We developed a colorectal cancer risk prediction tool (‘CRISP’) to provide individualised risk-based advice for colorectal cancer screening. Using known environmental, behavioural, and familial risk factors, CRISP was designed to facilitate tailored screening advice to patients aged 50 to 74 years in general practice. In parallel to a randomised controlled trial of the CRISP tool, we developed and evaluated an evidence-based implementation strategy.Qualitative methods were used to explore the implementation of CRISP in general practice. Using one general practice in regional Victoria, Australia, as a ‘laboratory’, we tested ways to embed CRISP into routine clinical practice. General practitioners, nurses, and operations manager co-designed the implementation methods with researchers, focussing on existing practice processes that would be sustainable. Researchers interviewed the staff regularly to assess the successfulness of the strategies employed, and implementation methods were adapted throughout the study period in response to feedback from qualitative interviews.The Consolidated Framework for Implementation Research (CFIR) underpinned the development of the interview guide and intervention strategy. Coding was inductive and themes were developed through consensus between the authors. Emerging themes were mapped onto the CFIR domains and a fidelity checklist was developed to ensure CRISP was being used as intended.Between December 2016 and September 2019, 1 interviews were conducted, both face-to-face and via videoconferencing (Zoom). All interviews were transcribed verbatim and coded. Themes were mapped onto the following CFIR domains: (1) ‘characteristics of the intervention’: CRISP was valued but time consuming; (2) ‘inner setting’: the practice was open to changing systems; 3. ‘outer setting’: CRISP helped facilitate screening; (4) ‘individual characteristics’: the practice staff were adaptable and able to facilitate adoption of new clinical processes; and (5) ‘processes’: fidelity checking, and education was important.These results describe a novel method for exploring implementation strategies for a colorectal cancer risk prediction tool in the context of a parallel RCT testing clinical efficacy. The study identified successful and unsuccessful implementation strategies using an adaptive methodology over time. This method emphasised the importance of co-design input to make an intervention like CRISP sustainable for use in other practices and with other risk tools.The online version contains supplementary material available at 10.1186/s13012-022-01205-8. Contributions to the literatureThis implementation study was conducted in parallel with an RCT which demonstrates an ability to conduct an implementation study in an isolated practice setting while testing the feasibility of the tool itselfThe CRISP tool which was being implemented was improved and designed with the feedback from practice nursesImplementation strategies were identified through trial and error in real timeWe have developed an evidence-based implementation strategy that can be used to prospectively implement tools into general practiceColorectal cancer is the third leading cause of cancer mortality in Australia causing 5255 deaths in 2019 . For thoIn 2012, we developed and validated an Australian colorectal cancer risk prediction model which included several lifestyle factors, family history, medications and screening history  We conducted a Phase IIA/feasibility study testing the proposed methods for a randomised controlled trial (RCT) . As a rehttps://crisp.org.au/crisp-clinic.The CRISP tool is described in detail within the efficacy trial protocol  and the Systematic reviews by the Cochrane Effective Practice and Organisation of Care group report several implementation strategies that have been effective in successfully translating health interventions into clinical practice . EvidencTo develop an evidence-based implementation intervention capable of supporting the adoption of CRISP in general practice, in a sustainable way,To determine methods for scaling-up the implementation of CRISP into general practices in Australia, andTo develop an approach to implement risk assessment tools for other cancers into general practice.In this qualitative study, we aimed to evaluate implementation methods to support the adoption of CRISP in a ‘real world’ clinical practice setting (Phase IIC). Specifically, we aimed toThis qualitative study used semi-structured focus groups and individual interviews of practice staff including receptionists, practice managers, nurses, and general practitioners. A constructivist paradigm was used as participants construct their understanding through experience , using icharacteristics of the intervention ; (2) the inner setting ; (3) the outer setting , peer pressure, external policy and incentives); (4) the characteristics of individuals involved , and (5) the process of implementation   on his desktop.” (Practice nurse 4)In practice, CRISP is hosted on an external website, and to make it easier to access, the practice staff created a shortcut to the website on their desktop. However, when CRISP was updated on the website, the shortcut had to be manually updated which occasionally caused problems (Quotation 1c).1d “Just wondered if there’s some way of putting it into the EMR software so you could actually remember it like the geriatric depression thing…and things that you can just take down” (Practice nurse 3)To overcome this, the nurses suggested embedding CRISP into the patient EMR software as they had with other risk calculators (Quotation 1d). They also felt this would encourage them to use it more regularly as well.1e “So, we were talking about how having the tool autofill some things. I know it’s been possible with us in the other program we were using so maybe we can try that? It would save a few minutes.” (Practice nurse 4)CRISP sessions also took a non-trivial amount of time to complete in the context of consultations which often required other clinical activities. The nurses identified opportunities to save time including auto-populating patient details from the practice’s EMR system (Quotation 1e).2a “...not gone off our radar at all or enthusiasm, it’s simply not been able to fit this in.” (Practice nurse 3)2b “There could have been people we missed out on, if I was travelling well for time then I could use the CRISP tool with the patients but no I didn’t catch everyone, sometimes it is impossible to fit something extra into a consultation.” (Practice nurse 2)The general practice was continually adjusting their priorities which impacted the use of CRISP. Despite the tool being designed to take about five minutes to complete, competing demands on practice staff’s time limited their capacity to use CRISP regularly . This was especially obvious during ‘flu vaccination season, during which vaccination consultation was prioritised over a CRISP consultation. Practice nurses did not have shifts covered when they went on annual leave, so at times the practice was not all full capacity, adding further workload pressure.2c “Cervical screening because my nurse team usually have 30 minutes for cervical screening, usually. That’s probably been an opportunity where we really have been able to do it at the CRISP tool.” (Practice nurse 2)2d “The care plans [chronic disease management plans], you know 30 minutes is very tight if you know the client well and the paperwork is fairly well organised, you could possibly fit it in there.” (Practice nurse 4)Practice nurses were constantly thinking of ways to identify opportunities to use CRISP during patient visits to overcome the time barrier (Quotation 2c and 2d).2e “[We have a] new billing system - some patients will be charged an out-of-pocket fee of $20” (Practice nurse 1)2f “Patients who aren’t on a care plan must pay out of pocket for their visit to [the practice] now, they may be hard to recruit” (Practice nurse 1)Throughout the duration of the project, the clinic experienced substantial internal changes that affected their use of CRISP. The clinic transitioned from being a fully bulk-billing clinic to charging patients out-of-pocket fees for many services (Quotation 2e and 2f). While preventative health checks continued to be bulk-billed, the billing change resulted in it not being used as frequently as it could have been.2g “All four [of the nurses] have been championing this... They’ve been absolutely on board with it” (Practice nurse 2)Although the practice faced many changes, the general culture of the staff was positive - they remained flexible and agile, and continued to be open to change (Quotation 2g).3a “I find it helpful in terms of trying to dial people away from colonoscopies because we have a lot who are captured by specialists who… they’ve had a colonoscopy and are immediately booked in for another one.” (Practice nurse 3)3b “They want to know are they iron deficient, are they anaemic, and that’s how they are prioritising so the referral’s going to enter the public system and face a lengthy wait and if you’re raising with the patient, “based on your history we think colonoscopy is the way to go but you may have to wait 9 months to a year for an interval colonoscopy” (Practice nurse 3)CRISP has been designed to increase risk appropriate screening, and this includes encouraging average risk patients to screen with the National Bowel Cancer Screening FIT kit rather than undergo unnecessary colonoscopies. The nurses recognised this was a benefit to using the tool as it not only provided individualised risk but also methods for communicating recommended screening advice (Quotation3a). Also, nurses were aware of the long waiting periods to access colonoscopies through the Australian public healthcare system (Quotation 3b). They understood that CRISP’s ability to steer average-risk patents towards FIT also had the potential to reduce the pressure on the public healthcare system by preventing unnecessary colonoscopies.3c “… people throw the NBCSP kit in the bin, so I do think this will be useful once we start approaching more people and people get used to being asked questions about it…” (Practice nurse 1)The CRISP tool and discussions about colorectal cancer screening prompted patients to participate in the National Bowel Cancer Screening program ; (Quotation 3c).3d “If I actually have a demonstration kit to show the patients how to use it I probably will but if I need to leave the consultation room to try and find one I might not use it or show them how to do it…this could be another barrier to implementation” (Practice nurse 2)Practice nurses also wanted NBCSP FIT kits to help explain their use to patients and further promote colorectal cancer screening uptake. Researchers SM and JM ordered sample kits from the NBCSP for each of the practice nurses (Quotation 3d).At the time of this study, the NBCSP had not been fully implemented as a biennial program. General practices were expected to use local pathology companies to provide FIT kits to patients who were not up to date with colorectal cancer screening. In discussions with the local pathology providers, we realised that the FIT kit instructions being offered by the practice nurses were written for the older guaiac-based test. That is, the instructions were not applicable to the immunochemical FIT kit being used. These instructions included dietary restrictions and a more complicated sample collection, which might have deterred patients from undertaking the test. The instructions for the immunochemical test were updated in the practice to eliminate this barrier to colorectal cancer screening.4a “Why is calcium a problem? I take calcium for my osteoporosis… I thought I better stop taking my calcium tablets if it will increase my risk.” (Practice nurse 2)4b “So HRT increases your risk of breast cancer if you take it for a certain period of time and calcium has some risks as well. So that’s one thing that we clarified with you and also the more information when it comes to the analgesic stuff as well.” (Practice nurse 1)4c “When you say NSAIDs, is Panadol an NSAID?” (Practice nurse 3)4d “… if someone has had significant bowel disease, like ulcerative colitis and diverticulitis the tool shouldn’t be used for them, right? (Practice nurse) [Researcher response: “That’s actually a very good question. If someone has diverticulitis they can be included if it is not significant as it is very common].” (Practice nurse 3)Practice nurses were unaware that ‘risk factors’ in the CRISP tool included some factors that increased bowel cancer risk, but also others which reduced risk (Quotations 4a and 4b). Furthermore, there was some misunderstanding about which patients might not be suitable for CRISP due to additional risk factors such as inflammatory bowel disease. This highlighted the need for comprehensive information and training to ensure correct use of the tool. (Quotations 4c and 4d).4e “I think we have to give them a bit of time to get their heads around this. One of the GPs who was overwhelmed coped two patients who had a history of polyps from me.” (Practice nurse 2)4f “Because the GPs must dig in the patient’s records, they don’t feel they can manage this. Patients are booked for one item per consult, the GPs have said that they want the patients to come back.” (Practice nurse 2)4g “…the GPs felt overwhelmed talking to the patients about the tool as they have very limited time to do so” (Practice nurse 2)Nurses reported that there were barriers for GPs to discuss the CRISP recommendations when they received the printed CRISP report. They felt overwhelmed if their patients presented with significant/multiple health issues, and a discussion of bowel cancer screening was also expected (Quotation 4e and 4f). Time pressures felt by GPs prohibited them from fully embracing CRISP (Quotations 4g).The overall culture of the practice meant that practice staff were open to changing and adapting to the implementation of CRISP. As a teaching clinic, practice staff were familiar with exploring new methods. Nurses were motivated and enthusiastic but recognised that this might not be the case in every clinic (Quotation 5a). They were open to introducing the tool into existing processes and were very forthcoming with new ways to incorporate CRISP into their clinical workflow. This included, for example, flagging specific patients aged 50 to 74 years old who were eligible for screening, and incorporating CRISP into chronic disease patients’ care plan appointments, which are typically longer than other consultations (Quotation 5b).The practice nurses were comfortable using the fidelity checklist alongside CRISP with their patients, and confident the tool was being used as it was intended (Quotation 5c).5a “If you look at a lot of things that happen in our clinics, I think we’re probably fairly motivated here. Not that other nurses aren’t but it depends on what they’re doing with the… the quicker and the … simpler you make it, the easier it will be to continue to apply in the longer-term basis.” (Practice nurse 2)5b “But you’re quite right, because we flag things on our care plan, particularly the breast screening, cervical screening, FIT, that is the time to flag and I thought to myself, I rebook for this, I give you the phone number for that, I’ve got time I’ll even do it on the phone in my room immediately.” (Practice nurse 4)5c “We usually go through a bit of a fidelity checklist which I know we’ve done that with you before and it seems like that was all… everything was being used in the right way.” (Practice nurse 1)5d “Mine have been fairly straight forward if I’m lucky.” (Practice nurse 4)5e “I found that the patients I asked were very keen and liked going through the tool, it’s great at bringing awareness tool and gets people talking about their risk” (Practice nurse 3)Overall, the practice nurses thought that CRISP was easy to use, and they reported that patients enjoyed working through it because it raised awareness. (Quotation 5d and 5e).CRISP is a personalised colorectal cancer risk prediction tool to increase risk appropriate screening . This stOverall, clinic staff responded positively to the tool. Training the staff to use CRISP was beneficial. This helped them understand how personalised colorectal cancer risk estimates and could facilitate appropriate screening. This included understanding why the use of an FIT kit by patients at average risk might prevent unnecessary colonoscopies . CRISP fThe CFIR framework was utilised to qualitatively evaluate our approaches to implementation, from which we determined that CRISP was well understood, liked by patients and staff, and utilised appropriately. We also determined that CRISP required training and had to adapt to changes in the clinic structure. Time and conflicting priorities were a barrier, and at times CRISP was overlooked in favour of more pressing issues such as flu vaccination and patient-driven needs. The comprehensiveness of CFIR and the ongoing engagement with the practice staff was beneficial in evaluating all aspects of CRISP implementation.Many colorectal cancer risk prediction tools have been developed and validated internationally –31 and, There were several limitations to this study. Firstly, general practice is an ever-changing environment, and the clinic where we implemented CRISP was no different. In Australia, there is a shortage of rural GPs; they move frequently and work in these areas for a specified amount of time . AlthougCRISP was only implemented into one general practice and due to the diversity of clinics, the implementation framework this project generated may not be fully scalable to another clinic. We attempted to minimise this by using implementation methods that are common to many general practices; however, each practice has its own inner setting characteristics that can affect the success of implementation. The evolving nature of general practice poses a challenge to the long-term implementation of tools such as CRISP.The final limitation was the absence of quantitative data. We did not intend to recruit a sample large enough to measure any changes over time in response to using CRISP. This would be an important addition to a larger implementation study. Logging of quantitative CRISP session data  was not in scope for this research but may be useful in the future to understand and assess uptake of risk prediction tools.This research has identified a method for implementing CRISP in general practice by applying co-design in situ within a clinical setting. Our learnings have significant value—while the findings presented here involve a risk assessment and screening tool for colorectal cancer, a leading cause of cancer mortality, they may also be applicable to the implementation of tools for other diseases. The methodology we used for evaluating implementation methods in general practice demonstrated the necessity for ongoing training, fidelity checking, and understanding the context of the clinic and related health services external to the clinic. The use of co-design and engagement over time with clinic staff was beneficial for establishing, reviewing, and adapting implementation methods for the CRISP tool. Although general practices vary, the methodology used to implement CRISP could be used across many general practices, used on a larger scale and supported by quantitative evaluation. We suggest this method could be used on a larger scale and supported by quantitative evaluation.Additional file 1: Supplementary file A. Interview guide.Additional file 2: Supplementary file B.TIDIeR checklist for the intervention.


Some estimates place the proportion of human malignancies attributable to viruses at between 15 and 20 percent. Viruses including the human papillomavirus are considered an interesting but controversial etiological risk factor for breast cancer. HPV infection is anticipated to be an early trigger in breast cancer carcinogenesis, followed by cumulative alterations over time (“hit and run” mechanism) through synergy with other environmental factors. The association between HPV and breast cancer has not yet been verified. There are very conflicting data on the presence of HPV DNA in breast cancer samples, and we lack a clarified, exact mode of HPV transmission to the breast. In our review article we analyzed the up-to-date knowledge about the association of HPV and breast cancer. Furthermore, we summarized the available original research published since 2010. In conclusion, the complexity and inconsistency of the available results together with the relatively low prevalence of HPV infection requires extensive research with much larger studies and exact and unified diagnostic methods are required to better understand the role of the HPV in breast carcinogenesis. The number of newly diagnosed cases of breast cancer (BC) has been predicted at 2.3 million in 2020 with mortality close to 685,000. BC incidence and mortality are predicted to rise to 4.4 million and 2.1 million, respectively, by 2070 [Viruses are considered an interesting but controversial etiological risk factor for breast cancer, which can precipitate tumorigenesis via synergy with other environmental factors. There is evidence that viral DNA from human papillomaviruses (HPV), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), and human herpesvirus type 8 (HHV-8) can be found in breast cancer samples as well as in healthy tissue samples ,7. HowevIn our review article, we focused on the oncogenic potential of HPV and its link to breast cancer. We searched the PubMed database using the keywords “breast cancer”, “HPV”, and “human papillomavirus”. In the literature search, all literature up to October 2022 was included, with no restriction on the publication date.Two main hypotheses have been proposed about how HPV can infect mammary gland cells; however, the specific mechanism is still unknown. According to the first hypothesis, HPV is transmitted to the mammary glands via the lymphatic or blood system through mononuclear white cells present in women with cervical dysplasias. In the case of malignancy, cells from a primary tumor can be transported by plasma flow. In addition, HPV virions can be transferred from the site of initial infection to other organs . Other sDe Villiers et al. , who posOne hypothesis is based on the complex mechanism of endocytosis and cellular transport associated with alpha HPV genus  . The α6 Another hypothesis is based on the activity of the extracellular vesicles (EVs). The ascending pattern of HPV infection spread is still a target of much discussion. The presence of HPV DNA in serum-derived EVs was found in patients with HPV DNA-positive squamous cell carcinoma of the middle rectum and BC patients . EVs incMany types of cells, tissues and bodily fluids produce EVs, including nucleic acids, proteins, non-coding RNA and viral nucleic acids . FurtherThe genome of HPV consists of double-stranded circular DNA reaching the length of around 8000 base pairs, containing from eight to nine open-reading fragments (ORFs). It consists of three functional regions: a long control region (LCR), early region (E ORF) and late region (L ORF). The approximately 1 kb long section of the LCR has no coding potential and contains sections regulating viral transcription and tissue tropism. Early region is represented by 6–7 ORFs , whereas the late region consists of only two ORFs (L1 and L2). All HPVs contain conserved core genes included in replication (E1 and E2) and viral capsid establishment (L1 and L2), with more diversity in other genes , which determine maturation and release of the virus, escape from immune system and regulation of cell cycle .The majority of HPV genomes (86–100%) detected in breast tissue are in an integrated form with a low copy number (0.00054–9.3 copies/cell) . In BC, Molecular changes in BC initiation may therefore occur via a “hit and run” mechanism. This theory proposes that HPV initiates or contributes to the development of cancer, but in some cases vanishes from tumor cells (possibly due to immune surveillance) before the disease is diagnosed ,61. The As a result of the integration of the HPV genome into the host genome, chromosomal instability and carcinogenesis may be induced . HPV E6 E6 promotes p53 degradation via its association with E6-AP, an auxiliary protein in the ubiquitin proteolytic pathway . In addiSeveral cellular pathways are involved in the transformation of mammary epithelial cells associated with HPV E6 and E7 proteins. These proteins inhibit pRb, p53, NFX1 and BRCA1, leading to an upregulation of nuclear factor kappa B and nuclear factor kappa E pathways ,100,101.Viruses contribute to carcinogenic processes by a variety of mechanisms, including chronic inflammation, disruption of cellular regulatory mechanisms and resistance to apoptosis. Chronic inflammation is associated with an increase in cytokines such as transforming growth factor beta (TGF-1) and interleukins (IL), resulting in a proliferation of breast tumors . FurtherThere is also the possibility that HPV could function synergistically with the estrogen receptor (ER) signaling pathway. Specifically, Wu et al. demonstrated that the E2 protein cooperates with nuclear receptor coactivators in order to enhance the ERE-dependent transcriptional activity of ERα . TherefoA well-established relationship exists between HPV and cancers of the uterine cervix, anogenital region, head and neck, and skin. A variety of other cancers have been reported to be associated with HPV, including glioblastoma, colorectal, lung and breast cancers; however, its pathogenic role remains ambiguous and controversial ,114,115.No definitive link has been established yet between HPV and BC. Although the presence of HPV alone is insufficient to cause breast cancer development, it is anticipated to be an early trigger followed by cumulative alterations over time. HPV DNA-containing cells have been identified even in the tissues surrounding BC  .Multiple studies used primers for the detection of dozens of HPV types, including lrHPV and the whole spectrum of hrHPV types . HPV16 iA study conducted by Band et al. in 1990 suggested that HPV infection may be associated with BC. In their study, HPV was found to immortalize normal human mammary epithelial cells and to decrease the need for growth factors in these cells . By idenA study conducted by Haghshenas et al. revealed that 23.6% of breast cancer cases were infected with HPV . AccordiIn the analysis of 37 case–control studies with 3607 BC cases and 1728 controls, there was conclusive evidence that BC risk was increased in cases of HPV positivity (summary odds ratio (SOR) = 6.22, 95%CI: 4.25–9.12) . The calFewer studies showed no HPV in breast cancer tissue ,129. HowHPV DNA is more frequently present in BC tissues compared to benign breast tumors or normal breast tissues . SeveralA number of studies have shown an increased incidence of breast cancer among patients with a history of cervical dysplasia, indicating a possible link between uterine cervix infection and breast glandular tissue infection . Almost Due to low viral load of HPV and a variety of diagnostic techniques, it is often difficult to determine whether HPV is present in tissue samples. These diagnostic techniques include in situ hybridization (ISH), different subtypes of polymerase chain reaction (PCR) and next-generation sequencing (NGS) . ConflicA possible role for oncogenic HPV types in breast carcinogenesis was suggested after Band et al. showed that plasmids containing HPV 16 and 18 could immortalize mammary epithelial cells . The facHPV should be more prevalent in breast cancer cases than in normal samples;Exposure to the HPV should precede disease outcome;Multidisciplinary research should be conducted to replicate the association between HPV and breast cancer;A dose–response relationship should exist between exposure levels and the incidence of BC;Viral causality should be explained in terms of the mode of transmission and the natural history and pathology;The virus can infect and transform mammary epithelium and induce cancer in an animal model;The virus can induce cancer in an animal model by infecting and transforming the mammary epithelium;Preventing HPV infection should reduce the incidence of cervical cancer.Despite the fact that approximately 30 years have passed since this hypothesis was first proposed, its definitive conclusion remains unknown . There wThe results of the study of Murtaza et al. failed to establish a casual association between HPV and BC according to the Bradford Hill criteria . In their study, HPV was proposed as a cause–effect agent or at least as one of the co-factors involved in the pathogenesis of BC .With the current state of the knowledge, how we can be sure that we are not looking at false negative or positive results? It is possible to obtain false negative results due to formalin-induced DNA fragmentation , DNA quaThe possible presence of HPV in unusual body tissues, such as the female breast, remains elusive. Data regarding the presence of HPV DNA in tumor samples from patients with BC are very inconsistent, and there is no clear indication of how HPV is transmitted to the breast. However, the presence of HPV in BC cannot be denied. An interesting fact is that HPV DNA can be found in healthy and benign breast tissue, which may prove useful in observing if those chosen patients develop BC in the future. These findings lend support to the hypothesis that the HPV contributes to the development of breast cancer. Similar to the above hypothesis, the use of the HPV vaccine may contribute to a reduction in BC cases. To date, no study has attempted to correlate HPV vaccine use with BC incidence, as such studies would require a much longer period of time to be conducted. For a better understanding of the role of the HPV in BC, extensive studies with a larger sample size and unified diagnostic methods are required, considering the complexity involved and the relatively low prevalence of HPV infection in BC lesions.


The human papillomavirus (HPV) vaccine was approved in 2006 and has been shown to decrease vaccine-related HPV types in the oropharynx. Its impact on the incidence of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) has not been examined. We investigated the impact of HPV vaccination on the incidence of HPV-related OPSCC in the US among male and female adults from different age groups.The US Cancer Statistics 2001–2018 database and the National Cancer Institute (NCI)’s Surveillance Epidemiology and End Results (SEER) program were used in this study. OPSCC incidence was age-adjusted to the US standard population in 2000. Cause-specific 5-year survival probability was calculated using 60 monthly intervals in SEER*Stat software.Incidence of HPV-related OPSCC was much higher in males than in females. Age-adjusted annual incidence of OPSCC was significantly lower in 2014-2018 than in 2002-2006 among males 20-44 years old  and among females 20-44 years old , but increased in both 45-64 year old and 65+ year old males and females. Joinpoint regression revealed a significant joint in the HPV-OPSCC incidence trend for 20-44-year-old males in 2008 at which time the incidence began to decrease. Except for 20-44 year old females , cancer-specific 5-year survivals significantly improved for males and females of all age groups.HPV-related OPSCC was much more common in males. Incidence of HPV-related OPSCC declined among young adults during the vaccination era compared with pre-vaccination era. Cancer-specific 5-year survival was significantly improved in young males but not in young females. A persistent oral human papillomavirus (HPV) infection places a person at risk of developing oropharyngeal squamous cell carcinoma (OPSCC) –3. OPSCCPrior estimates of OPSCC incidence in the US were largely based on the National Cancer Institute (NCI)’s Surveillance Epidemiology and End Results (SEER) program, which maintains a nationally representative sample of cancer patients –12. HPV-We used the USCS database 2001–2018, which combines data from the Centers for Disease Control and Prevention (CDC)’s National Program for Cancer Registries (NPCR) and the SEER program . Both coWe classified OPSCC cases potentially due to HPV based on anatomic sites identified by ICD-0-3 as follows: base of tongue (C01.9), lingual tonsil (C02.4), overlapping lesion of tongue C02.8), soft palate (C05.1), uvula (C05.2), tonsil , vallecula (C10.0), anterior surface of epiglottis (C10.1), oropharyngeal wall (C10.2-10.3), branchial cleft (C10.4), pharynx (C14.0), Waldeyer ring (C14.2), or other oropharynx site . Invasive squamous cell cases were defined using ICD-O-3 histology codes 8050-8086 and 8120-8131 and only microscopically confirmed cases were included , soft pa.The SEER*Stat statistical software package (version 8.3.8) and SAS for Windows version 9.4 (SAS Institute) were used to conduct the analyses. Differences with two-tailed P values < 0.05 were considered statistically significant. OPSCC incidence rates were calculated as the number of cases per 100,000 persons and were age-adjusted to the 2000 US standard population. The Tiwari method was used to determine the confidence intervals (CI) . Annual Cancer-specific five-year survival probability was calculated using 60 monthly intervals in SEER*Stat software. Data used were from SEER’s 18 registry areas. SEER*Stat software used expected life tables instead of a cohort of cancer-free individuals, assuming that the cancer deaths were a negligible proportion of all deaths. Individuals who died of causes other than OPSCC were considered censored when we estimated cancer-specific survival. Cox proportional hazard models were fitted to compare differences in 5-year survival probability across time by stage at diagnosis, controlling for age at diagnosis and race/ethnicity. Hazard ratios (HRs) and 95% CIs were estimated from the Cox model. Kaplan-Meier curves were plotted to show differences in cumulative probability of death across time. We had successfully used the same software package and similar methods to examine incidence of cervical cancer and breast cancer, and cancer-specific survival –23.There were 229,264 adult males and 55,108 adult females diagnosed with HPV-related oropharyngeal squamous cell carcinoma (OPSCC) from 2001 to 2018. Among these male patients, 4.6% were 20-44 years old, 61.7% were 45-64 years old, and 33.7% were 65+ years old, while among these female patients 5.4% were 20-44 years old, 50.8% were 45-64 years old, and 43.8% were 65+ years old  compared with the pre-vaccination era (2002–2006). Previous research established that changes in HPV prevalence are the primary driver behind increased OPSCC incidence , 9, 10. –2018 comPrevious literature showed increasing trends in the incidence of HPV-related OPSCC among middle-aged and elder adults in the US –12. We aOverall, our data show that HPV-related OPSCC was much more common in males. This likely reflects the higher oral HPV infection rates observed among men , 30. AmoThe findings of our study have important health implications. Our observed increasing trends in HPV-related OPSCC among middle-aged and elder males and females call for interventions to reduce risk factors for this type of cancer. Notable actionable risk factors include oral HPV infection , 30 and via p16 immunohistochemistry). Modern estimates of HPV DNA-positive tumor sites classified as HPV-related based on anatomic site are merely 70%; therefore, these anatomic site-based classifications may have led to the occasional misclassification  and the Surveillance, Epidemiology, and End Results (SEER) databases (2001-2018).This study did not involve human subjects and did not require approval by the Institutional Review Board of The University of Texas Medical Branch.Study concept, methodology, data analyses, manuscript preparation, and funding (FG). Data analyses and manuscript preparation (MC). Manuscript preparation (MS). Study concept and manuscript preparation (BK). Study concept, methodology, data analysis, manuscript preparation, and funding (AB). All authors contributed to the article and approved the submitted version.Dr. Guo is currently supported by an award (K07CA222343) from the National Institutes of Health, National Cancer Institute (NIH/NCI). This study was supported by an award to Dr. Berenson (PP200005) from the Cancer Prevention and Research Institute of Texas (CPRIT). The content is solely the responsibility of the authors and does not represent the official views of the NIH/NCI or CPRIT.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.


L1 gene from different HPV genotypes by bioinformatics software, an innovative technique based on the nested- high resolution melting was designed with three approaches and using conventional PCR, qPCR, and diagnostic standards. HPV-positive samples identified by microarray along with diagnostic standards were evaluated by qPCR-HRM and discordant results were subjected to sequencing and analyzed in silico using reference types. In addition to screening for human papillomavirus, nested-qPCR-HRM is one of the modified HRM techniques which can discriminate some genotypes, including 6, 16, 18, 52, 59, 68 and 89. Despite the differences in diagnostic capabilities among HRM, microarray and sequencing, a number of similarities between HRM, and sequencing were diagnostically identified as the gold standard method. However, the bioinformatics analysis and melting temperature studies of the selected region in different HPV genotypes showed that it could be predicted. With numerous HPV genotypes and significant genetic diversity among them, determining the virus genotype is important. Therefore, our goal in this design was to use the specific molecular techniques with several specific primers to increase sensitivity and specificity for discriminating a wide range of HPV genotypes. This approach led to new findings to evaluate the ability of different approaches and procedures in accordance with bioinformatics.The prompt detection of human papillomavirus and discrimination of its genotypes by combining conventional methods in new molecular laboratories is essential to achieve the global call of eliminating cervical cancer. After predicting the melting temperature of an approximately 221 bp region of the  The cofactor role of HPV and other risk factors was proven in the progression of cervical cancer3. The genotyping of human papillomaviruses is challenging due to their high diversity. Determining the genotypes of this virus plays an essential role in breaking the infection transmission cycle, treatment management, vaccine development, which will result to avoid cancer occurrence. As mentioned in the study of Bonde JH et al., human papillomavirus genotyping discriminates the risk of cervical intraepithelial neoplasia grade 3 or worsens (CIN3 +) or with more intensity to a clinically significant degree, irrespective of cytology result4. Furthermore, Arroyo Mühr, LS, et al. stated that when cervical cancers were analyzed by both PCR and deep sequencing, HPV was present is > 92% of cervical cancer samples5.Human papillomaviruses (HPVs) are responsible for various human pathologies, being the primary agent for the second most prevalent cancer among women, cervical cancer6.The severe danger of HPV for women and its relation with different cancers necessarily require genotyping by a susceptible and rapid method7.The genotyping of HPV in various studies was performed via the nucleotide sequencing, DNA Chip, hybridization, reverse line blot (RLB) assays, generic and type-specific probes, liquid bead microarray assay (LBM) based on Luminex technology, etc.8; whereas microarray provides genotype-specific information which can detect the presence of mixed HPV infection, but both techniques are expensive, and they are not available everywhere. Compared to two methods mentioned above, the hybridization DNA-RNA method could detect 13 high-risk human papillomaviruses, but without their type specificity10. RLB assay has a limit in repeatability and is laborious to set up, in addition to the high expenses11.DNA sequencing is the gold standard assay for the accurate viral typing12.There are other quick and accurate methods, such as probe-based methods, but they are also expensive and cannot be used as a routine screening method due to lack of affordability by the general public13. Thus, the combination of two techniques, qPCR and high-resolution melting (HRM), can be used for genotyping. Moreover, when microarray and sequencing are compared with qPCR and HRM, microarray and sequencing could only detect 250 copies of the virus out of 1000, while qPCR could detect as few as ten copies of the virus14.Because it is challenging to design multiple compatible primer sets for the genotype-specific PCR, the maximum number of human papillomaviruses detectable in a single assay is relatively limited15. Especially for targets with high homology, such as HPV, additional details of the melting temperature, such as those obtained in real-time PCR, are valuable to reduce false positivity.An advantage of qPCR is that some of the detection chemistries which are used to allow for a post-PCR high-resolution melting curve analysis provide additional information on the specificity17. The high sensitivity and specificity HRM, along with the melting temperature prediction, has high importance for different genotype diagnoses; indeed, this technique can distinguish various temperatures with the disputes of 0.2–1.1 from each other19.HRM method, which decreases costs, could be a suitable replacement for other genotyping methods and a powerful technique to use dye chemistry, resolution instruments, and data analysis20.One of the crucial issues in HRM diagnostic approaches is accurately identifying the gene region among different types of strains. This method, due to the difference in melting temperature alteration of gene region and common primers, could discriminate species and genotypes22.HRM is one of the most powerful methods to study genetic variation such as SNPs, insertions, and/or deletions in the genome. The fundamental aspect of method is that a minor variation in the nucleic acid sequence leads to a detectable alteration in the melting curve, and differences in the amplified PCR sequences23. This can lead to missing fragments that have an identical melting point20.The advantage of HRM method is the amplification of short DNA fragments with high resolution and accuracy. However, some limitations, such as the mismatch of intercalated dyes to ambiguous sequences, like dimer primers and nonspecific products, should not be ignored17. The quick diagnosis of human papillomavirus could prevent the development and progression of cervical cancer. Moreover, the determination of viral load in women with positive HPV requires a highly sensitive method24. The determination of viral load in women is of great interest because it is associated with disease prognosis. Analysis of human papillomavirus viral load and genotype improves prediction of invasive cervical cancer25.This feature has a vital role in the genotyping and other applications, including finding genetic variation, mutation screening, methylation analysis, etc.We used molecular methods, including qPCR and HRM, to screen, genotype and discriminate HPV. Our goal is to design a novel approach for a rapid, sensitive, specific, and cost-effective diagnosis of HPV genotype in the cervical cancer samples.Cervical tissue DNA was successfully extracted with genomic DNA concentrations ranging from 50 to 387 ng/µl. Moreover, the mean A260/A280 ratio obtained for all DNA samples was approximately 1.82, which indicates pure DNA (mean standard deviation = 0.05). The beta-actin gene was observed for all samples as an internal extraction control with a band size of 120 bp.L1 region using degenerate primers FRG5 and MY09 could detect 100 HPV-positive samples revealing different genotypes. Another primer pair, FRG5/FRG2, amplified 215–221 bp of HPV L1 and corresponding amplicons generated up to seven different HPV genotypes .Consensus PCR amplifying 486 bp of the HPV L1 region  revealed the highest similarity to HPV 16 type when performing blast. Bioinformatics analyses of the 218 bp and 215 bp FRG5/2 amplicons from HPV16 and HPV53 showed a 30.28% nucleotide difference, which was significant enough to not be considered a sequencing or blasting error . The two genotypes of human papillomavirus at the salt concentration of 0.2 in the FRG5/2 diagnostic region have the same melting temperature, differences in 61 bases, and 72.77 percent identity.As shown in Table 5BA and G43 isolates, which were identified as HPV 59 and 43, respectively, were isolated by the microarray method. After sequencing, the reading result of both isolates was HPV 6. The first approach to examine these two samples showed the same melt curve, but in the second approach, the graphs were different Fig. .Figure 8Due to the importance of identifying low-risk genotypes, high-risk genotypes, and mixed infections, a rapid and cost-effective method is needed to address HPV genotyping obstacles.26. According to World Health Organization (WHO), human papillomavirus is currently the most common sexually transmitted disease in terms of the unprotected sex, and the most important consequence of the virus is cervical cancer28.Cervical cancer is the second most common malignant tumor in women and seriously threatens women's health. Although cervical cancer is preventable, more than 500,000 women worldwide are diagnosed with cervical cancer, and more than 250,000 women die of cervical cancer each year29. Therefore, in addition to the screening methods, discrimination among HPV-DNA genotypes and determining the number of copies could be important, which was the main topic of our study. It is known that the number of copies of HPV infection determines the duration and severity of the illness30.Diagnosing precancerous cells that are part of cervical cancer screening can be invaluable. Therefore, cervical cancer screening for eligible and high-risk individuals, as well as the discrimination against HPV genotypes, is crucial in the prevention, diagnosis and early treatment of the diseaseHence, considering the above mentioned factors, melting-based curves, especially HRM, can be used as an alternative method in the simultaneous identification and discrimination of HPV genotypes. Furthermore, the acquisition of a novel technique by targeted selection of primers gives acceptable results and may be useful for discriminating between different genotypes.31, astroviruses32, C. meleagridis23, and Yersinia pseudotuberculosis33. Besides, melting curve alteration in HRM method discriminated Iranian Leishmania parasites of L. major, L. tropica and mixed infection in the study of Ghafari SM et al.34. In addition, Mosawi SH et al., on asymptomatic malaria status in eastern Afghanistan, could distinguish P. vivax, P. falciparum, and mixed infections using high-resolution melting analysis35. The differentiation of Mitragyna speciosa from allied Mitragyna species was performed using DNA barcoding‑high‑resolution melting analysis by Chayapol Tungphatthong et al.36.Previous studies showed that HRM method could diagnose different subtypes of influenza AThis study could identify important epidemiological and carcinogenic HPV genotypes using the seminested-HRM approach. These findings were very promising because they provided acceptable results with less time and cost than conventional methods in the market.37. Preliminary studies on temperature melting analysis of these three genotypes with insilico assays showed that bioinformatically, it is impossible to separate HPV18 and HPV6 despite the difference in six nucleotides among these two genotypes in studied gene region with a temperature difference of 0.03 °C. However, HPV16 could be distinguished with a Tm = 82 °C but a temperature difference of 0.7 °C from HPV6 and HPV18. Moreover, in the firs approach of the study, non-considering in-vitro results, there was consistency with the bioinformatics analysis. The melting temperatures of HPV18, HPV16, and HPV6 were 81.67 °C, 81.04 °C, and 81.52 °C, respectively. HPV18, a high risk type being the second most common type found in cervical cancer and, HPV6—a low-risk genotype being the most common genotype in our study population37, did not show a unique profile and therefore were not distinguishable from one another. However, five HPVs, 16, 52, 59, 66 and 89, with unique characteristics were distinguished. To address this issue, and in light of the findings by Lee et al., we employed an unlabeled probe for HPV18 in the second method with the goal of differentiating HPV18 from HPV6, although the results were surprising. In contrast to Lee et al. investigation, .'s in which an unlabeled HPV-18 probe resulted in an extra melting peak for HPV18 that separated it from HPV4566, unlabeled probes for HPV6 had additional melting points; therefore, HPV6 and HPV18 could indefinitely separate, where this contradiction is still unknown.The separation of two main carcinogenic genotypes, HPV18 and HPV16, as well as the predominant genotype, HPV 6, in the research region, was a significant hurdle in our investigationAlthough the results of our approaches with the sequencing method as the gold standard have shown our high success to design this assay, comparing the samples identified using the microarray method with our study method has provided new challenges. However, the discrepancy among the microarray results and these two methods is still debatable because DNA microarray method has sufficient accuracy to detect known HPV subtypes simultaneously.38.This challenge occurred for the isolates which were reported as HPV 11, 45, 83 and 84 using the microarray method, whose melting temperature curves were similar to the HPV 59 diagnostic standard in the semi-nested-qPCR-HRM method in the first and third approaches which were genotyped by the sequencing method as HPV59. According to these views, incorrect genotyping or inaccurate differentiation between low-risk genotypes (HPV11 and HPV84) and high-risk genotypes  might have irreversible effectsAnother notable point is that although all HPV-DNA samples from isolates G40, G11 and G42 were genotyped by sequencing as HPV11, they did not show identical melting curves. This may be because, in only G40, HPV11 was a single infection, but in G11 and G42, HPV-DNA was a mixed infection. The predominant genotypes of these isolates were identified as 59 and 45, respectively.In the case of G11 isolates, the presence of HPV-DNA 11 and 59 was genotyped after sequencing. In approach one, the different HRM pattern of this isolate compared to other isolates that confirmed the presence of HPV-DNA 59, despite having the same peaks, could prove the hypothesis of mixed infection. This justifies the discrepancy between the microarray results with the nested-high-resolution melting and sequencing methods.In the third approach, the patterns were almost identical to approach one, with the addition of GP primers, confirming the difference between G11/HPV-DNA isolation and HPV59 as a pure diagnostic standard. Comparing all three methods, nested qPCR-HRM, Sanger sequencing, and microarray, showed that nested qPCR-HRM approaches almost corresponded to Sanger sequencing as the gold standard diagnostic.39. However, several HPV genotyping assays have recently been reported that are capable of typing a relatively large spectrum of HPV genotypes, but they cannot be automated or deployed in a high-throughput platform43.These contradictions were observed in other studies; for example, in the study of Alexander Harlé et al. one sample had HPV 6/11 DNA, which was detected with conventional PCR and not with the Cobas assay. Besides, one sample had HPV 16 DNA detected with Cobas assay and not with conventional PCR, one sample had HPV High-Risk DNA which was detected with conventional PCR and not with Cobas assay, one sample had HPV 16 DNA detected with Cobas assay and HPV 16 and HPV HR DNA with conventional PCR, one sample had HPV 16 DNA detected with Cobas assay and not with conventional PCR and one sample had HPV 18 DNA detected with Cobas assay and not with conventional PCR. The different reason could be the absence of consensus probes designed by the Cobas assay44, and we anticipated that we would be able to clone more genotypes as the diagnosis standard, the discrepancy among the results obtained by the microarray method and the sequencing prevented us from achieving this goal. Genotypes cloned in this study represent common low-risk and high-risk HPVs in the Middle East45. In this regard, the study of Lee et al. showed eight HPV genotypes 16, 18, 39, 45, 52, 56, 58, and 68 with a prevalence of over 75% in Asia, Europe, and the United States. Considering the limited genotypes of HPV preserved in our study, large-scale typing was limited for different HPV genotypes. It is expected that by obtaining more genotypes in the future, this method could be further evaluated and analyzed.In population-based cervical screening, human papillomavirus (HPV) types 16, 18, 31, 33, 45, and 52 are associated with 85% of HPV-associated cervical cancersIn conclusion, we evaluated the validation of HPV genotyping via Tm value and HRM analysis of nested real-time PCR, which displayed the differential melting curves of different human papillomaviruses.This approach has the potential to improve the discriminating of seven HPV genotypes, including HPV 16 and HPV 18, as cervical cancer carcinogens. The assay may be suited for routine analysis to detect HPV DNA in molecular laboratories as an alternative to the Pap smear test and enables effective treatment management, which is very practical for the success of implementation of women's health programs in low-resource regions.To set up this technique, it is necessary to check all the diagnostic standards that are foreseen in the kit with the all of samples which are in the workflow and to determine the HPV genotype using HRM technique, a decision is made based on the comparison of diagnostic standards Tm with the sample Tm. It is both simple and quick to perform which was shown to have high sensitivity and specificity. Furthermore, when used to screen samples, it can significantly reduce the cost and time.37. Informed consent was obtained from all women who were referred for conventional Pap tests to the medical centers of Qom Province. For optimization of the qPCR-HRM performance, known HPVs DNA were obtained from the previous study or microarray method in clinical laboratory37 , Iran, and all experiments were performed in accordance with relevant guidelines and regulations. A total of 57 HPV positive samples were randomly selected from 486 Pap smears analyzed in a previous studyCervical tissue DNA was extracted based on manufacturer’s protocol DynBio kit . The quantity and quality of extracted DNA were measured by a Nanodrop One Spectrophotometer  and the beta-actin gene, respectively Table .Establishing an effective qPCR-HRM approach requires choosing an area with a highly conserved region with the least interspecific variation for primer design and an unconserved region with the most intraspecific variation for pathogenic agent differentiation based on melting temperature.L1) was preferred for assay design. The suggested primers were selected for ordering and establishment of the semi-nested qPCR-HRM method  were used for the three approaches mentioned below.For HPV detection, with the aim of screening and investigating HPV prevalence, 2 µl of the sample were subjected to PCR in a 20 µl reaction mixture volume using the general FRG5 forward and MY09 reverse primers on a conventional thermal cycler  with the PCR program described in the reference 37. 29/57HPV-positive samples were sequenced  and submitted to GenBank (MG825048-MG825062 and MZ305435- MZ305448)37. All 7 plasmids were genotyped by Sanger sequencing  and then used in all approaches as diagnostic standards for HPV genotypes and amplification controls.In the next step, to construct the diagnosis standard, identified HPV genotypes were amplified by conventional PCR using FRG5/2 primers. The 221 bp PCR product was cloned in the PTG19 vector according to the manufacturer's protocol of the Sinaclon kit In the first approach, qPCR and HRM were performed in a single run by FRG5/2 primers in a reaction mix containing 4 μl 5 × Hot FIREPOL EvaGreen HRM Mix-Rox , 2 μl of diluted PCR product of FRG5/MY09 primers or plasmid, and 5 pmol of each primer with double distilled water to a total volume of 20 µl. The reaction conditions included an activation step at 95 °C for 15 min followed by 45 cycles of 95 °C for 20 s, 50 °C for 30 s, and 72 °C for 30 s. HRM was carried out over the range from 60 to 95 °C, with an increment of 0.3% °C for 15 s.6.The second approach was performed with asymmetric qPCR and HRM by FRG5/FRG2 primers and UP18 probe. Briefly, the reaction mix contained 4 μl 5 × Hot FIREPOL Eva Green HRM Mix-Rox , 2 μl of diluted PCR product of FRG5/MY09 primers or plasmid, 2 μl of FRG2 (5 pmol/µl), 0.4 μl FRG5 (2/5 pmol/µl) and 2 μl probe UP18 (5 pmol/µl). The amplification protocol was predenaturation for 15 min at 95 °C followed by 65 cycles of denaturation for 15 s at 95 °C, annealing for 20 s at 46 °C, and extension for 30 s at 72 °C. HRM was continued from 60 to 95 °C, with an increment of 0.3% °C for 15 sIn the third approach, the qPCR-HRM technique was performed with FRG5, FRG2, and GP5 primers. The qPCR mix reaction and amplification protocol were performed according to the first approach.Leishmania (without an HPV DNA insert)46 was used as a negative control.In all approaches, deionized water was used as non-DNA blank control, and a plasmid containing the 225 bp ITS2 region of Each approach in the first phase was set up by identified diagnostic standards and then evaluated alongside microarray samples of patients.To investigate intra-assay and interassay reproducibility, all reactions were performed in duplicated form on Applied Biosystems StepOnePlus  and LightCycler 96  in two different labs. Moreover, assay performance was assessed by comparing CLART HPV4  and the microarray method.All statistical analyses were performed with MedCalc statistical software  and SPSS software .The study protocol was reviewed and approved by Ethics Committee of Qom University of Medical Sciences (IR.MUQ.REC.1396.40), Iran.


A screening test is done to confirm the presence of the virus in the cervix. Many screening tests are available today for accurate diagnosis or confirmation of the condition being suffered from. The main goal of screening is early detection and making certain lifestyle changes to deduce the potential harm of the disease and start the treatment as soon as possible. A thorough study of the already published articles by scholars, professors, and doctors is carried out to conclude the necessity of cervical cancer screening and early detection.Cancer has been one of the major illnesses faced by people over many generations. Despite the advancements made in medicine, there are still many problems faced by humankind. Every year a large number of people are diagnosed with cervical cancer. It is the most common type of cancerous condition prevalent among females, especially females over thirty years of age. Like any other cancer, cervical cancer also occurs because of the rapid uncontrolled division of the body cells. A primary reason for its development is the longstanding infection of certain types of  The fourth most common cancer in women worldwide, cervical cancer kills 85% of its victims in low- and middle-income countries . In deveHuman papillomavirusPapillomaviruses are pervasive, have been found in a wide range of species in addition to humans, and are unique to the hosts they infect. If treatment is delayed, HPV infection can occasionally develop into a premalignant lesion that becomes more advanced and aggressive . NumerouEarly detection and screeningEarly identification of cervical cancer (CC) has decreased the disease's mortality and morbidity, and both structured and opportunistic pap smear-taking has been shown to minimize CC incidence rates . EffectiTwo sections that indicated a tendency toward less comprehensive screening were highlighted when key guidelines-making organization unified their recommendations for cervical cancer screening in 2012: 1) Regardless of the age at which sexual activity first begins, regular cervical screening really shouldn't start until age 21, and 2) The recommended frequency for regular cytology testing would be every three years, with the option to transition to cytology plus HPV testing ("co-testing") every five years starting at age 30. These modifications were brought about by a greater comprehension of how the sexually transmitted infection of human papillomavirus (HPV), which is quite common, contributes to cervical cancer development . AssociaPap/CISOE-A, a modified Papanicolaou classification system, or the Bethesda classification system, is used to classify cervical smears . It has The Pap smear methodThe classic Pap-smear is made in a straightforward and uncomplicated manner; biological material first from the squamocolumnar connection in the cervix is gently scraped off with a brush or scraper and spread onto a microscope slide measuring approximately 25 to 50 mm. Below a microscope, the cells are dyed, fixed, and subsequently visually inspected . CytotecVaccination and preventionIdentifying hrHPV as the essential factor in carcinoma development has resulted in ground-breaking improvements in preventing cervical cancer, including preventive HPV vaccination. Given the fundamental evolutionary biology of HPV infection and cervical malignancy, the necessity of infection prevention in adolescent students who have been the least likely to be exposed, and the high levels of effectiveness and consequent expenditure of HPV vaccine ,24. IdenCervical cancer avoidanceThere are two types of cervical cancer prevention: primary and secondary. Safer sexual behaviors, such as using condoms correctly and consistently to avoid cervix HPV infection, are essential to the primary prevention of the disease. The recently released HPV vaccinations Cervarix  and Gardasil  may also help with the primary prevention of cervical cancer . A custoEducating peopleHealth-seeking behavior is impacted by awareness of the many cervical cancer indications, symptoms, and risk factors. Furthermore, the chance of contracting human papillomavirus (HPV) may be substantially decreased given their awareness of and favorable attitudes toward cervical cancer vaccination ,5. NumerBarriers and affecting factorsPrograms for cervical screening in underdeveloped nations were not previously given attention. Prior to developing community-based screening programs, it is crucial to investigate variables and challenges related with cervical screening uptake . The varThe Pap smear test is a crucial and effective way to check for cervical cancer. Precancerous lesions are treated, and women are screened for the condition using Pap smears globally. In certain industrialized nations, Pap smear screening has shown impressive success in lowering cervical cancer mortality and incidence. Wherever screening quality and reliability are good, the cases of this type of cancer can be decreased by up to 90%. However, numerous women have never had a Pap smear in advanced countries, which accounts for 80% of all new cases. Screening programs must be appropriately implemented to lower the disease rate and severity of cancer in India. The study found that college students generally have a poor grasp of cervical cancer, HPV, and HPV vaccination. This comprehension is significantly connected to the student's age and biology-related educational background. There is an urgent need for educational intervention, both formal and informal, for both girls and boys with backgrounds in biology and non-biology, as well as for parents who wish to change their attitudes against HPV vaccination. To do this, print and audio-visual media may both be crucial. It is advised that the National Immunization Program include HPV vaccination so that it may be integrated with the screening (via pap/HPV DNA) and national cancer control programs, reducing the hurdles of vaccine cost and poor awareness for efficient prevention.


To investigate the differences in HPV genotypes and clinical indicators between cervical squamous cell carcinoma and adenocarcinoma and to identify independent predictors for differentiating cervical squamous cell carcinoma and adenocarcinoma.A total of 319 patients with cervical cancer, including 238 patients with squamous cell carcinoma and 81 patients with adenocarcinoma, were retrospectively analysed. The clinical characteristics and laboratory indicators, including HPV genotypes, SCCAg, CA125, CA19-9, CYFRA 21–1 and parity, were analysed by univariate and multivariate analyses, and a classification model for cervical squamous cell carcinoma and adenocarcinoma was established. The model was validated in 96 patients with cervical cancer.P < 0.05). Logistic regression analysis showed that SCCAg and HPV genotypes (high risk) were independent predictors for differentiating cervical squamous cell carcinoma from adenocarcinoma. The AUC value of the established classification model was 0.854 (95% CI: 0.804–0.904). The accuracy, sensitivity and specificity of the model were 0.846, 0.691 and 0.899, respectively. The classification accuracy was 0.823 when the model was verified.There were significant differences in SCCAg, CA125, CA19-9, CYFRA 21–1, HPV genotypes and clinical symptoms between cervical squamous cell carcinoma and adenocarcinoma (The histological type of cervical cancer patients with persistent infection of high-risk HPV subtypes and low serum SCCAg levels was more prone to being adenocarcinoma. When the above independent predictors occur, the occurrence and development of cervical adenocarcinoma should be anticipated, and early active intervention treatment should be used to improve the prognosis and survival of patients. Cervical cancer is the most common malignant tumour of the female reproductive system, and it ranks fourth in both the number of new cases of cancer and the proportion of cancer deaths among women in the world. In 2020, there were approximately 604,000 new cases and 342,000 deaths worldwide, so cervical cancer poses a serious threat to the lives and health of women worldwide . SquamouScreening and Treatment Guidelines for Cervical Precancerous Lesions released by the World Health Organization (WHO) clearly recommended human papillomavirus (HPV) DNA testing as the preferred screening method for cervical cancer [In 2021, the second edition of the l cancer . Persistl cancer . When HPl cancer . HPV spel cancer . Therefol cancer , 19, butAt present, clinical indicators such as tumour markers are often used in relevant studies on the identification of cervical squamous cell carcinoma and adenocarcinoma. Such indicators are easy to obtain clinically and play an important role in the differential diagnosis and prognosis prediction of tumours –25. LehtThe inclusion criteria of this study were as follows: (1) the surgical method was radical hysterectomy and pelvic lymph node dissection, and cervical cancer was confirmed by postoperative pathology combined with immunohistochemistry; (2) preoperative chemotherapy, radiotherapy, coning or other treatments were not performed; and (3) the preoperative clinicopathological data were complete. The exclusion criteria were as follows: (1) other malignant tumours or major diseases; and (2) rare histological types of cervical cancer, such as adenosquamous carcinoma, clear cell carcinoma, or small cell carcinoma.In this study, a total of 415 cases of cervical cancer admitted to the Department of Gynecology of the First Affiliated Hospital of Chongqing Medical University from January 2018 to September 2021 were enrolled according to the inclusion and exclusion criteria. The 319 cases (238 cases of squamous cell carcinoma and 81 cases of adenocarcinoma) from January 2018 to December 2020 were used as the primary cohort for retrospective analysis and establishing a clinical classification model; 96 cases (76 cases of squamous cell carcinoma and 20 cases of adenocarcinoma) from January 2021 to September 2021 were used as the validation cohort to verify the model effect. The demographics information of the study population are shown in Table General clinical features and laboratory indicators included age, clinical symptoms, smoking history, parity, gravidity, menopausal status, BMI, red blood cell count , white blood cell count , platelets , neutrophil percentage , lymphocyte percentage , SCCAg , CA125 , CA19-9 , CYFRA 21–1 , carcinoembryonic antigen , and HPV genotypes.The above indicators were collected after the patients were admitted to hospital and before radical hysterectomy. Patients' blood routine data  and tumour marker data  were obtained by blood samples drawn from veins. The patient's HPV-DNA test was carried out using cervical secretions and exfoliated cells of the cervix. The specific method was as follows: a disposable cervical sampler special cervical brush was placed in the cervical opening, rotated 5 times clockwise, put into 2 ml cell special preservation solution, fully rinsed, and then broken along the crease of the brush handle, leaving the brush head for examination.The clinical symptoms were assessed as follows: contact bleeding, irregular vaginal bleeding, asymptomatic or other. For the HPV risk subtypes, high risk refers to HPV negative or only HPV alpha 7 positive; medium risk refers to both HPV alpha 7 and HPV alpha 9 being positive; and low risk refers to positive only for HPV alpha 9 or other HPV genotypes , 19.t tests. If not normally distributed, the measurement data are expressed as the median (interquartile range), and significant differences between two groups were analysed using the Mann–Whitney U test. The qualitative data are represented as n (%) and were compared using the chi-square (χ2) test or Fisher's exact test. Spearman correlation analysis was used for correlation analysis. Independent predictors of cervical squamous carcinoma and adenocarcinoma were analysed by binary logistic regression. The Hosmer–Lemeshow test was used to analyse the goodness-of-fit of the model. Receiver operating characteristic (ROC) curves and calibration curves were drawn to evaluate the prediction efficiency of the model. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS statistical software version 22.0. The measurement data conforming to a normal distribution are presented as the mean ± standard deviation (SD), and the comparison between the two groups was performed by two independent sample P < 0.05). The SCCAg level was significantly higher in cervical squamous cell carcinoma than in adenocarcinoma, and the difference was statistically significant (P < 0.001). The main clinical symptoms of the cervical squamous cell carcinoma group were contact bleeding (54.2%), followed by irregular vaginal bleeding (27.7%). The clinical symptoms of the adenocarcinoma group were mainly contact bleeding (45.7%), followed by asymptomatic or other symptoms (32.1%), and there was a significant difference between the two groups (P < 0.05). Regarding the HPV risk subtypes, the high-risk subtype (61.7%) was more common in the adenocarcinoma group, while the low-risk subtype (86.1%) was more common in the squamous cell carcinoma group, and there was a significant difference between the two groups (P < 0.05) . The distribution of clinical indicators and HPV risk subtypes between squamous cell carcinoma and adenocarcinoma is shown in Table The levels of CA125 and CA19-9 in the cervical adenocarcinoma group were higher than those in the squamous cell carcinoma group, while the level of CYFRA 21–1 was lower, and the differences were statistically significant  and HPV risk subtypes  were independent predictors for distinguishing cervical squamous cell carcinoma from adenocarcinoma. For each unit increase in the SCCAg level, the risk of the cervical cancer subtype being adenocarcinoma decreased by 0.671 times. Patients infected with high-risk HPV subtypes were 19.722 times more likely to develop adenocarcinoma of cervical cancer than those infected with low-risk HPV subtypes. The details are shown in Table Binary logistic regression analysis was performed using the significantly different indicators between cervical squamous cell carcinoma and adenocarcinoma combined with clinical experience indicators as independent variables and the classification of the two as dependent variables. It was found that SCCAg (P = 0.963) indicated that the model had a high goodness of fit. A ROC curve was drawn for the classification model of cervical squamous cell carcinoma and adenocarcinoma. The area under the curve (AUC) was 0.854 , and the model accuracy rate was 0.846. The prediction probability under the maximum Youden index (0.59) was taken as the cut-off value (cut-off = 0.277), and the sensitivity and specificity of the model were 0.691 and 0.899, respectively, as shown in Fig. According to the multivariate analysis of HPV risk subtypes and clinical indicators, a clinical classification model mainly based on SCCAg and HPV risk subtypes was established. The nonparametric Hosmer–Lemeshow test results P = 0.96 indicateP > 0.05). Among them, there was no significant difference in parity or BMI between cervical squamous cell carcinoma and adenocarcinoma, which was consistent with previous research results [In current study, the age and distribution proportion of histological subtypes of the study population were consistent with previous studies and known knowledge . In our  results , 30. How results , 31.P < 0.05), which was consistent with previous research results [P = 0.084), which was consistent with previous research results [P < 0.05). This result may be because CYFRA 21–1 is a product of cytokeratin 19, which is mainly distributed in squamous and monolayer epithelial cells [P = 0.009, OR = 0.671, 95% CI = 0.497–0.905). SCCAg is a subcomponent of TA-4 extracted from cervical squamous cell carcinoma, and its serum level can be used as one of the auxiliary indicators for the diagnosis, efficacy evaluation and prognosis prediction of cervical squamous cell carcinoma [In the study of tumour markers, we jointly studied SCCAg, CA125, CA19-9, CYFRA 21–1 and CEA. The results showed that the levels of CA125 and CA19-9 in cervical adenocarcinoma were higher than those in squamous cell carcinoma, while the level of SCCAg in cervical squamous cell carcinoma was higher than that in adenocarcinoma; the difference was statistically significant . That is, cervical adenocarcinoma is more likely to show no HPV infection or only HPV alpha 7  positivity, whereas cervical squamous cell carcinoma is more likely to show only HPV alpha 9  positivity or positivity of other HPV genotypes. This result may be because HPV 18, as the most common genotype of HPV alpha 7, is most associated with cervical adenocarcinoma, while HPV 16, as the most common genotype of HPV alpha 9, is closely related to cervical squamous cell carcinoma [P < 0.001, OR = 19.722, 95% CI = 9.252–42.040). Patients with high-risk cervical cancer who are HPV negative or only infected with HPV alpha 7 have a worse clinical prognosis [In the correlation study of HPV risk subtypes and the identification of cervical squamous cell carcinoma and adenocarcinoma, the results of this study showed that the HPV-negative rate of cervical adenocarcinoma was approximately 22.2%, while that of cervical squamous cell carcinoma was approximately 3.8%. This is consistent with the results in previous studies that approximately 20%-30% of patients with cervical adenocarcinoma were HPV negative, while only approximately 5% of patients with cervical squamous cell carcinoma were HPV negative –38. Morearcinoma . In furtrognosis . Our resP < 0.001), and the model accuracy was 0.846. The prediction probability under the maximum Youden index was taken as the cut-off value, and the sensitivity and specificity of the model were 0.691 and 0.899, respectively. Nonparametric Hosmer–Lemeshow test results (P = 0.963) indicated that the model had a high goodness of fit, suggesting that the classification model had good differentiation and calibration abilities and could distinguish cervical squamous cell carcinoma and adenocarcinoma well. In addition, the data of cervical cancer patients from January 2021 to September 2021 were used for model validation, and the classification accuracy was 0.823, indicating the good stability and reproducibility of the model.At the same time, this study established a clinical classification model for the differential diagnosis of cervical squamous cell carcinoma and adenocarcinoma based on SCCAg and HPV risk subtypes, and the ROC curve was drawn. The AUC of the model was 0.854  As a retrospective study, this study has a certain selection bias. For example, the sample size of the smoking group was small, which makes it difficult to evaluate the difference in smoking history between cervical squamous cell carcinoma and adenocarcinoma. (2) Imaging features were not included in this study, and we only explored the differences in clinical features and laboratory parameters between cervical squamous cell carcinoma and adenocarcinoma. In the future, we will combine imaging features for further research.In conclusion, HPV risk subtypes and SCCAg are independent predictors for differentiating cervical squamous cell carcinoma from adenocarcinoma and can play a good role in classification. The histological type of cervical cancer patients with persistent infection of high-risk HPV subtypes and low serum SCCAg levels is more prone to being adenocarcinoma, while the histological type of cervical cancer patients with persistent infection of low-risk HPV subtypes and high serum SCCAg levels tends to be squamous cell carcinoma. Clinical attention should be given to the occurrence and development of cervical adenocarcinoma, and early intervention should be given to improve the prognosis and survival of patients.