–Whitney p-value of equal or less than 0.05 was considered to be statistically significant. The statistical analyses were performed using the Statistica® software program, version 13.0 (StatSoft).The log-rank test was used to assess the equality of survival distributions across different strata. The hazard ratio for death among patients with HPV− and HPV+ was determined. A The general clinical characteristics of 127 patients is presented in p = 0.745). The male gender was predominant in the both groups. There were 51 (75.1%) and 36 (61.0%) males in the HPV− and HPV+ groups, respectively (p = 0.133).Both groups were comparable regarding the age and gender structure. The mean age was 60.85 ± 7.48 (37–79) and 60.36 ± 9.67 (30–80) years in the HPV− and HPV+ groups, respectively (p = 0.010). The palate location was not observed in HPV+ patients, and this location was noted in 10 (14.7%) HPV− patients.The tonsil was the most common OPC location in both groups, but the incidence of the tumor location was different depending the HPV status. The tonsil location was the most frequent (47 (79.7%)) in HPV+ patients, and this location was noted in only 44 (64.7%) HPV− patients (p = 0.054). The tumor depth was similar in both groups. It should be emphasized that the HPV+ patients had the more advanced nodal status compared to HPV− patients .Concerning the histopathological findings, in both groups, G2 grading and T2/T3 staging were the most common. G2 grading was reported in 34 and 21 patients who were HPV− and HPV+, respectively. G3 grading was more frequently noted in HPV+ patients compared to HPV− ones .The significantly higher regional advancement of the HPV+ tumors was associated with the difference in the treatment regimen in the both groups. RCT was significantly more frequently used in the HPV+ patients compared to HPV− patients  and after treatment  was comparable in HPV− and HPV+ groups, respectively, but the Ret decrease was significantly greater in HPV+ patients compared to HPV− ones . A lower WBC0 was reported in HPV+ patients compared to HPV− participants , while the difference in WBC1 between both groups was not statistically significant . The WBC decrease was observed in both groups. CLC1 (p = 0.004) and CMC1 (p = 0.012) were significantly lower in HPV+ patients, while these pre-treatment parameters were comparable in HPV− and HPV+ groups . A significantly lower PLT1 count was recorded in HPV+ patients compared to HPV− ones , while PLT0 was similar in HPV+ and HPV− groups . It was associated with a greater PLT decrease in HPV+ patients (p = 0.218).Most pre-treatment (0) and post-treatment (1) laboratory results were comparable in both groups. Moreover, a decrease of the most parameters following RT/CRT was noted in our study. Only ret-hemoglobin (RetHb) increased after the treatment in HPV+ patients. Therefore, a significantly higher RetHb1 level was noted in HPV+ patients (35.01 ± 1.43) compared to HPV− patients (33.50 ± 3.38). A reticulocyte count (Ret) before  were analyzed.p = 0.039). In HPV+ patients, the incidence of tonsil location was comparable in both NLR subgroups . There was no significant difference in terms of the other clinicopathological parameters between the two NLR groups regardless of HPV status . In a comparison of the determined grading, G2 was the commonest grading in both subgroups. In HPV+ patients, histological grading was comparable in both MLR subgroups. The other clinicopathological parameters were similar in both MLR subgroups . This difference was not noted in HPV− patients. The other clinicopathological parameters were comparable in both PLR subgroups . In HPV+ patients, G3 grading was more frequent in the low SII group compared to patients with the high SII . Moreover, smoking was seen significantly more frequently in the low SII HPV+ subgroup compared to patients with the high SII   . All comp = 0.0008 and p = 0.0009, respectively) (p > 0.05). Therefore, the treatment regimen did not impact survival in our patients (OS and DFS in both HPV groups are presented in ctively) . The trepatients .p = 0.048), a higher PLT , and a higher MLR . A higher WBC  marginally predicted inferior OS in HPV− patients.In UVA, poor prognostic factors for OS in HPV− patients were as follows: A higher CMC , a higher MLR , and a higher PLR  were poor prognostic factors for OS.In MVA, a higher CMC , a higher NLR , and a higher SII . A higher Ret was associated with higher OS ,In UVA, poor prognostic factors for OS in HPV+ patients were as follows: A higher WBC , and a higher SII  were poor prognostic factors for OS.In MVA, a higher CMC , and higher PLR , whereas a higher RBC  predicted better DFS.In UVA, poor prognostic factors for DFS in HPV− patients were as follows: A higher PLT  was a poor prognostic factor for DFS, whereas a higher RBC , a higher MLR  were associated with higher DFS.In MVA, a higher PLR , and a higher SII , whereas a higher CLC , a higher CMC  predicted higher DFS.In UVA, poor prognostic factors for DFS in HPV+ patients were as follows: A higher NLR  was a poor prognostic factor for DFS, whereas a higher CLC  was associated with higher DFS. A higher CMC  marginally predicted higher DFS in HPV+ patients.In MVA, a higher Hb , and a higher SII . A higher Hb  predicted better OS in HPV−patients.In UVA, poor prognostic factors for OS in HPV− patients were as follows: A higher PLR  and a higher SII  were poor prognostic factors for OS. A higher RBC , a higher WBC  were associated with better OS.In MVA, higher PLT  was a poor prognostic factor for OS in HPV+ patients.In UVA, only a higher CLC  was a poor prognostic factor for OS in HPV+ patients.In MVA, also a higher CLC  and a higher SII , whereas a higher Hb  and a higher RBC  were associated with better DFS.In UVA, poor prognostic factors for DFS in HPV− patients were as follows: A higher NLR  was a poor prognostic factor for DFS, whereas a higher RBC  was associated with higher DFS.In MVA, a higher PLR  was a poor prognostic factor for OS in HPV−+ patients.In UVA, only a higher PLT  was the only poor prognostic factor for DFS.In MVA, also a higher PLT  ,13,14,159 /L. Anemic patients demonstrated comparatively decreased LRC, FDM, and OS. Additionally, patients with simultaneous PLT elevation and anemia had significantly worse oncologic outcomes for LRC, FDM, and OS than those with anemia or platelet elevation alone or those with no alteration. It should be emphasized, that Shoultz-Henley’s study did not stratify patients according to HPV status [9 /L were promising predictors of prognosis in patients with operable HNSCC. Explanation of the association between increased PLT and poor survival in OPC patients is the theory that PLTs direct tumor cell growth, vascular invasion, hematogenous dissemination, immune system evasion, and creation of metastasis site [In our study, PLT count was a significant poor prognostic factor for both OS and DFS survival in HPV− patients. This association of the higher PLT count with inferior survival in OPC patients is in accordance with observations of other authors. Shoultz-Henley et al.  evaluateV status . Gorphe V status  investigV status . In our sis site . There asis site . Rachidisis site  reportedsis site . This phCharles et al.  comparedThe SII was the second independent prognostic factor for both OS and DFS in HPV+ patients in our study. The significant prognostic SII cut-off in the HPV+ group was >448.60 for OS and 462.58 for DFS. The SII as a prognostic factor for survival in cancer patients is less frequently described. Gao et al. described the SII, NLR, and PLR as independent prognostic factors for OS and DFS in patients with surgically resected esophageal squamous cell carcinoma (ESCC). The optimal cut-off values for the prediction of survival were 479.72 for the SII, 2.27 for NLR, 117.07 for PLR, and 0.19 for MLR . Thus, tThe SII in the role of an adverse prognostic factor was described in various cancers as follows: oral squamous cell carcinoma , esophagHuang et al.  comparedp = 0.0008 and p = 0.0009, respectively). Thus, HPV status was a very strong prognostic factor for OS and DFS. Survival of HPV+ patients was better despite the higher regional disease advancement. These results are in full accordance with the literature data. Patients with HPV-related OPC have a better prognosis and longer survival compared to patients without HPV-related OPC with typical risk factors  [Generally, our study showed significantly better OS and DFS in HPV+ patients compared to HPV− ones (l abuse) ,44. A bel abuse) ,6. Moreol abuse) ,45. It al abuse) ,47. In ol abuse) . The assl abuse) ,50,51,52p > 0.05). Thus, treatment strategy had no impact the final results. Additionally, the impact of RT and CRT on survival has been assessed in univariate (UVA) and multivariate (MVA) Cox analysis. In both UVA and MVA, for HV- and HPV+ patients, the treatment strategy was not a significant prognostic factor. There are studies with a comparative analysis of survival depending on various parameters in head and neck cancer patients despite the statistical difference of the treatment strategies between compared groups in the literature [Our patients were treated under standard department protocols definitively with radiotherapy (stage I-II) and chemoradiotherapy (stage III-IV). The difference in treatment strategy was determined by tumor stage rather than HPV status. The fact that more patients with HPV-related tumors presented more advanced stages was associated with a different tumor biology and clinical outcome. There was a significantly higher number of more advanced N2–3 tumors in HPV+ patients compared to HPV− ones. RT was significantly more frequent in N0–1 tumors, whereas CRT was more common in N2–3 tumors in an analysis of all patients together. The greater nodal involvement is characteristic for HPV+ OPC patients, and it has been confirmed by numerous studies. Moreover, according to the literature, typically HPV OPC presents in a younger, healthier population with a different set of risk factors and good prognosis for survival. Moreover, the majority of analyses showed that patients with HPV+ tumors had significantly better responses to treatment than those with HPV− tumors. HPV− OPC patients are usually older, with numerous comorbidities ,53,54. Tterature ,31,55.To our knowledge, there is only one study regarding a similar topic. In this study, Huang et al.  investigThe single center observation and retrospective analysis of a prospectively collected database are limitations of our study. A prospective randomized multi-center study is needed to understand the biology of our observation and potentially to identify new therapeutic targets based on our findings.The higher NLR and SII are significant adverse prognostic factors for HPV+ OPC patients because they are significantly associated with both inferior OS and DFS in this group, whereas the higher PLT is a significant adverse prognostic factor for HPV− OPC patients because it is significantly associated with inferior OS and DFS in this group. Further studies are needed in order to validate our findings. The knowledge of differences in immune parameters between HPV− and HPV+ OPC patients can be useful for the identification of new targeted immunotherapy in the HPV+ OPC treatment.