An estimated 42,000 people currently living with chronic spinal cord injury (SCI) are veterans. SCI was a common combat-related injury in the World Wars and Vietnam era and now affects more than 11% of military personnel injured in Operation Iraqi Freedom and Operation Enduring Freedom. The Veterans Benefits Administration primarily offers financial compensation for disabilities sustained or re-aggravated during military service, called service-connected disability compensation. With the overwhelming cost of living with an SCI, this monthly financial compensation can provide service-connected veterans and their families with access to additional supportive resources  and maintain their quality of life (QOL). Little is known about personal, health, functional, and QOL outcomes associated with service-connected and nonservice-connected status for veterans living with an SCI.The aim of this study is to compare the ways in which Veterans Affairs’ (VA) service-connected and nonservice-connected status may be associated with health and functional outcomes, choice of health care provider, and overall QOL for veterans living with an SCI and their caregivers.This cross-sectional qualitative study will gather data using retrospective chart reviews, semistructured interviews, and focus groups. After obtaining institutional review board (IRB) approval, purposeful sampling techniques will be used to recruit and enroll the following key stakeholders: veterans living with an SCI, family caregivers, and SCI health care providers. Concurrent data collection will take place at 2 sites: Veterans Administration New Jersey Healthcare System and Northern New Jersey Spinal Cord Injury System.This study was funded in July 2015. IRB approval was obtained by November 2016 at both sites. Enrollment and data collection for phase 1 to phase 4 are complete. A total of 69 veterans, 18 caregivers, and 19 SCI clinicians enrolled in the study. Data analyses for these phases are underway. In phase 5, the follow-up focus group activities are scheduled. The final results are expected by the end of 2019.The factors that contribute to veterans living with SCI seeking and not seeking VA disability compensation benefits are not well understood in rehabilitation research. Triangulation of these data sources will allow us to compare, contrast, and integrate the results, which can be used to develop clinical guidelines, caregiver training, and patient education programs.DERR1-10.2196/14039 The Donnected ,6. For eAs the cost of living with an SCI can be insurmountable, the monthly financial compensation provided to service-connected veterans living with SCI through the VBA can be used to offset the loss of wages. In addition, veterans who are service connected specifically for their spinal cord condition  may also qualify for additional grants funded through the VBA. These grants promote functional independence by providing resources for the aid and attendance required to maintain the veteran in the least restrictive setting; for example, a VA automobile allowance or specially adapted housing grant where the goal is to help veterans participate in their home life, employment, and social activities that might otherwise be inaccessible and maintain positive quality of life ,8-10.Despite VA’s efforts to reduce the financial burden associated with successful rehabilitation, independent living, and community integration through disability benefits, a portion of veterans living with SCI have nonservice-connected disabilities because their disabilities were not incurred or aggravated by their military service ,6. On thThe Department of Veterans Affairs (VA) estimates approximately 450 newly injured veterans and active-duty members receive rehabilitation at VA’s SCI centers annually ,11,12.Rehub and spoke system. The VA SCI/D System of Care includes 25 regional SCI/D Centers (known as hubs) that provide comprehensive range of care including, inpatient and outpatient rehabilitation, specialty care, and coordinated lifelong continuum of care delivered by interdisciplinary teams [The primary goal of VA SCI and Disorders (SCI/D) services is to restore functioning, reduce secondary complications, and promote the health and sustainability of functional independence to maximize QOL after injury . The VA ry teams . After rry teams . Thus, iry teams ,17.National Institute on Disability, Independent Living, and Rehabilitation Research, Administration for Community Living, US Department of Health and Human Services, supports innovative projects and research in the delivery; demonstration; and evaluation of medical, rehabilitation, vocational, and other services to meet the needs of individuals with SCI. The Northern New Jersey Spinal Cord Injury System (NNJSCIS) was established as a SCIMS in 1990. The NNJSCIS provides a comprehensive continuum of state-of-the-art care for persons with spinal cord injury and their families from the time of injury through rehabilitation and return to the community.The SCI Model Systems (SCIMS) are specialized programs of care in SCI that gather information and conduct research with the goal of improving long-term functional, vocational, cognitive, and QOL outcomes for individuals with SCI. SCIMS, sponsored by the service connected [average impairment in earning capacity [scheduler ratings), with a higher percentage of rating equaling a greater functional impairment and amount of disability compensation awarded. Typically, a VA disability rating is derived from an algorithm that combines the individual scheduler ratings of each compensated disability [VA provides monthly disability compensation benefits to veterans who develop medical conditions and disabilities related to military service; that is, who are deemed onnected ,18-19,20onnected  followincapacity -23,18-19sability ,6. Theresability ,6. The asability ,6. Unliksability -23,18-19invisible wounds) and external factors  [Little is known about the differential impact of service-connected status on the health status, functional outcomes, QOL, and health care utilization patterns of veterans living with SCI. Interestingly, veterans service-connected for PTSD have been found to report high rates of medical impairment, psychiatric symptomatology, and utilization of medical and mental health services ,23,18. Fenefits) . These fThe proposed study intends to address this gap in the literature using qualitative research methods to explore the perspectives of service-connected and nonservice-connected veterans living with SCI, family caregivers as well as SCI clinicians about factors that contribute to these veterans’ health status, functional outcomes, and health care utilization. On the basis of the aforementioned literature, individual/personal factors, socioeconomic, family, and health system factors will be explored. Individual factors such as demographic characteristics , cultural beliefs, socioeconomic status , and health risk behaviors  have been found to the impact on health status, functional outcomes, and health care utilization in veterans living with SCI . Family This investigation will be guided by the framework of the International Classification of Functioning, Disability, and Health (ICF) ,26 to exThe proposed project will use qualitative methods to examine the factors associated with outcomes for service-connected and nonservice-connected SCI. Qualitative methods have the advantage of allowing us to address these aims in a manner that is meaningful to individuals who are actively involved in SCI veteran rehabilitation: veterans living with SCI, family caregivers, and SCI clinicians. We propose the following aims:To describe veterans living with SCIs’ reasons for seeking versus not seeking service-connected disability compensation and the factors that influence their choice.To explore the impact of service-connected disability compensation on health status, functional outcomes, QOL, and medical decision making .To explore the impact of service-connected disability compensation on the family caregivers and households of veterans living with SCI.To explore SCI clinicians’ perspectives on the impact of service-connected disability compensation status on the provision of adequate long-term health care and rehabilitation for veterans living with SCI.To develop a set of practice and policy recommendations about the impact of service-connection status of veterans living with SCI on clinical and policy guidelines, family interventions, caregiver training, and patient education programs.The proposed work is significant because it will provide new knowledge about veterans living with SCI with and without service connected disability compensation in the realm of family caregiver support, access to community resources, personal factors, and health behaviors, including patient-provider relationships and their impact on health status, functional outcomes, and QOL. The findings will describe areas of care considered priorities for veterans living with SCI and families that must be clearly integrated into clinical care to support the successful maintenance of health status, functional outcomes, and QOL.The study protocol, research team and data collection instruments were reviewed and approved by the institutional review board (IRB) at each study site as well as US Army Medical Research and Materiel Command`s Office of Research Protections, Human Research Protection Office.experts to explore the health status, functional outcomes, and QOL of veterans living with SCI with different types of disability benefits and who decide to receive their health care from VA centers and non-VA.This cross-sectional qualitative study will use a community-based participatory approach to examine the reasons for seeking disability compensation benefits, factors associated with outcomes, and choice of health care facility among veterans living with SCI with and without service-connected disability benefits. Community-Based Participatory Research represents a collaborative process between researchers and community partners; builds on the unique strengths, knowledge, and resources within a given community by employing local knowledge in the understanding of health problems and their potential solutions; and facilitates collaboration throughout all phases of the research .We will hub and spoke system of care, private care sector of SCI care, caregiving, and SCI research.To achieve these aims, we assembled a highly productive and interdisciplinary team of SCI/D researchers and community advisors with expertise in qualitative research methods, VA SCI The study will collect data from 3 groups of participants involved in sustaining the health status, functional independence, and QOL of veterans living with SCI who are impacted by the type of disability benefits and health care utilization patterns:An equal number of veterans living with SCI receiving clinical services or who have previously participated in research at the East Orange Campus of the VA New Jersey Health Care System (VANJHCS) or Northern New Jersey SCI Model System (comprised the Kessler Foundation and Kessler Institute for Rehabilitation) will participate in phase 1 (n=15 per site) and 2 (n=30 per site) of the study (described below).Inclusion criteria are being male, veterans (or served on active duty service in the US Armed Forces), at least 18 years of age, SCI that occurred at least 1 year ago, and receiving health care/rehabilitation at the participating VANJHCS and/or a participating Spinal Cord Injury Model System site or Spinal Cord Injury Model System (SCIMS).Exclusion criteria are inability to communicate because of neurological impairment ; identifying as a female. We limited the scope of the study to patients to male veterans living with SCI because there are not likely to be sufficient numbers of female veterans living with SCI to be able to recruit a meaningful sample of them, given a larger proportion of people with SCI and veterans are males and thus beyond the scope of this project.primary caregivers. Family caregivers who may be eligible to participate in the focus groups or interviews will be identified by clinical staff  or an SCI veteran who participated in a semistructured interview. A member of the research team will contact the family caregiver to inform them about the study and determine if they meet the eligibility criteria to provide additional information and make arrangements to obtain informed consent before the start of focus group or interview data collection.SCI often results in physical limitations such that receiving assistance from others is critical to maintaining health and facilitating full societal integration. The help received ranges from assistance with basic daily activities such as bowel and bladder management and dressing, as well as instrumental activities of daily living, including managing household finances, shopping, or transportation. In the United States, most home care is provided through informal mechanisms by family members , and almClinicians’ perceptions are important because they may affect patient-provider relationships, the course, and the outcome of treatment. Clinicians have knowledge of the medical and functional consequences of SCI and experience providing training to veterans living with SCI and their family caregivers to plan for adjusting to home life and community reintegration. SCI clinicians at each participating center will be informed of the study through written (flyers and emails) and oral communications with local study leaders and/or supervisory staff. We will recruit a sample of 10 clinicians at both sites that includes staff members who have experience providing direct care or services to veterans living with SCI at each of the research sites. The SCI clinical staff will include physiatrists, nurses, social workers, physical, recreation, and occupational therapists who have at least 2 years of experience providing care to SCI. Clinicians who are interested in participating and wish to be contacted by the research team will receive additional information and, if interested, give informed consent before the start of focus group data collection.Each group of participants has real-world experiences and clinical knowledge that will inform the content of the key practice recommendations that can be readily integrated into clinical guidelines, family interventions, caregiver training, and patient education programs.Two study sites that serve veterans living with SCI in New Jersey will conduct the proposed study: VANJHCS and Northern New Jersey Spinal Cord Injury System. A description of each research site is provided in After obtaining approval from the IRB at the study sites, purposeful sampling strategies will be used to identify and recruit potential study participants. A research coordinator (RC) will recruit potential participants using advertisements, brochures, and referrals from SCI registries at each collaborative site. If we do not get an adequate recruitment response, we will implement the snowball recruitment technique that involves asking participants to inform and encourage friends, colleagues, and other peers to participate .In recent years, data saturation has become the gold standard by which purposive sample sizes are determined in qualitative research . TheoretOnce each participant completes oral and written informed consent and is scheduled for interview or focus group session, he/she will receive a telephone reminder 2 weeks before the interview or focus group as well as a written letter 1 week beforehand. The day before the event, the consultant will make 1 last round of confirmatory phone calls. Participants will be compensated for their time. In addition, to maintain enrollment and participation, we will provide transportation cost for a portion of veterans living with SCI and family caregiver on an as-needed basis.independently interpret the reasons and impact of the disability benefits on veterans living with SCIs’ . Results from each phase will be analyzed separately and then merged to inform the content of the subsequent phases as well as the set of practice recommendations that can be readily integrated into clinical guidelines and family interventions. Three methods of qualitative data will be collected: chart review, semistructured interviews, and focus groups. These 3 qualitative data collection methods will be implemented over 4 phases of sequential qualitative data collection outlined in comprehensive annual history/physical exam with specialty assessments, offering an annual evaluation is mandated for patients with SCI in the Veterans Health Administration (VHA) [The RC will be trained to use a standardized chart abstraction instrument to gather demographic, clinical, and disability benefits data. The chart review will be designed to help prepare and supplement data gathered in subsequent phases by providing data that will (1) inform the development of discussion questions for the participant interviews and focus groups; (2) confirm the veterans living with SCI disability status and rating; and (3) documentation of health status, functional information, and patient/family education logs gathered during the most recent annual evaluation of 30 veterans living with SCI (15 per site). An annual evaluation was defined as a on (VHA) .Semistructured interviews will be aimed at capturing service-connected and nonservice-connected veterans living with SCI perspectives on ways in which their financial compensation (or lack thereof) impacts their health status, functional independence during community reintegration, QOL, and their utilization of health care. The sample will be equally split between study sites (n=30) veterans. We will modify items from the benefits coverage inventory, a measure that has been used in previous research to assess rehabilitation/independent living benefits received after discharge in 5 areas: housing, personal care assistant, transportation; outpatient therapies , and equipment (durable and nondurable). The measure asks about who pays for these items . We will work with the CAB to develop semistructured interview questions that relate to service-connected disability benefits. The semistructured interview will discuss veterans living with SCIs’ likes and dislikes of the being service-connected or nonservice-connected—their perceptions about whether disability benefits may be viewed as a barrier to independence, and difficulties with bureaucracy for some veterans living with SCI. The interview will ask veterans living with SCI to express their experiences about unexpected barriers associated with seeking service-connected financial compensation. Key questions will focus on their health status, maintenance of their functional independence during community reintegration, health care, and rehabilitation experiences. The individual semistructured interview allows for rapport and confidence building at a sensitive time after injury so that more honest opinions and attitudes may be revealed more readily than in a group setting. The interviewer can answer respondent questions, probe for additional answers, and observe visual cues. To facilitate access to veterans living with SCI who are unable to come to the research site for an interview, we will use the VA’s real-time video health tool—Clinical Video Telehealth that is a technology that is frequently used in the VA to promote video communication between patients and providers  clinical factors of veterans living with SCI who seek different types of disability compensation benefits ; (2) describe their perception about the relationship between disability benefits and health status, functional outcomes and health care utilization among veterans living with SCI; (3) identify VA and private-sector health care system issues related to disability compensation benefits, which are obstacles for veterans living with an SCI; and (4) identify solutions to address these concerns.To design a useful set of practice recommendations, we will analyze results from phases I to IV separately and then merge them to prepare the content materials for phase V. The chart review, interview, and focus group data will be prepared for analysis by converting the raw data  into partially processed data , which will then be coded and subjected to an analytic theme. The analysis will focus on the key research questions and include the following steps:Read each transcript in an editing style to augment an initial codebook template developed from the ICF guidelines and interview guide.Read and reread highlighted portions to develop keywords .Divide the themes, patterns, and categories into groups by the research questions.Examine the convergence/divergence by completing the following steps:Convergence will be examined by determining what themes fit together to develop the internal homogeneity and external heterogeneity.Internal homogeneity will be determined by analyzing the themes to see which are more similar, and external heterogeneity was determined by analyzing the themes to see which ones are distinctly different from each other .These processes will enhance the credibility of the research. Divergence will reveal some of the patterns within the categories and helps to make connections among the themes for categorical saturation .Qualitative data collected in phases I to IV will generate its own findings to add additional information to our understanding about the reasons veterans living with SCI seek different types of disability benefits and how these factors that are associated with health status, functional outcomes, and QOL. Triangulation is methodological approach that contributes to the validity and reliability of qualitative data collection when multiple method and sources are employed . Triangugroup-to-group validation [Using the themes generated from the qualitative analyses, we will work with the CAB to summarize to identify the most frequently cited factors  that impact veterans living with SCIs’ decision to seek service-connected disability benefits  and across samples of participants—a process that is known as lidation .There are a number of steps outlined in the literature used to develop targeted recommendations for clinical guidelines, developing recommendations for patient and family educational resources, and policy-level interventions. First, the research team will generate a summary of the most frequently cited factors identified from the data as recommendations to the CAB. Examples of potential interventions include revisions to existing patient and family education material related to programs and services available from the VA for veterans with SCI; educational interventions for SCI clinicians to make them more aware of resources available from the VA that are correlated with positive clinical practice outcomes for veterans with SCI veteran, and their families, caregivers, and other clinicians; procedures to help clinicians identify veterans living with SCI at civilian hospitals and connecting these veterans and their families with knowledgeable VA advocates  and VA disability compensation and health care resources.targets of change. After identifying the factors that are both important and modifiable, the CAB and research team will decide which factors will be targeted for recommendations for interventions. Finally, the CAB and research team will select promising strategies or key recommendations to develop an intervention action-point document. The intervention action-point document will be a road map toward the operationalization of an educational intervention for SCI, families, SCI clinicians, and policy recommendations for the VBA.Second, the CAB and research team will have monthly consensus meetings, which will be used to evaluate aspects of the most frequently cited individual, family, SCI clinician, and systemic factors generated from the data: importance to veterans living with SCI and modifiability. Determination of importance can be obtained in several ways, such as the CAB rating on the degree of importance, and assessing socially significant implications of the qualitative findings. The CAB and research team will collaboratively rate the modifiability of the individual, family, SCI clinician, and systemic factors from the qualitative data by asking the question, “Can this problem easily be addressed?” or “Can this solution be implemented pragmatically?” The collaborative will rate these individual, family, SCI clinician, and systemic factors as (1) factors that can be completely changed, (2) factors that may be modified, but we are unable to change them completely, and (3) factors that are nonmodifiable. Third, the CAB and research team will select Content of the intervention action-point document will be evaluated assessed for their appeal, clarity, and appropriateness for the target users. We will recruit veterans living with SCI and family caregivers who participated in phases I to III. We will recruit approximately 5 veterans living with SCI and 5 family caregivers. Veterans living with SCI and family caregivers will be asked if they are willing to participate in future groups or interviews. Members of the research team will recruit those who agreed using personal phone calls. If we do not get an adequate response from the previously identified participants, advertisements, brochures, word of mouth, and the aforementioned snowball technique will be used. An honorarium will be offered to compensate participants for their time and transportation.The project was funded in July 2015, and recruitment was completed in October 2018.A total of 69 male veterans living with an SCI participated in medical chart review and interview phases of the study. The mean age of the sample was 59.5 years . Most of the veterans self-identified as non-Hispanic white , married , had some college education , and unemployed . In terms of military experience, approximately one-third served in the army  and primarily in the Vietnam era . The majority of veterans sustained their injury after military service , and were living with paraplegia  for an average of mean 15.0 years  of the veterans self-identified as nonservice connected and 36% (25/69) were service connected for SCI or another disability . A total of 18 caregivers participated in focus groups and interviews at the 2 sites. All of the caregivers were females, the mean age of caregivers was 64.8 years (SD 10.8) and reported caregiving for their loved one for approximately, 15.9 years (SD 11.4). A total of 9 SCI clinicians participated in a focus group at Veterans Administration New Jersey Healthcare System and 10 at the SCIMS site. Comparable clinical professions  were represented in each focus group and average of 11.5 years (SD 8.8) working in their current positions.Preliminary content review of narrative data suggests that veterans living with an SCI and caregiver participants varied in their reasons why they did not apply for VA disability compensation, including a lack of knowledge and misinterpretations about the VA disability compensation eligibility and health care coverage from the VBA and VHA, respectively.independently interpret the reasons and impact of service-connected compensation (or lack thereof) on the health status, functional outcomes, QOL, and health care utilization of veterans living with SCI. Preliminary findings suggest a small proportion of participants receive VA service-connected disability compensation benefits. Participants’ responses indicate that veterans living with an SCI and their caregivers may not be fully aware of their eligibility for VA disability compensation, making it more difficult to make an informed decision about pursuing VA disability compensation benefits. To make an informed decision about eligibility for VA disability compensation benefits, veterans living with SCI should be connected with an experienced veterans benefits advocate (ie veterans service officer). Study findings will be used to generate a set of practice recommendations to the clinical guidelines, family interventions, caregiver training, and patient education programs that can be tested in a future large-scale multisite quantitative study to devise targeted community-based interventions.This qualitative study will use a community-based approach to derive information from veterans living with SCI, family caregivers, and SCI clinicians about their day-to-day experiences with being service connected or nonservice connected status. Of particular interest is how these groups


Specific HPV genotypes have been recognized as risk factors inducing head and neck cancers (HNC). The aim of this study was to validate a real-time PCR assay to detect accurately High Risk HPV DNA in Formalin Fixed Paraffin Embedded (FFPE) and oral cytobrush samples and compare the results with conventional PCR. Repeatability, reproducibility and limit of detection of Cobas assay were estimated for oral cytobrush and FFPE samples of patients with HNC. 53 samples of patients with a HNC were then used for assay comparison with conventional PCR. Finally, 26 samples of patients with anogenital neoplasia cancer were analyzed as control and assays comparison. Among the 53 samples of patients with HNC, 12 (26.7%) were HPV positive, 33 (73.3%) were HPV negative and 8 (15.1%) were non contributive with the Cobas assay. Among the 26 samples of patients with anogenital neoplasia, 15 (57.7%) were HPV positive and 11 were HPV negative (42.3%). One sample was found with an HPV 16 and HPV 18 co-infection. Only 3 samples were found with discrepant results. Cobas assay was found suitable for routine HPV detection with a very good repeatability and reproducibility for all HPV genotypes (CV < 0.6% and <0.4% respectively). Sensitivity and specificity for Cobas assay were 91.7%  and 96.9%  respectively. Ten nanograms of DNA were sufficient for the detection of HPV 16, HPV 18 and HPV in FFPE and oral cytobrush samples. Cobas assay was found comparable to conventional PCR and can detect accurately and rapidly HPV DNA in FFPE and oral cytobrush samples for the management of HNC and other types of HPV-associated neoplasia. HPV are classified into high-risk (HR) and low risk (LR) according to their oncogenic potential, as shown in Suppl Table 2.Specific types of human papillomavirus (HPV) are recognized as major oncogenic factors for the development of a large subset of head and neck squamous cell carcinomas. More than 245 HPV types have been described as pathogens in human and at least 40 of them have a tropism for anogenital mucosa3. In parallel, HPV has been recognized as a potent risk factor inducing head and neck cancers, especially oropharyngeal squamous cell carcinomas in young male nonsmokers4. An international meta-analysis shows that HPV 16 is found in 87% of oropharyngeal cancers and in 68% of cancers of the oral cavity5.Over the last decade, the incidence of head and neck cancer has increased, despite the decreasing risk factors like tobacco smoking and alcohol consumption6. Cervical cytobrush sampling is the gold standard for the detection of dysplasia and cancers of the cervix. Unfortunately, there is no clinical routine used to detect oral or oropharyngeal dysplasia before their progression. Only a thorough clinical examination allows identification of suspicious lesions and biopsy is the only way to confirm diagnosis and search HPV infection. The most widely used method for the cervix is a liquid cytobrush sample: liquid-based cytology method using residual cell suspensions. This sample allows the cytology assessment of the sample and the research for an HPV infection. Nevertheless, this assay has not been designed or validated to assess oral cytobrush or Formalin Fixed Paraffin Embedded (FFPE) samples.Several studies indicate that oral and genital HPV infection are not independent: same subtypes are found in oral and genital tract in a same women or in sexual partners7. FFPE tumor biopsies can be used for HPV detection or genotyping, but DNA can be altered or fragmented during the pre-analytical process and this may cause assays failure8. Few assays have been described for the detection and genotyping of HPV in FFPE9. The most common used and current validated assay is using PCR followed by a gel migration 10 but requires several manual steps and more time than a real-time PCR.HPV status represents an independent prognostic factor in head and neck carcinoma, most probably related to the fact that HPV positive tumors are more sensitive to radiotherapy than HPV negative tumorsThe aim of this study was to validate a real-time PCR assay to detect quickly and accurately HR HPV in FFPE head and neck tumor samples and oropharyngeal cytobrush samples. We validated this assay according to ISO 15189 directives and French accreditation comity (COFRAC) recommendations. In this study, we adapted the CE-IVD marked Cobas HPV assay, which is validated for the detection and genotyping of HPV in cervix cytobrush samples, for the detection and genotyping of HPV in FFPE tumors and oral cytobrush samples. We finally analyzed with both conventional PCR and Cobas real-time PCR assays 53 FFPE and oral cytobrush samples of patients with head and neck cancer in double blind conditions and compared the results. Twenty-six consecutive samples of patients with anogenital neoplasia were also analyzed with both assays and used for confirmation.Samples with a possible DNA amplification were set as “interpretable” and samples with a too low DNA quality and no amplification as “non-interpretable”. Among the 53 head and neck assessed samples 45 had interpretable results with Cobas assay. HPV DNA was found in 12 samples (26.7%), 11 HPV16 (91.7%) and one HR HPV (8.3%). DNA was amplified but no HPV DNA was found for 33 samples (73.3%). Among the 53 assessed samples 44 had interpretable results with the conventional PCR and DNA amplification was not possible for 8 samples with Cobas assay and 9 with conventional PCR. HPV DNA was found in 12 samples (27.3%), 11 HPV16 (91.7%) and one HPV 6/11 (8.3%). No HPV DNA was found for 32 samples (72.7%).Among the 26 anogenital neoplasia samples, HPV DNA was found in 15 samples (57.6%) and no HPV DNA in 11 samples (42.3%) with both assays. Cobas assay detected HPV16 DNA in 9 samples (60.0%), HPV 18 DNA in 5 samples (33.3%) and HR HPV DNA in 2 samples (13.3%). Conventional PCR detected HPV16 DNA in 8 samples (53.3%), HPV 18 DNA in 4 samples (26.7%) and HR HPV DNA in 5 samples (33.3%). Sample #3 was found with HPV 16 and HPV 18 with both assays  were 88.9%  and 93.0%  respectively Table .Table 2C12. There are currently no clinical guidelines differentiating the treatment of HPV positive and HPV negative cancers14. However, research suggests that HPV expression is correlated with increased response rates to conventional treatments as radiotherapy, chemotherapy and radiochemotherapy17. Moreover, patients with HPV-associated head and neck squamous cell carcinoma appear to be the most appropriate subgroup to benefit from minimally invasive trans-oral surgery18. HPV status of these tumors will probably determine their management, thus HPV detection in head and neck cancer is highly important.HPV infection is now clearly associated with an increasing incidence in oropharyngeal squamous cell carcinoma. The young adult population, non-smoker and non-drinker, is particularly exposed to these HPV-related cancersThe Cobas real-time PCR assay validation data proved that this method is suitable for the detection of HPV DNA in oral cytobrush and FFPE samples with a very high repeatability and reproducibility. Moreover, the assay was really sensitive and able to detect HPV DNA with a low DNA input of 10 ng. Robustness of the assay was also very good because no impact on the final result has been found when sample, mix or coenzyme volumes were modified.19 and laryngeal papillomatosis which may lead to carcinogenesis21. More recently, different studies showed that these two types are also involved in the development of anal dysplasia and cancers especially in men who have sex with men23. Two samples had HPV 16 DNA and one sample had HPV 18 DNA with Cobas assay and not with conventional PCR assay. We think that this result could be explained by a lower sensitivity of the conventional PCR assay, or could also be false positive; the Cobas HPV assay is based on TaqMan probes technology which has been reported to have no or few false positive results25. The last discrepant sample had only HPV 16 DNA with Cobas assay and HPV 16 and HPV HR DNA with conventional PCR. This discrepancy suggests that the sample could contain a double contamination with HPV 16 and another HPV with a genotype detected with the consensus primers of conventional PCR and not present in the consensus probes design of the Cobas assay.Only 12 HNC samples were positive, thus we chose to complete our study with 26 consecutive samples from patients with anogenital neoplasia. We assumed that most samples will be positive according to the HPV prevalence in this disease. We decided to not select only positive samples to also estimate the specificity of the Cobas assay in this cohort. Cobas Real-time PCR assay results were comparable to conventional PCR assay with a good sensitivity and specifiity for head and neck samples  and anogenital neoplasia FFPE samples . Some discrepancies were found in our study. One is related to the HPV types. HPV 6/11 DNA has been detected with conventional PCR and not with Cobas assay. This difference is explained by the design of the Cobas assay which is only based on the HR-HPV detection and HPV 6 and 11 are associated to low-risk. Whereas HPV 6 and 11 are considered as low risk HPV, they have been described as an etiological factor in the development non tumoral lesions, such as genital condylomas8.Finally, in our study Cp were lower for FFPE samples for the detection of High Risk HPV and HPV 16 than oral cytobrush samples and higher for HPV18. These results can be explained by the DNA quality used in this study. Quality of DNA extracted from FFPE tissues depends on the duration of fixation and sample conservation whereas DNA of oral cytobrush samples have been extracted less than one week after collectionOur study has some limitations: First of all, the detection of HPV in our samples is limited by the assays we used. Cobas assay can only detect high risk HPV and conventional PCR can only detect high risk and 6/11 HPV, thus the term “absence of high risk HPV DNA and 6/11 low risk HPV DNA” would be more exact than “no HPV DNA”, especially for samples which are non-tumour or non-neoplasia. Moreover, HPVs are fast evolving viruses with different variants found in different populations. Cobas assay uses concensus probes for HR HPVs and since no sequencing has been done in our study, we can’t affirm that Cobas assay may be able to detect all variants subtypes.26. Moreover, the sensitivity has been evaluated using quantity of DNA but not with HPV copy number, thus our study only allows an estimation of the limit of detection of the assay. We assume that the DNA we used for this determination contained human and HPV DNA. Hence, it would make more sense if the internal controls with known copy numbers could be used to gauge the sensitivity on the basis of copy numbers rather than total DNA concentration.Another technical limitation of the Cobas assay is the absence of copy number determination. It has been described that the copy number of the HPV infection will determine the duration and intensity of the therapies that will have minimal side effects but yet effective29. Finally, a capture NGS based assay has recently been described which allows the detection of 245 genotypes and the identification of viral insertion signature in human genome30.This second assays comparison allowed showing that Cobas assay results were comparable to conventional PCR results. Another limitation is that 100% of our samples found with HPV DNA were HPV16 and HPV18, which is consistent with HPV prevalence in European countries. Samples with other high risks HPV would have been useful to demonstrate that Cobas assay was able to detect other genotypes. Moreover, Cobas assay does not allow the exact genotyping of non 16 or 18 HPV. Several described assays like SPF10-PCR DEIA LiPA25, Inno-LiPA, Linear Array or Onclarity allow the identification of more genotypes even on FFPE samplesIn conclusion, we validated Cobas HPV test for the detection of HPV in FFPE samples and oral cytobrush samples. This assay is suitable for routine analysis and allows the detection of HPV DNA with a comparable sensitivity to conventional PCR.Fifty-three samples from 44 patients  treated for a head and neck at Institut de Cancérologie de Lorraine, were prospectively collected for this study.Among the 53 samples, 19 were oral cytobrush samples and 34 were FFPE tumor samples. Samples characteristics are detailed in Suppl Table ® . Brush was introduced in oral cavity and brushed 5 times on internal side of the cheek, mobile tongue and anterior pillar of the soft palate or on the tumoral lesion. The brush-head was then conserved in Thinprep®  at room temperature. Twenty-six consecutive FFPE samples from patients with anogenital neoplasia were retrospectively used for confirmation  according to the manufacturer protocol.For FFPE samples, two 5 µm-thick serial sections were cut from each paraffin block and collected in Eppendorf® were centrifugated at 2000 rpm for 10 minutes at room temperature. The samples were then washed with PBS and centrifugated again at 2000 rpm for 10 minutes at room temperature. Supernate was eliminated and sediment was treated using 70 µL of proteinase K. DNA was finally isolated using Roche Cobas DNA Sample preparation Kit according to manufacturer protocol.Cytobrush samples collected in ThinPrep31. DNA concentration was finally measured using NanoVue spectrophotometer .DNA quality was assessed using GAPDH housekeeping gene amplification  and gel migration as previously described33. A total of 50 µL of reactional mix constituted of 16.9 µL of HPV MMX, 8.1 µL of HPV Mn/Mg and 25 µL of samples was added in a 96 wells plate. β-globin was used as housekeeping gene. Internal controls  were used on each plate. PCR consisted in 50 cycles (93 °C and 56 °C) followed by 30 seconds of cooldown to 40 °C and 10 seconds at 25 °C. Data were finally assessed using LightCycler 480 SW 1.5.0 software (Roche Diagnostics)  was assessed using Cobas HPV test  based on TaqMan probes technology and Cobas z480 (Roche Diagnostics). The Cobas HPV test is a CE-IVD validated assay for the detection of HPV in cervical cytobrush samples but is not designed for the detection of oral cytobrush or FFPE samplesRepeatability, reproducibility, sensitivity and robustness were assessed for the validation of Cobas real-time PCR assay for oral cytobrush and tumor samples.Repeatability was measured for each HPV genotype  and for β-globin for oral cytobrush samples and tumor. For each sample  crossing point-PCR-cycle (Cp) was measured 10 times during the same run. The mean, the standard deviation and coefficients of variation (CV) were calculated for each genotype and sample type.One oral cytobrush sample  and one FFPE sample  have been analyzed 15 times  and Cp for HPV and for β-globin were measured to estimate the reproducibility of the assay. The mean, the standard deviation and coefficients of variation (CV) were calculated for each genotype and sample type.Limit of detection has been tested using control dilution from 0.4 ng/µL to 6 ng/µL for each HPV genotype and for each type of samples , equivalent to a total DNA input of 10, 20, 30, 40, 50, 75, 100 and 150 ng. Samples from patients with HNC and a known oral HPV infection were used for this determination.Finally, robustness was evaluated using variations of MMX reagent from 14 to 20 µL and Mg/Mn reagent from 7 to 12 µL.Specific primers for HPV 6/11, 16, 18, 33 and consensus  were used for the detection of HPV presence in samples. All primers are detailed in Suppl Table TM gel loading buffer  at 100 Volts for 2 hours. A total volume of 10 µL of each sample was finally used for HPV assessment and a DNA size marker (Roche Diagnostics) was present on each gel. Gels were finally analyzed using Gel DocEQ . Conventional PCR with electrophoresis procedure are then mentioned as “Conventional PCR” in this manuscript.Migration was assessed using 1% agarose gel with 0.5X TBE and BlueJuiceRepeatability and reproducibility were evaluated by the coefficient of variation (CV) calculated by dividing the standard deviation by the mean.Conventional PCR assay was set as the gold standard for this study. The sensitivity for Cobas assay corresponded to the number of positive samples with Cobas assay divided by the number of positive samples with the conventional PCR assay. The specificity for Cobas assay corresponds to the number of negative samples with Cobas divided by the number of negative samples with the conventional PCR assay. The 95% confidence intervals for sensitivity and specificity were computed.Statistical analyses were performed with SAS software version 9.2 . The assay comparison has first been assessed using only head and neck tumor samples  and has been confirmed using 26 FFPE anogenital neoplasia samples.Supplementary tables


Premalignant stages in cervical cancer have been studied extensively, while little is known about premalignant stages in TSCC/BOTSCC and the role of HPV. Here we analyzed differences in gene and protein expression between high‐grade dysplasia and invasive cancer in both HPV‐positive (HPV+ and HPV− TSCC/BOTSCC tumor sections. Differential gene expression was studied utilizing nanoString RNA‐panels and genes of interest were validated on the protein level by immunohistochemistry.High‐grade dysplasia and invasive carcinoma were laser microdissected from HPV+ tumors showed significantly different expression between high‐grade dysplasia and invasive cancer and 33 genes in the HPV− tumors. Five out of the nine most significant pathways showed similar increased activity in invasive cancer as compared to high‐grade dysplasia in both HPV+ and HPV− tumors. Lastly, significant differences in protein expression was confirmed for SPARC, psoriasin, type I collagen and galectin‐1 in both HPV+ and HPV− tumors.Forty genes in the HPV+ and HPV− TSCC/BOTSCC.This is to our knowledge the first study disclosing differences and similarities in gene expression between dysplastic and invasive HPV It is debated if high‐grade dysplasia exists in HPV+ TSCC/BOTSCC, there is also a need for pathological invasion markers in HNSCC. Here we present similarities between HPV+ and HPV‐ high‐grade dysplasia, suggesting HPV+ high‐grade dysplasia is an entity. We also identify possible invasion markers in HNSCC. AJCCAmerican joint committee on cancerBOTSCCbase of tongue squamous cell carcinomaCINcervical intraepithelial neoplasiaDCISductal carcinoma in situECMextracellular matirxFFPEformalin fixed paraffin embeddedHNSCChead and neck squamous cell carcinomaHPVhuman papillomavirus−HPVhuman papillomavirus negative+HPVhuman papillomavirus positiveHSILhigh‐grade squamous intraepithelial lesionsIHCimmunohistochemistryLCMlaser capture microdissectionLSILlow‐grade squamous intraepithelial lesionsOPSCCoropharyngeal squamous cell carcinomap16INK4a tumor suppressor proteinp16SPARCsecreted protein acidic and rich in cysteineTILtumor infiltrating lymphocyteTSCCtonsillar squamous cell carcinoma1+) TSCC and BOTSCC are generally younger and do not usually present with the classical head and neck squamous cell carcinoma (HNSCC) risk factors, for example, smoking and alcohol abuse.Previous studies by us and others have demonstrated an epidemic increase in the incidence of tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC), and this has been attributed to an increase in human papillomavirus (HPV) infection.+ TSCC or BOTSCC.The absolute majority of all carcinomas develop in a similar manner, starting with dysplasia followed by high‐grade dysplasia/cancer in situ, which eventually progresses into an invasive cancer and thereafter becomes metastatic. High‐grade dysplasia/cancer in situ refers to the earliest stage of cancer when the cancerous growth is still contained by the basal membrane and has not invaded the surrounding tissues or spread to other organs in the body.Increased knowledge of genes involved in tumor invasion mechanisms and identification of new progression markers could potentially in the future improve and personalize treatment and/or facilitate development of new strategies for early detection and preventative measures.+ TSCC and BOTSCC. Instead, a handful of studies have been performed on premalignant changes in HPV+ cervical carcinomas. In two independent studies by Gius et al and den Boon et al, differences in gene expression in the different stages of premalignant lesions (CIN1‐3) leading up to cervical cancer were examined, and both studies show specific changes displayed from stage‐to‐stage.+ and HPV negative (HPV−) precancerous and invasive tumors were possible in these studies, since >99% of all cervical carcinomas are HPV+.Due to its rarity, no studies have, to our knowledge, been performed on differences between premalignant and invasive lesions in HPVMoreover, it is imperative for the patient treatment and prognosis to distinguish dysplasia/cancer in situ from an invasive carcinoma. Whearas an invasive tumour diagnosis most often will result in heavy oncological treatment and/or radical surgery, a diagnosis of cancer in situ/severe dysplasia diagnosis may lead to surgical extirpation, but not with extensive margins, and only clinical follow‐up. Normally, the distinction between premalignant and invasive disease is unproblematic, but there are cases, especially in head neck cancer pathology when the diagnosis is vague. Therefore, some invasion markers, for example, Laminin‐5,+ and HPV− preinvasive TSCC and BOTSCC lesions and invasive tumors by laser capture microdissection (LCM) of the malignant epithelial cells followed by gene analysis utilizing the PanCancer Progression Panel (NanoString). In addition, we also aspired to identify usable clinical invasion markers by visual verification of obtained RNA data on protein level by immunohistochemistry (IHC).In this study we aimed, for the first time, to examine and better understand differences between HPV22.1INK4a (p16) expression,+p16+) were identified from the cohort and 11 HPV DNA negative and p16 negative (HPV−p16−) tumors, thus driven by other factors, were selected. From these 24 samples, the six HPV+ and HPV− patient samples with highest quality and satisfactory amount of RNA were used in the RNA expression assay (please see below). Differentially expressed genes were then validated in all 24 samples by IHC. For more details about the study subjects see Supplementary Tables In this study 24 TSCC and BOTSCC  biopsies were selected from a larger consecutive cohort diagnosed at Karolinska university hospital 2007‐2015.2.2Hematoxylin/eosin stained FFPE tumor sections were histologically examined by three independent researchers  and selected for containing sufficient amounts of high‐grade dysplasia/cancer in situ as well as invasive cancer Figure . FFPE bi2.3+p16+ tumors and six HPV−p16− tumors) with sufficient RNA concentrations (>2 ng/μL) were selected for the multiplex gene expression assay. Due to low RNA concentrations from FFPE material, the nCounter Low RNA Input Amplification Kit (NanoString Technologies) was used for the cDNA conversion and multiplex target enrichment steps. Thereafter the hybridization reactions were set up using provided master kit and progression primers needed for the nCounter PanCancer Progression Panel (NanoString Technologies). The gene expression assay was performed according to manufacturer's instructions utilizing the nCounter® Sample Prep Station with FLEX configuration (NanoString Technologies) as well as the nCounter® Digital Analyzer 5s (NanoString Technologies) for reading the samples. For data analysis the nSolver Analysis Software (version 4.0) was used, including the nCounter Advanced Analysis add‐on software (version 2.0.115). More specifically, fold changes and P‐values were calculated using nCounter default settings. Pathway scores were calculated using measurements of genes included in 34 different pathways, using the nCounter Advanced Analysis Software.Samples were kept on ice whenever possible and RNA was extracted immediately upon laser microdissection to ensure best RNA quality, using the RNeasy FFPE kit (Qiagen) according to manufacturer's instructions. RNA concentration was measured on Qubit 4 Fluorometer (Thermo Fisher Scientific) using the Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific). Twelve samples . In total, 40 genes in the HPV+ tumors and 33 genes in the HPV− tumors showed differential expression (P < .05), comparing high‐grade dysplastic epithelium and invasive cancer. Significant genes are presented in Figure + and HPV− tumors. Of these genes, 10 were common for both HPV+ and HPV− tumors. More specifically, the genes COL3A1, COL1A1, COL1A2, SPARC, COL6A3, AEBP1, COL6A2, and VIM were significantly up‐regulated, whereas S100A7 and TACSTD2 were significantly down‐regulated, in invasive cancer as compared to the dysplastic epithelium  receptor interaction, cellular growth factor, collagen family, and ECM structure , SPARC (SPARC), galectin‐1 (LGALS1), IL1‐RA (IL1RN), psoriasin (S100A7) and periplakin (PPL)) were selected for evaluation by IHC as potential invasion marker candidates. The transcripts of these six proteins showed with few exceptions either an increase  or decrease  between high‐grade dysplasia and invasive cancer    Figure .P = .02)  Figure .P = 0001)  Figure .4+ and HPV− TSCC/BOTSCC. Some of the most significant differential mRNA expressions were found for COL1A1, SPARC, and LGALS1 that were stronger expressed in invasive carcinoma compared to high‐grade dysplasia, and the opposite was shown for S100A7, which was stronger expressed in high‐grade dysplasia. These findings were also confirmed on a protein level by IHC. Moreover, we show that there are many similarities in gene expression and signal pathway activities between HPV+ and HPV− tumors—especially in genes related to ECM, highlighting the importance of the tumor micro milieu.In this study we report, for the first time, differences in gene expression between high‐grade dysplasia/cancer in situ and invasive HPV+ TSCC/BOTSCC is rarely detected and pure premalignant stages have not been found, but common in HPV− TSCC/BOTSCC. Some authors have even postulated that HPV+ premalignant phases do not exist in TSCC/BOTSCC,+ TSCC/BOTSCC dysplasia on a molecular level. Mooren et al, studied presence of p16INK4a expression in tonsillar dysplasia and found that p16INK4a positivity correlated to HPV status.+ and HPV− TSCC/BOTSCC. Notably, numerous similarities in differential expression were here observed between HPV+ and HPV− TSCC/BOTSCC dysplasia and invasive cancer, suggesting that HPV+ dysplasia in tonsils/base of tongue is a distinguishable entity from invasive HPV+ TSCC/BOTSCC. Therefore, it is very likely that premalignant stages of HPV+ TSCC/BOTSCC exist. Why such stages have not been described so far,+ dysplasia, an assumption that is not quite supported by data presented above. Hence, larger cohorts may be needed to identify premalignant phases in tonsils/base of tongue—possibly also by utilizing markers described here, such as psoriasin.As noted in the introduction, malignant invasive epithelial tumors develop from a dysplastic epithelium and detection of dysplasia has been utilized in different screening programs, for example, in cervical cancer. However, dysplasia in HPV+ and HPV− invasive carcinoma. Interestingly, we also observed a higher stem cell associated score in invasive cancer compared to dysplasia, which was especially observed in the HPV+ samples while present to a lesser extent in the HPV− samples. Notably, a study by Zhang et al showed that the cancer stem cell pool was much higher in HPV+ OPSCC as compared to HPV− cancer.+ tumors in general have a very good prognosis.On the signaling pathway level, it is not surprising that pathways involved in tumor progression, such as metastasis response, ECM receptor interaction, ECM structure, collagen family, and cellular growth factor pathways are up‐regulated both in HPVCOL1A1, COL1A2, COL3A1, where in our study found to be the most significantly increased ones in invasive cancer as compared to dysplasia in both HPV+ and HPV− tumors. Moreover, the proteins encoded by these genes belong to a family of proteins that strengthen and support many tissues in the body, being the most abundant part of the ECM.On the transcript level, the collagen genes S100A7 encodes the protein psoriasin part of the S100 family containing calcium‐binding motifs and is an important cell mediator for, for example, cell survival and maturation and has been associated to tumor progression and survival.IL1RN encodes the interleukin‐1 receptor antagonist (IL‐1RA) and is a natural inhibitor of the IL‐1 receptor. Expression of IL‐1RA protein has been suggested to be able to decrease tumor growth, metastasis, and angiogenesis in mouse models.IL1RN gene expression is lower in invasive cancer compared to dysplastic epithelium, especially in HPV+ TSCC/BOTSCC. Interestingly, similar results have previously also been published in CIN3 vs. invasive cervical carcinoma.Lastly, in order to validate our data and in search for new pathological invasion biomarkers, we assessed six gene transcripts for protein expression and tissue distribution by IHC and were able to validate four out of six transcripts at the protein level. However, while the relative expression in dysplasia and invasive carcinoma often corresponded to the trend of transcriptional data, the absolute expression varied considerably between samples. Therefore, using any of these markers as markers of invasion in a clinical setting would probably need further tuning of staining interpretation.+ TSCC/BOTSCC.There are some limitations in this study. First, tumor areas containing high‐grade dysplasia/carcinoma in situ were often scanty and decreased upon serial sectioning in some cases. Hence, the RNA concentration was very low after extraction. Due to low RNA concentrations and fragmented FFPE material, the NanoString platform using tumor progression RNA panel was utilized. However, using a more “global” method, such as RNAseq, would have yielded more data and a better picture of the transition between dysplasia and invasive tumor. Secondly, few patient samples were included in this study, reflecting that dysplasia is uncommonly detected in HPV+ and HPV− TSCC/BOTSCC. However, larger studies are needed to distinguish pure premalignant from invasive tonsillar/base of tongue neoplasias.In conclusion, this is the first study to our knowledge, examining and disclosing differences in gene expression between dysplastic and invasive HPVEthical approval and consent to participate was conducted according to ethical permission 2005/431‐31/4 from the Ethics Committee at Karolinska Institute, Stockholm, Sweden. The study was performed in accordance with the Declaration of Helsinki.The authors declare that they have no conflict of interest.L.H., A.N., and T.R. formulated the research question and came up with the study design. Sample selection and collection was done by A.N., P.F., and L.H. L.H. and A.N. performed the tissue microdissection, RNA extraction, and gene expression assay. A.Ä.R., L.M., and R.G.U. performed the IHC stainings. L.H., A.N., and L.M. evaluated the IHC stainings. L.H. and A.N. analyzed, summarized, and interpreted the data and wrote the manuscript which was revised and approved by all co‐authors. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Adenocarcinoma in situ (AIS) of the uterine cervix is a preinvasive lesion of the invasive adenocarcinoma. We analyzed the cervical screening results leading to detecting the AIS lesions including the co-existence of AIS lesions with high-grade squamous intra-epithelial lesions (HSIL) and invasive carcinoma. Women who were diagnosed and received treatment for AIS at Chiang Mai University Hospital between January 1, 2007 and August 31, 2016 were retrospectively reviewed. The inclusion criteria were the women who had pathological diagnosis of AIS obtained from cervical punch biopsy or excisional cone biopsy with either loop electrosurgical excision procedure (LEEP) or cold-knife conization (CKC). The patient characteristics, diagnostic work-up and treatment details were reviewed, including the cervical screening results prior to the diagnosis of cervical AIS, pathologic results of excisional cone biopsy and hysterectomy specimens.During the study period, 75 women with AIS pathology undergoing excisional cone biopsy with either LEEP (n=62) or CKC (n=13) were identified. The abnormal cytologic screening leading to detection of AIS was the squamous cell abnormality accounting for 57.3%. Abnormal glandular cytology accounted for 37.3%. The most common abnormal cervical screening results was HSIL cytology (n = 25) followed by AIS cytology (n = 13). Normal cytology was noted in 4 women in whom 3 were positive for HPV 18 and 1 had AIS on the endocervical polyp. AIS coexisted with HSIL and invasive carcinoma were detected in cone biopsy specimens in 21 (28%) and 29 (38.7%) patients, respectively. The majority of cervical screening results leading to detection of cervical AIS was the squamous cell abnormality accounting for 57.3% in which, HSIL cytology was the most common. Abnormal glandular cytology accounted for only 37.3%. Diagnostic cone excision is recommended if AIS lesion is noted in cervical biopsy specimen since nearly 40% have coexisting invasive lesions. Cervical cancer is the second most common cancer in females worldwide and the most common in low and middleincome countries . In general, squamous cell carcinoma and adenocarcinoma are the 2 major histological types of cervical cancer. Adenocarcinoma, accounting for 10 20% of all cervical cancers, has been increasing in the last two decades, unlike those of squamous cell carcinoma which is proportionally decreasing . Adenocarcinoma in situ (AIS) of the uterine cervix has been recognized as a pre-invasive or precursor lesion of invasive adenocarcinoma due to its morphological futures, which are similar to those of invasive adenocarcinomas. AIS lesion frequently coexists with invasive adenocarcinoma on excisional specimens and has similar human papillomavirus (HPV) types as have been detected in invasive adenocarcinoma . AIS lesion of the uterine cervix is almost always asymptomatic and is generally not visible upon gross examination. It is typically detected after a diagnostic work-up for abnormal cervical cytology screening. The average age of patients with AIS is approximately 5 years younger than those with early invasion, supporting the potential for cytologic screening to prevent this disease. Most cases of invasive adenocarcinoma develop from an AIS precursor located within or just proximal to the transformation zone. There is a window of approximately 5 years between clinically detectable AIS and early invasive adenocarcinoma, indicative of an opportunity for screening and detection before progressing to invasion . The ability to detect its precursor, AIS, would certainly decrease the incidence of invasive adenocarcinoma. However, limitations of conventional methods in detecting glandular lesions exist. Pap cytology had a sensitivity of only 45 %in detecting glandular diseases . Since Pap cytology commonly fails to detect the AIS lesions, accordingly, this retrospective study was conducted to determine the screening results leading to the detection of cervical AIS, in addition, the co-existence of high-grade squamous intra-epithelial lesions (HSIL) and invasive carcinoma was also evaluated.After approval of the Research Ethics Committee of Chiang Mai University Hospital, women who were diagnosed and received treatment for adenocarcinoma in situ (AIS) of the uterine cervix between January 1, 2007 and August 31, 2016 were retrospectively reviewed. The data resources for review were medical records in the colposcopy clinic of Obstetrics and Gynecology Department and the computerized database of Chiang Mai University Hospital. The patient characteristics, diagnostic work-up and treatment details were reviewed, including the age ,parity, contraception methods ,HIV status, number of sexual partners ,smoking history, cervical screening results prior to the diagnosis of cervical AIS, pathologic results of cervical punch biopsy, cone biopsy and hysterectomy specimens. The 2001 Bethesda System for nomenclature of cervical cytology was used to report the cytology results . The inclusion criteria were the women who had pathological diagnosis of AIS obtained from cervical punch biopsy or diagnostic excision procedure with either loop electrosurgical excision procedure (LEEP) or cold-knife conization (CKC). Women who were pregnant or had history of gynecologic cancer were excluded. Management of women with glandular cell abnormality on cytology and AIS on cytopathology are showed in The statistical analysis was performed using SPSS version 21.0 . The descriptive data were presented as percentage / range or means + SD, as appropriate. Mann-Whitney U test, Student’ t test, chi-square test, and Fisher exact test were used for statistical analysis when appropriate. P value of less than 0.05 was considered statistically significant. All statistical tests were of two-sided significance.During the study period, 75 women with AIS pathology who underwent excisional cone biopsy with either LEEP or CKC were identified. The mean age of the women was 45.9 years with a range of 26-66 years. Eight (10.7%) women were nulliparous. Anti-HIV testing was negative in all patients. No contraception was used in 23 (30.7%) women, while 27 (36%) used oral contraceptive pills, 7 (9.3%) used injectable contraception, 16 (21.3%) had tubal sterilization and 2 (2.7%) used condom contraception. Nineteen (25.3%) women were postmenopausal.HPV testing was performed in conjunction with cervical cytology in 9 women and 8 was positive (6 for HPV 18 and 2 for unknown high-risk HPV type). Among 4 women with normal cytology, 3 were positive for HPV 18 and 1 had AIS on the endocervical polyp. Positive HPV 18 was found in 2 women with AGC cytology and 1 with adenocarcinoma on cytology. HPV testing was positive for unknown type in 1 women with ASC-US cytology and another one with HSIL cytology. AIS coexisted with HSIL and invasive carcinoma were detected in cone biopsy specimens in 21 (28%) and 29 (38.7%) patients, respectively. The resection margins of the cone biopsy specimens were positive in 51 (68%) patients and were not significantly different when compared between LEEP specimens and CKC specimens (p = 0.74)Our study showed that among 75 women with a diagnosis of AIS of the uterine cervix, the most common preceding cervical cytology screening leading to the detection of this glandular disease was the squamous cell abnormality accounting for 57.3 %. The glandular cell abnormality including the AIS cytology accounted for only 33.3%. These findings were different from the previous retrospective studies reporting the glandular cell abnormality on cervical cytology prior to the detection of AIS ranging from 50 to 69 % and squamous cell abnormality ranging from 26 to 31 % . Among squamous cytologic abnormality, the most common were HSIL cytology ranging from 76 to 79 % . HSIL cytology (33.3%) was also the most common abnormal screening results in our study followed by the AIS cytology accounting for 17.3%. The lesions of AIS preferentially extend into the endocervical canal and occur in the deeper portions of the endocervical clefts beneath the transformation zone rather than at the surface as with the squamous lesions. Consequently, cytological screening frequently fails to detect the glandular lesions. Accordingly, Pap cytology in patients with AIS may be normal or squamous cell abnormality depending on the associated squamous lesions.Of interest, normal cytologic screening was noted in 4 (5.3%) patients with AIS lesions in our study. Of these 4 patients, 3 had positive HPV testing for HPV 18, the remaining 1 had AIS on the endocervical polyp. Normal cervical cytology was found approximately 4 % in women with pathological diagnosis of AIS . AIS lesions have similar type of HPV as have been detected in invasive adenocarcinoma of the cervix . The most common HPV types found in AIS lesion are HPV 16 and 18 accounting for more than 90% . In a meta-analysis of HPV prevalence in glandular lesions of the cervix, the HPV 16 and HPV 18 positivity rates in AIS lesion were 49% and 52%, respectively . It was shown that HPV type specific risks for AIS and adenocarcinoma are not the same as for squamous cell carcinoma, with HPV18 being consistently observed to be pref-erentially associated with AIS . HPV 18 is more frequently detected in adenocarcinoma than in squamous cell carcinoma . Currently, it is well recognized that HPV testing is more sensitive than cytology in cervical cancer screening especially AIS and adenocarcinoma . Concurrent HPV testing resulted in early detection of adenocarcinoma, 17 of 27 (63%) women with adenocarcinoma were found to have HPV-positive but cytology-negative screening results . Pap smear had a sensitivity of only 43% in detecting AIS lesions. In 2 clinical trials of quadrivalent HPV vaccine, HPV testing combined with cervical cytology was used to evaluate the efficacy of HPV vaccine. AIS lesions occurred in 22 women in whom 21(95.5%) had positive HPV testing while only 2 had abnormal glandular cytology. Furthermore, colposcopy failed to detect AIS in all cases . Colposcopic appearance is not specific to identify the lesions of AIS. Colposcopic evaluation and sampling are more difficult to detect AIS lesion because of the location of lesions within the endocervical canal or beneath the transformation zone. The findings of low sensitivity of cytology in screening glandular diseases strongly support the addition of HPV testing to cytology to improve screening performance. We suggest to keep in mind that glandular diseases may be present when manage HPV 18-positive women regardless of cytology results.AIS coexisted with HSIL and invasive carcinoma, in our study were found in 21 (28%) and 29 (38.7%) patients, respectively. In the meta-analysis of 33 studies, approximately 55% of women with AIS were found to have a coexisting squamous neoplasia . The co-existence of both glandular and squamous neoplasia reflects the similar etiology between the 2 types of cervical cancer precursors. Because of the location of the AIS inside the endocervical canal and in the glandular clefts, the Pap cytology of both squamous and glandular lesions may indicate only squamous cell abnormality. In the study by Andersson et al., (2013) AIS was detected in 64.3% of the patients due to accompanying squamous cell abnormalities, but in only 27% due to glandular cell abnormalities. This may be due to the relatively low sensitivity of cytology screening which is ineffective in reducing the incidence of adenocarcinoma. When glandular and squamous neoplasia coexist, the squamous component is more likely to be noted colposcopically because of its prominent features, especially when the squamous lesions are extensive or high grade. Glandular lesions may be adjacent to the squamous lesion, be sandwiched between 2 squamous lesions, lie under or above the squamous lesion. Thus, the squamous lesion is frequently diagnosed from colposcopy-guided biopsy, with AIS being detected incidentally in a subsequent excisional cone biopsy or in a hysterectomy specimen. Invasive carcinoma, if detected concurrently, should be further evaluated and managed as appropriate.When AIS is found on punch biopsy or when AIS is suspected cytologically or colposcopically but not proven histologically, a diagnostic excisional cone biopsy must be performed. The purpose is to confirm the diagnosis, assess the extent of lesion and exclude the coexisting invasive carcinoma. In our study, we preferred LEEP rather than CKC. We noted that overall , the cone margins were positive in 68% of the patients and were not significantly different when compared between LEEP specimens and CKC specimens. In the systematic review of 35 studies on excision procedures for AIS, it was shown that LEEP had a clinically significantly higher rate of positive cone margins when compared with CKC (51% vs. 30%) . In general gynecological practice, CKC is favored over LEEP for women with AIS due to its greater depth and larger volume of specimens, more easily interpretable resection margins and lower rate of positive margins . However, the Updated Consensus Guidelines of the American Society for Colposcopy and Cervical Pathology (ASCCP) allowed any diagnostic excision method for AIS lesion with carefully keeping specimen intact, margin interpretable, avoiding fragmentation of the specimens including ‘‘top-hat’’ serial endocervical excisions. This may require the use of larger loops than those employed to excise visible squamous lesions . The risk of residual diseases after excisional cone biopsy in our study was significantly associated with the resection margin status in patients undergoing LEEP. The risk of residual diseases in the CKC group seemed to be higher in patients with involved cone margins but not statistically significantly when compared with those who had negative margins. However, the number of patients (13) in this group was too small to conclude. Systematic review of 35 studies on excision procedures for AIS showed that the risk of residual disease was significantly associated with cone margin status, i.e. 16.5% vs. 49% in negative and positive cone margins, respectively. In addition, the risks of residual invasive carcinoma in subsequent surgical specimens after a negative and positive cone margins were 0.6% and 5.9%, respectively . The risk of residual lesions in patient with AIS is quite high despite negative cone margins because the pattern of this glandular neoplasia is often multifocal and located beyond the proximal end of the endocervical cutting edge of the cone. Accordingly, it is recommended that women with AIS who have completed childbearing should undergo hysterectomy to reduce the risk of adenocarcinoma.Among 61 women undergoing subsequent hysterectomy in our study,33 (54%) had residual diseases in hysterectomy specimens. The risk of residual lesions appeared to be higher in women with abnormal ECC results (69.2%) comparing with that of normal ECC results (39.3%), however, the difference was not significant. Of note, nearly 40% of patients had residual disease despite normal ECC results. Performing endocervical sampling is generally recommended after colposcopy and excisional cone biopsy in managing women with glandular cell abnormality on cytopathology, although the benefit of such procedure is conflicting in prediction of residual disease . ECC result is considered as a part of evaluating margin status, i.e. abnormal ECC is a positive margin. Our findings support that negative ECC result after excisional cone biopsy does not guarantee the absence of disease in the residual cervix. Because the nature of AIS lesions are frequently located deep in endocervical clefts and invaginated within the cervical stroma, the lesions might not be scraped during ECC. As a result, normal ECC result does not necessarily ensure that the lesion has been completely excised. The strength of the present study was the inclusion of patients who were treated at a single institution with same management protocol and all the cytopathologic specimens were examined by expert gynecologic pathologists. However, the limitation of the study was the retrospective by nature, some data were not available, e.g. patient and physician preferences that might affect the selection of cone biopsy methods (LEEP vs. CKC). Due to the small number of patients with this rare disease, the clinical significance of cone margin status in women undergoing CKC could not be concluded. In addition, the treatment outcomes especially recurrence after definitive treatment for AIS were not available.In conclusion, the majority of cervical screening results leading to detection of AIS, the pre-invasive glandular lesion of invasive adenocarcinoma of the uterine cervix was abnormality of squamous cells accounting for 57 % in which, HSIL cytology was the most common. Abnormal glandular cytology accounted for only 37 %. Diagnostic cone excision is recommended if AIS lesion is noted in cervical biopsy specimen since nearly 40% have coexisting invasive lesions.The authors have no conflict of interest to disclose.


Cervical cancer is the most common genital malignant tumor in women worldwide. However, the reliability of different detection methods may vary according to populations and epidemics. This study analyzed factors relevant to high-risk human papillomavirus (hrHPV) infection among rural Uyghur women aged > 30 years and evaluated the value of different screening methods for cervical precancerous lesions.From July 2015 to May 2016, 225 rural Uyghur women aged > 30 years were recruited from local health clinics throughout Pishan, Xinjiang, China. HrHPV DNA testing, colposcopy, biopsy of cervical precancerous lesions, and surveys were conducted. The results of different screening methods were compared, and factors associated with hrHPV infection were analyzed.P = 0.753) for the HPV test and 0.995  for colposcopy. Factors associated with HPV infection included widowhood , P = 0.005) and ≥ 3 sexual partners in the past 5 years , P < 0.001). .The rates of hrHPV infection and cervical epithelial lesions were 9.3 and 1.8%, respectively. The area under the ROC curve was 0.538 (95% CI: 0.292, 0.784; Among rural Uyghur women aged > 30 years, the main factors for HPV infection include marriage and frequent sexual intercourse. Colposcopy has a higher screening value for cervical epithelial lesions than hrHPV testing. Cervical cancer is the most common malignant genital tract tumor in women and has shown an increasing incidence in recent years. The age of onset for cervical cancer tends to be young. Every year, approximately 500,000 new cases of cervical cancer occur worldwide, 90% of which are in developing countries . China, The development of cervical cancer is continuous and progresses from cervical intraepithelial neoplasia (CIN) to early invasive cancer and then to more established cancer, which involves a long and reversible precancerous lesion stage. If CIN can be recognized early and then properly treated during this precancerous lesion stage, the conversion from CIN to early invasive cancer  and, thus, the development of cervical cancer can be prevented . StandarSome scholars have proposed the utilization of pathogeny screening, such as high-risk human papillomavirus (HPV) testing, as an early screening strategy , 12. HPVIn this study, high-risk HPV DNA testing, colposcopy, cervical biopsy, and questionnaires were utilized to analyze the factors related to high-risk HPV infection and to evaluate the clinical value of applying different methods, using histopathology as the gold standard, to cervical precancerous lesion screening among normal rural Uyghur women in Xinjiang. The results may provide a reference for the prevention and treatment of cervical cancer in this population, as well as in populations in the regions with similar medical circumstances.In this study, a cross-sectional survey was conducted from July 2015 to May 2016 in Pishan County, Hotan Prefecture, in the Xinjiang Uyghur Autonomous Region of China. Information and motivation were provided by local health care workers in individuals’ homes to recruit local eligible women to participate in the survey. The inclusion criteria were as follows: women from rural areas, age older than 30 years, Uyghur ethnicity, and a history of sexual intercourse. The exclusion criteria were pregnancy or termination of pregnancy less than 3 months prior to the study; a past history of cervical cancer or unknown uterine tumors; a history of hysterectomy, cervical conization, radiotherapy or chemotherapy; cervical dysplasia; no sexual intercourse; mental illness or other serious disease; a long-term out-of-home residence; and women belonging to a nomadic population.This study was approved by the Ethics Committee of the Xinjiang Uyghur Municipal People’s Hospital, and written informed consent was obtained from each participant.All examinations of women participating in the study were conducted during nonmenstrual phases. These evaluations included a physical examination, a questionnaire, high-risk HPV DNA testing, colposcopy, and tissue biopsy. Respondents were not allowed to use vaginal medication or perform vaginal washing 3 days before the examination and could not engage in sexual behavior within 24 h. A detailed flow chart of the study design is shown in Fig. One-on-one interviews were conducted by trained investigators. The interview was performed according to a simple self-designed questionnaire that included basic demographic information, family background, marital status, childbearing status, gynecological conditions, and family history.A trained gynecological clinician used a speculum to widen the vagina of each subject and fully expose the cervix, cleaned away secretions using sterile saline cotton balls or dry cotton swabs and inserted a sampling brush  into the cervical orifice, rotating it clockwise 3–5 times. The sampling brush was slowly withdrawn and immersed in an HPV DNA sampling tube , and the cap was tightened for storage. The sample was then sent for immediate detection; samples that could not be tested immediately were stored at 4 °C for testing within 2 weeks.HPV DNA testing was conducted using the second-generation hybrid capture method to detect HPV-16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, and 68, employing a DML 2000 gene recombination signal amplifying system and a DML 2000 microplate reader (Digene Company). The tubes were shaken well and numbered, and five drops of the indicator were applied to the lysate, followed by mixing. Approximately 1000 μl, 500 μl, and 500 μl of the lysate was applied to the negative control (2 ml), positive control (1 ml) and specimen, respectively, to maintain a consistent lysate concentration among the groups, and the samples were mixed. The solutions were subsequently placed in a water bath at 65 °C for 45 min, and all solutions appeared purple. The prepared probe B (25 μl) was added to a 96-well plate, and the lysate controls and samples (75 μl) were then added to the wells. Adhesive film was attached, and the samples were oscillated at 1100 rpm/min for 3 min. The solutions turned yellow and were then incubated at 65 °C for 60 min. Thereafter, the tape was removed, and the solution was cooled for 5 min. Approximately 100 μl of the solution was transferred to the capture wells, and tape was attached. Oscillation at 100 rpm/min was performed for 60 min. The liquid was then removed, and detection reagent 1 (75 μl) was added to the wells. The wells were subsequently capped, and the plate was placed at 20–25 °C for 30 min. The wells were then covered with absorbent paper, and the liquid was removed via rapid inversion. The plate was blotted on absorbent paper until dry. After washing six times, the sample was placed on absorbent paper, and blotting and drying were performed again. Approximately 75 μl of detection reagent 2 was subsequently added to each well, and the samples were incubated in the dark at 20–25 °C for 15 min. Finally, the results were read with a DML 2000 system. An HPV DNA concentration ≥ 1.0 pg/ml was considered HPV positive.After vaginal secretion sampling (for HPV DNA testing), colposcopy  was performed immediately by trained full-time practitioners with the subjects in a lithotomy position. The cervix was exposed with the aid of a disposable speculum. After secretions were cleared, the diopter and focal length of the colposcopic eyepiece were adjusted to display a clear image, and each area to be inspected was exposed. The appearance and color of the cervix were observed at low magnification, and the morphology of blood vessels was assessed using a green filter and high magnification. The cervix was compressed for 1 min with 3–5% glacial acetic acid solution (applying 1% compound iodine solution evenly to the cervix if necessary) to improve visualization of the cervical epithelium: the cervical squamous epithelium, columnar epithelium, and the color, appearance, and blood vessels of the transformation area were observed. Normally, abnormal or diseased regions are not colored or are mustard yellow in color. In cases categorized as CIN-I, the acetowhite epithelium was thin, with an irregular boundary and rare embedded and dotted blood vessels, and iodine coloring showed a negative result or resulted in yellowish coloring. For CIN-II, the acetowhite epithelium was thick and gray, with dotted and embedded blood vessels, and the coloring test showed a negative result or resulted in mustard yellow coloring. For CIN-III, the acetowhite epithelium was dense and gray, with irregular thick dotted blood vessels, and the coloring test showed a negative result or resulted in mustard yellow coloring. Live tissue samples from abnormal cervical regions were sent for pathological examination; if no suspicious lesions were found, tissues from the 3, 6, 9, and 12 o’clock sites of the transformation area were routinely obtained for biopsy, or endocervical curettage was performed. All living tissue samples were fixed with formaldehyde and labeled with the subject’s information before being sent for pathological examination.Pathological diagnoses included the following: (1) normal cervical epithelium ; (2) CIN-I ; (3) CIN-II ; (4) CIN-III ; and (5) cervical squamous cell carcinoma (SCC) .P < 0.05 was considered to indicate statistical significance.A database was established using EpiData through double data entry and verification. The data were processed and statistically analyzed using SPSS 19.0. The histopathological findings were used as the gold standard, and the sensitivity, specificity, accuracy, positive likelihood ratio, and Youden’s index of the different detection methods were calculated. The effects of the different screening methods for cervical precancerous lesions were compared, and receiver operating characteristic (ROC) curves were drawn. The factors associated with HPV infection were analyzed through univariate and multivariate stepwise logistic regression analyses. A total of 225 eligible women were enrolled in the study, with an average age of 43.6 ± 8.1 years; the minimum age was 31, and the maximum age was 74 years. The characteristics of these enrolled women are summarized in Table The colposcopic results showed that there were 217 normal cases, 6 cases of CIN-I, 1 case of CIN-II, and 1 case of CIN-III in our study population. The biopsy results identified 221 normal cases, 3 cases of CIN-II, and 1 case of CIN-III, and the corresponding prevalence rate of high-grade CIN (II and III) was 1.8%.CIN II and III were recorded as positive pathological results (+), while other pathological findings were recorded as negative (−). The pathological results were used as the gold standard, and the comparison between the HPV results and the gold standard is shown in Table P = 0.753, while the area under the ROC curve for colposcopy was 0.995 , P < 0.001. Further details are shown in Fig. Similarly, for analyses of the colposcopy results, CIN II and III were recorded as positive pathological results (+), while other pathological findings were recorded as negative (−), and the pathological results were used as the gold standard. The comparison between the colposcopy results and the gold standard is shown in Table 2 test was performed to compare the HPV results and colposcopy results, and a significant difference was observed between them , having ≥3 sexual partners in the past 5 years , P < 0.001) , One of the measures currently used to improve the prevention and management of cervical cancer in women is enhancement of etiological analysis and prevention, where various factors are analyzed to determine the factors that are primarily associated with the disease; these factors are then targeted in prevention efforts. Another approach is to perform early, standardized screening in women in high-risk areas to identify and eliminate the factors leading to cervical cancer, such as precancerous lesions, with the goal of reducing the incidence of cervical cancer and prolonging patient survival through early detection, diagnosis, and treatment.There were three main findings of this study regarding the screening for cervical precancerous lesions in rural Uyghur women through HPV testing and colposcopy. First, the rate of high-risk HPV infection in rural Uyghur women aged > 30 years in Pishan County was 9.3%, and the high-grade CIN (II and III) rate was 1.8%. Second, the colposcopy results were significantly consistent with those of cervical biopsy in the diagnosis of CIN; additionally, colposcopy exhibited a high sensitivity and specificity, showing an area under the ROC curve of 0.995, indicating the diagnostic value of this approach. High-risk HPV DNA testing exhibited poorer performance in detecting CIN; although it showed high specificity (90.95%), the sensitivity was low (25.00%), and the area under the ROC curve was only 0.538, with no significant difference. Therefore, in the absence of pathological biopsies, colposcopy is a better approach for determining whether there are precancerous lesions in Uyghur women. Finally, the results of the stepwise logistic regression analysis showed that the relevant factors for high-risk HPV infection in rural Uyghur women were their widowed status, and their number of recent sexual partners .There are a variety of approaches that are currently used for early cervical cancer screening. HPV detection is an effective method of cervical cancer screening, showing a high sensitivity in screening for CIN-II and CIN-III, and may become the primary tool for cervical cancer screening in women over 30 years of age . HoweverColposcopy is a widely used screening method for cervical disorders and plays an important role in the determination of disease. This methodology has been rapidly developed in recent years and, combined with the use of acetic acid or iodine and optical amplification technology, provides a clearer picture of the abnormal areas of cervical lesions. Biopsy under microscope is the “gold standard” for the diagnosis of CIN and cervical cancer and is an indispensable second-line screening tool in cervical cancer screening. Luo et al. reported that the sensitivity of colposcopy in diagnosing CIN was 84.2%, while the specificity was 51.6%, indicating a high predictive value . In the At present, the international community follows a three-step screening method: cervical cytology or cytology combined with HPV detection is performed as the preliminary screening; cases that are suspicious or positive are referred to colposcopy; and colposcopic biopsy and pathological examination of the lesions are then performed. The China Cancer Research Foundation has proposed three programs: the preferred plan (TCT combined with HPV testing), the common plan , and the basic plan . When selecting the screening method, the optimal allocation of medical resources in different regions and the risk of disease development in different populations should be considered; costs, techniques, screening sites and other objective conditions should also be considered. Furthermore, good screening methods must include a rational scientific strategy for patients showing negative results in preliminary screening, to provide guidance regarding their need for follow-up and decide upon the review intervals to capitalize on the role of screening, in addition to reducing misdiagnosis and missed diagnoses and minimizing the threat of cervical cancer to the lives and wellbeing of women.In this study, biopsy revealed a detection rate of high-grade CIN (II and III) of 1.8%. In a previous investigation of 20,000 women in Shanxi, China, 410 (2.05%) of the women were diagnosed histologically with CIN lesions, including 317 (1.58%) diagnosed with CIN-I, 93 (0.50%) with CINII/III, and 11  with squamous cell carcinoma (SCC) . In anotThe development of cervical cancer is associated with various factors, including viral infection, sexual behavior, and education, and studies have shown that high-risk HPV infection is a major risk factor for cervical cancer , 31. MorIn conclusion, colposcopy was found to be more effective than high-risk HPV testing in screening for cervical precancerous lesions among rural Uyghur women in Pishan County, and this method can be popularized for cervical precancerous lesion screening in future preventative and curative practices. Factors related to sexual life, including marital status, were the main factors correlated with HPV infection among these women. However, as the conclusions of this study were mainly obtained through the investigation of Uyghur women in Pishan County, extrapolation of the findings should consider the representation of the samples and the differences between different regions. Furthermore, the factors associated with cervical precancerous lesions were not analyzed due to a small case number of high-grade CIN. To overcome this limitation, multi-center studies with a larger sample size are still needed.