The eighth edition American Joint Committee on Cancer (AJCC) Staging incorporates significant changes to the seventh edition in the staging of oropharyngeal squamous cell carcinomas (OPSCC). An important change was the inclusion of OPSCC associated with the human papilloma virus (HPV). Our goal is to compare the performance of both staging systems for patients with HPV-selected and unselected clinical characteristics for OPSCC.Using the Surveillance, Epidemiology, and End Results (SEER) database, 2004-2016, we identified patients with likely HPV-associated OPSCC based on surrogate markers , excluding those who underwent surgery. We re-classified these patients using seventh and eighth edition staging for HPV-selected OPSCC and compared the prediction performance of both staging editions for overall survival (OS) and disease-specific survival (DSS). We performed the same analysis for clinically unselected patients with OPSCC.Our analysis included 9554 patients with a median follow-up of 67 months. Comparing the eighth versus seventh edition for our HPV-selected cohort, clinical staging changed for 92.3% of patients and 10-year OS was 62.2%, 61.2%, 35.3%, and 15.5% for Stage I, II, III, and IV, versus 52.9%, 59.2%, 61.6%, 55.1%, 38.3%, and 15.5% for stage I, II, III, IVA, IVB, and IVC, respectively. A similar pattern was observed for 10-year DSS. The concordance statistics for our HPV-selected cohort were improved for both AJCC 7 (0.6260) and AJCC 8 (0.6846) compared with the unselected cohort, 0.5860 and 0.6457 for AJCC 7 and 8, respectively.The overall performance of discrimination improved from AJCC 7 to AJCC 8 for both clinically selected and unselected patients, but more notably for our HPV-selected cohort. Despite the lack of statistically significant differentiation between Stages I and II in AJCC 8 in either groups, markedly improved discrimination was observed between Stages I/II, III, and IV in the HPV-selected cohort. The 8th edition of the American Joint Committee on Cancer (AJCC) Staging Manual incorporates significant changes to the 7th edition in the staging of oropharyngeal squamous cell carcinomas (OPSCC). An important change was the inclusion of OPSCC associated with human papilloma virus (HPV). This article compares the performance of both staging systems for patients with HPV-selected and -unselected clinical characteristics for OPSCC. This article addresses the impact the new AJCC eighth edition for OPSCC staging has compared with the prior seventh edition. Our findings support evidence that the eighth edition provides better prognostication and discrimination between stages compared with AJCC 7 for both selected HPV-associated and unselected OPSCC patients. In comparison to prior publications using single-institution studies, ours is the only study to date using the SEER database to compare the performance of AJCC 7 and 8 for OPSCC. It is of interest that our findings applied regardless of attempting to clinically select patients for HPV relatedness.1 While cervical carcinoma was the most common HPV-associated malignancy, as of 2015, oropharyngeal SCC has now become the most prevalent HPV-associated malignancy.1 During this time, the incidence of OPSCC has increased despite a notable improvement in survival.2 This discrepancy is likely a reflection of the emergence of HPV-associated OPSCC as the predominant subtype of OPSCC.4 This trend was also paralleled by changes in tobacco use and sexual practices.4The Centers for Disease Control (CDC) has reported a significant change in the incidence and proportion of common human papilloma virus (HPV)-associated cancers within the last 2 decades. The incidence of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) increased at an estimated rate of 2.7% per year in men and 0.8% per year in women from 1999 to 2015.5 AJCC 8 incorporated HPV-associated OPSCC as a separate entity in terms of staging and prognosis. AJCC 8 was largely based on the multicenter cohort study by the International Collaboration on Oropharyngeal cancer Network for Staging (ICON-S), which showed similar overall survival (OS) for different nodal (N1-N2b) and tumor (T4a-T4b) categories, ultimately simplifying this tumor nodes metastases (TNM) classification system.6 Consequently, there are major TNM classification differences between the seventh and eighth editions of the AJCC Staging Manual, most notably in the T and N criteria.This change in behavior noted in HPV-associated OPSCC is reflected in recent changes in AJCC staging for this disease. While the seventh edition of the AJCC Staging Manual was agnostic to HPV status,7 T0 was removed from the non-HPV-associated disease given the strong correlation of unknown primary (T0) with HPV-associated disease.7 Lastly, T4a and T4b were combined in HPV-associated OPSCC given their similarity in outcome.7Within the T classification, HPV-associated carcinoma in situ was disregarded.7 It is important to consider the diagnostic differences between clinical and pathologic N categories. While clinical N classification can apply to all patients at diagnosis regardless of therapeutic intervention, pathologic N classification exclusively applies to patients who have undergone surgery. Regardless of the differences in nodal size or laterality, these 2 N classification systems are not designed to be compared since they apply to different clinical settings.N classification was partitioned into clinical and pathologic categories. For clinical N criteria, HPV-associated disease has similar prognostic implications for one or more ipsilateral lymph node (LN) <6 cm in size (previously subdivided into N1 and N2 in the seventh edition based on number and size). These were combined to cN1 in the eighth edition. Since the presence of contralateral or bilateral LNs is correlated with worse outcomes, bilaterality was upstaged to cN2. Finally, LN >6 cm, which foretold the worst prognostic findings, remained in the cN3 category. In contrast, pathologic N criteria in AJCC 8 considered the number of pathologically positive LNs, regardless of size or laterality, with ≤ 4 LNs conferring pN1 classification and >5 LNs a pN2 classification.We conducted a retrospective cohort study to stage patients identified in the SEER database with likely HPV-associated OPSCC using AJCC 7 and 8 criteria to compare their discrimination of OS and disease-specific survival (DSS).8 For the staging not available in the database , we constructed them based on individual T, N, and M categories in seventh (2010+) or sixth edition, utilizing the most recent edition T, N, M classification was available in.The Surveillance, Epidemiology, and End Results (SEER) 2004-2016 database collects cancer data from population-based cancer registries across the United States to derive information on survival and incidence. We utilized SEER to identify patients with suspected HPV-associated OPSCC and compared AJCC 7 and 8 staging in terms of their discrimination of outcome, namely OS for the 2 cohorts. To assess this, we completed a retrospective cohort study staging HPV-selected and non-selected OPSCC subjects based on TNM classification of the seventh and eighth AJCC editions, respectively. To clarify, we will refer to the suspected HPV-associated group as “selected” and the general OPSCC population in the SEER database as “non-selected.” Details on group staging can be found in 9–13 Mahal assessed over 12 000 patients from the SEER database with SCCHN diagnosed from 2013 to 2014 (similar to our median year of diagnosis 2010), describing white race  and male sex  with the highest incidence of HPV-positive OPSCC.9 Younger age <65 has also been more commonly seen in HPV-positive OPSCC as noted in Mahal’s population-based epidemiologic study with the age distribution peaking at 60-64 years old for all patients .9 Other studies have echoed these findings.12–15 On the other hand, the incidence of HPV-positive disease in OPSCC is increasing in the elderly population. This is notably represented in Thompson’s study in which the mean age of diagnosis increased from 55.2 years (2002-2010) to 58.5 (2011-2016).16 However, since our study’s mean date of diagnosis was ~2010, we decided to utilize the association with a younger patient population and implement an age restriction < 65 years old. Another HPV criteria were to target those with squamous cell disease at the base of the tongue and tonsils, as many prior studies have localized HPV-positive OPSCC to these locations.17–19 As Zamani noted in a population-based study with more than 2000 patients from 2000 to 2017, HPV-positive OPSCC were more often located in the base of tongue and tonsils than in the uvula, soft palate, or pharyngeal walls (HPV-positive 93% vs HPV-negative 58%).14 In addition, we also explored the performance of the 2 staging systems in a completely unselected group of OPSCC as a secondary confirmatory analysis.Given that HPV status was not available in our full patient subset, surrogate markers  were used as clinical surrogates for HPV-associated OPSCC. While this methodology cannot replace direct HPV testing, these demographic markers were carefully chosen to create a specific subset of likely HPV-positive disease. White males have the highest incidence of HPV-positive OPSCC, documented in multiple prior epidemiologic assessments.20 and more specifically has been shown to increase death and recurrence rates of OPSCC in HPV-positive patients.21 Ang et al. detailed this relationship by purporting that tobacco use changes the biological behavior of HPV-positive OPSCC and can subsequently decrease responsiveness to the standard of care therapy.22We searched the SEER database from 2004 to 2016 with limitations on sex, race, age, and disease location, as detailed above. Disease location was limited to the tonsil and base of the tongue. Patients were excluded for unknown/immeasurable T and/or N classification and were excluded if they had undergone surgery as a treatment modality. This was largely to focus our analysis on clinical rather than pathologic TNM classification. In addition to TNM categories, we collected risk factors (age and smoking status) as well as the type of treatment (chemotherapy and radiation). We considered excluding smokers to obtain a more refined subset of HPV-driven disease; however, we did not want to omit the portion of concomitant smokers and HPV-positive patients who represent an important trend toward worse outcomes. Studies have shown that smoking is a negative prognostic factor in HPV-positive patientsSummary statistics and demographics were represented as mean ± standard deviation (SD) and sample frequency and proportion. The seventh and eighth editions AJCC staging was constructed from individual clinical T, N, and M criteria based on their definition. OS was defined as months from the date of diagnosis to death for any cause or to the last date of follow-up. DSS was defined similarly to OS except that deaths specific to head and neck cancer were counted as events unless death was linked to other causes or patients were lost follow-up.23 Prediction and c-index calculations were performed in R packages survival, survcomp2, cph3. The significance level was set at P < .05.The comparison of AJCC 7 and 8 on OS was evaluated by the Cox proportional hazard model along with Kaplan–Meier method. The discrimination performance was measured and compared by concordance statistics (C-statistics) in the Cox model. Results were generated using SAS Macros.Between 2004 and 2016, 48 655 patients with OPSCC were identified in the SEER database. After including male gender, age < 65 years, and disease location isolated to tonsil and/or base of the tongue and excluding unknown/nonspecified T and/or N criteria, non-Caucasian patients, and those who underwent surgery as a primary treatment, 9554 patients were analyzed . DemograOur cohort of HPV-selected OPSCC patients was restaged from the seventh edition AJCC staging to the eighth edition as indicated. Details of the distribution of staging based on TMN for the seventh and eighth edition AJCC are available in Overall, the clinical staging changed for 92.3% of patients, with a majority of patients classified as Stage IVA (63.3%) using AJCC 7 compared with a predominance of Stage II (55.8%) with AJCC 8. In addition, Stage IV in the eighth edition was isolated to patients with metastatic disease, which was previously staged IVC in the seventh edition.In contrast to the survival curves based on the seventh edition, staging using AJCC 8 demonstrated a better differentiation of OS over the 10 years between Stage I/II, III, and IV . There wP = .039; P = .038). When employing the eighth edition criteria, Stage I demonstrated marginally improved survival compared with Stage II without statistical significance for OS or DSS. On the other hand, stages III and IV had statistically significantly higher hazard ratios compared with Stage I and were statistically different from each other.For disease staging, P < .001). This is in comparison to the survival concordance statistics for the unselected cohort of OPSCC patients (data not shown), where the C-indices were 0.5860 and 0.6457 for the seventh and eighth editions, respectively. This is represented in For our HPV-selected cohort, the C-index (95% CI) was statistically lower for seventh edition (0.6260 [95% CI 0.6098-0.6422]) compared with eighth edition (0.6846 [95% CI 0.6704-0.6987]) AJCC staging  and 0.6846 (95% CI 0.6704-0.6987) for AJCC 7 and 8, respectively. This shows that the concordance for AJCC 8 is better than AJCC 7 for all OPSCC patients regardless of HPV status, enforcing the improvements of AJCC 8 in this disease. Our findings are likely related to the fact that HPV-related OPSCC has become the predominant OPSCC disease but also raise the interesting question of AJCC 8 performance in HPV unrelated OPSCC. While our HPV-selected population is by no means inclusive, it likely encompasses a significant subset of highly predictive HPV-positive disease.24–28 where a large shift from Stage IV to lower stages was observed between AJCC 7 and 8 regardless of treatment. A few of these studies were also unable to prognostically differentiate Stages I and II OPSCC.26 This could be a feature of the similarly excellent OS and DSS of these early stages of the disease as well as the potential lack of observed discriminatory benefit for early-stage OPSCC. It also raises some questions regarding limitations of the SEER data for these earlier stages as well as our inability to confirm the HPV status in our study.Similar findings to ours in HPV-related OPSCC were confirmed in other validation studies,27 In a second study, Würdemann found statistically significant differences between Stages I and II and utilized combined HPV detection with DNA PCR and p16 in 150 patients who underwent surgery followed by concurrent chemoradiation.28 Finally, the third study by Gupta et al. included 218 p16+ OPSCC patients who had surgery as the primary form of treatment and compared both clinical and pathological staging of AJCC 7 and 8.24 While they too had overlapping confidence intervals for clinical Stages I and II using AJCC 8, this study could statistically differentiate all pathological stages. These studies are fundamentally different from ours as they directly assessed HPV status and were single-institution trials. Additionally, the studies by Würdemann et al. and Gupta et al. included different patient cohorts who were able to initially proceed with surgery. The latter also separated comparison of clinical and pathological staging and reported a statistical difference in pathological staging only but results similar to ours for clinical staging.In contrast, 3 prospective studies were able to differentiate outcomes between Stages I and II disease. Van Gysen et al. demonstrated a statistically significant difference in 5-year OS between Stages I and II in an Australian cohort of 153 patients with p16+ OPSCC treated with weekly cisplatin.29 In our analysis, we excluded patients who had surgery, limiting our assessment of pathological staging.Our results are in contrast to those of a larger population-based study utilizing the National Cancer Database (NCDB), in which Zhan et al. described 3745 surgically treated HPV-positive OPSCC patients with a dramatic downstaging of Stage IV using AJCC 7 to Stage I when applying AJCC 8 with good hazard discrimination of all eighth edition pathological staging.30 This outcome, however, was not observed with Stage I patients without lymph node involvement, previously staged I or II in AJCC 7. In fact, this population had worse outcomes with concomitant chemoradiation.30 This finding emphasizes the heterogenous collection of OPSCC included in Stage I, despite the different treatment options and that AJCC 8, while prognostically better, should not drive treatment recommendations. This incongruity is paralleled by worse outcomes in Stage I HPV-positive OPSCC patients with radiologic findings of extranodal extension (ENE), seen to have increased risk of distant metastasis and reduced disease-free survival.31 This elucidates the need for further efforts to improve staging, as evidenced by poor differentiation of Stage I and II and the known heterogeneity within this subgroup. This raises the question of treatment de-intensification within a subgroup of favorable patients; however, data from the NRG HN002 study showed that not all favorable disease benefits from de-escalation.32 An interesting observation that would warrant a further investigation is the stark similarity of 15.5% OS at 120 months for both Stage IVc in the seventh edition and Stage IV in the eighth edition, likely representing not only the poor prognosis of this final stage but also the direct association of AJCC 7’s IVc to AJCC 8’s IV staging. These observed discrepancies reflect the need for continued improvement to our AJCC staging guidelines, especially for clinical staging as those studies assessing pathological staging had more consistent differentiation among the different stages. Despite it being a controversial topic, there is evidence that ENE is a predictor of worse prognosis in HPV-positive OPSCC patients. Bauer et al. showed ENE-positive cases with worse 5-year survival for HPV-positive cases compared with ENE-negative with a hazard of death HR = 1.90; 95% CI: 1.35-2.67.33 Other studies have shown a similar correlation between ENE presence and HPV-positivity.35 Whether the inclusion of radiologic ENE in future staging will confer an advantage remains to be seen as conflicting reports exist as to the value of radiologic ENE specifically in HPV-related OPSCC. If this were to be the case, the inclusion of rENE could transition this small subset of Stage I patients with worse survival outcomes into more advanced staging.36 This ultimately could improve staging differentiation and impact treatment and subsequent deintensification.Some important considerations for the staging of our HPV-selected cohort focus on the weak performance of Stage 1 in AJCC 8 and the poor prognosis incurred with AJCC 8 IV staging. Within our AJCC 8 assessment, Stage I was difficult to distinguish from Stage II. While this has been seen with other similar studies mentioned above, this finding likely represents heterogeneity within Stage I. Yoshida et al. describe this finding in terms of treatment response, namely from the association of chemotherapy and improved survival amongst patients with Stage I HPV-positive OPSCC with lymph node involvement, previously staged III or IVa in AJCC 7.37 Another study by Liu et al. focuses on wild-type p53 with low levels supporting a favorable response to radiotherapy.38 Further understanding of how these genomic profiles can impact OPSCC outcomes is needed.40Our findings also raise the question of whether AJCC 8 ought to be examined as a possible staging of choice for OPSCC regardless of HPV status. There are emerging similarities between HPV-positive and HPV-negative disease that may justify more detailed genomic investigation. While HPV status is the most common and robust molecular biomarker for OPSCC, other molecular markers are being highlighted in their role in prognosis and disease behavior. One example is pAMPK activity, which portends a better prognosis for HPV-positive OPSCC, perhaps warranting consideration for de-escalation of therapy.41 Treatment data are also limited by underlying biases associated with receiving or not receiving chemotherapy and/or radiation, specifically selection bias, patient preference, physician recommendations, comorbidities, disease severity, and so on. Given these limitations, we are unable to compare outcomes based on treatment received or explain the discrepancy in hazard ratios for patients receiving these associated treatments. Furthermore, we cannot base treatment decisions or de-escalation on AJCC 8 as it stands currently, as evidenced by Stage I’s notable heterogeneity.30 In regard to smoking, there were 2 time ranges 2004-2007 and 2008-2010 that measured smoking status. We were unable to combine these 2 different variables, so just the status for the 2008-2010 time range was included to avoid any confusion. Due to how these values were calculated, they could not be included in the univariate analysis but are represented in Limitations of our study include the retrospective nature of data collection utilizing the SEER database with incomplete demographic information, treatment, and HPV status. This study relied partially on demographic factors and HPV-targeted anatomical sites as surrogate markers of HPV status, which was not an ideal proxy for this selected group. While women were excluded to create a more definite HPV-presumed subset, it is important to note women are an affected subgroup that will be important to consider in future studies. Because this was a SEER limitation, we attempted to bypass this constraint by collecting a specific, yet understandably not inclusive, a subset of patients which reflect the changes associated with the new staging guidelines. In addition, our treatment and demographic data were limited by the completeness of variables. SEER notably reports high specificity for treatment data with a high likelihood of receiving treatment if reported. However, overall sensitivities are much lower averaging about 88.0% and 80.1% for radiation and chemotherapy, respectively.In summary, ours is the only study to date using the SEER database that attempts to compare the performance of AJCC seventh and eighth editions for OPSCC. Our findings support evidence from other analyses that the eighth edition provides better prognostication and discrimination between stages compared with AJCC 7 for patients with a clinical presentation consistent with HPV-associated OPSCC. It is of interest that our findings applied regardless of attempting to clinically select patients for HPV-relatedness.oyab001_suppl_Supplementary_FiguresClick here for additional data file.oyab001_suppl_Supplementary_TablesClick here for additional data file.


Next-generation sequencing (NGS) yields powerful opportunities for studying human papillomavirus (HPV) genomics for applications in epidemiology, public health, and clinical diagnostics. HPV genotypes, variants, and point mutations can be investigated in clinical materials and described in previously unprecedented detail. However, both the NGS laboratory analysis and bioinformatical approach require numerous steps and checks to ensure robust interpretation of results. Here, we provide a step-by-step review of recommendations for validation and quality assurance procedures of each step in the typical NGS workflow, with a focus on whole-genome sequencing approaches. The use of directed pilots and protocols to ensure optimization of sequencing data yield, followed by curated bioinformatical procedures, is particularly emphasized. Finally, the storage and sharing of data sets are discussed. The development of international standards for quality assurance should be a goal for the HPV NGS community, similar to what has been developed for other areas of sequencing efforts including microbiology and molecular pathology. We thus propose that it is time for NGS to be included in the global efforts on quality assurance and improvement of HPV-based testing and diagnostics. Tests for the detection of human papillomavirus (HPV) infection in humans have evolved dramatically over the last decades. Initial low-throughput hybridization/blotting techniques prefaced broad-spectrum signal amplification assays, which were then replaced by rapid high-throughput target-amplification assays involving quantitative polymerase chain reaction (qPCR). The latter tests are capable of detecting individual HPV-genotypes and have become the mainstay of HPV-based screening and clinical testing . ArguablE7 gene conservation in cervical cancer development [Next-generation sequencing (NGS) of human or microbial genetic material is being applied increasingly in laboratory contexts, to facilitate research, population-based epidemiology, and recently, personalized patient diagnostics. NGS can be used as a highly sensitive method for HPV detection due to its ability to detect types at low copy number (even within multiple infections), novel types, and/or known types that are distantly related to primers/probes which may escape detection using standard molecular approaches ,3,4. Wheelopment .Although NGS has been used in HPV research for some years with different applications, as described above, NGS in clinical diagnosis is not yet extensively used. Lack of standardization and quality guidelines, as well as expense and requirement for ancillary laboratory infrastructure, may have slowed down the adoption of this technology in clinical laboratories. However, as is discussed later, NGS use could ultimately improve diagnosis and management of patients with HPV-driven lesions. This includes utilities ranging from a more sensitive detection of HPV to detection of true viral persistence , identifNGS is a technology based on massively parallel sequencing or “deep sequencing” of nucleic acid sequences. Nucleic acid sequences are fragmented with each fragment being amplified and sequenced multiple times, providing a depth of information which can deliver accurate data at the nucleotide level. NGS can sequence the entire genome of HPV or be limited to specific areas of interest ,14.There are several general approaches to sequencing, depending on the size  of nucleotide reads obtained and detection method employed. Illumina and IonTorrent instruments obtain reads that are approximately 250 base pair (bp) long, whereas Oxford Nanopore MinIon and PacBio obtain longer reads, potentially exceeding 10,000 bp in length .The whole NGS process requires several steps, which include initial sample identification, processing (nucleic acid extraction), viral enrichment , library preparation, sequencing, and bioinformatic analysis of the raw data . While sWe are active in the field of HPV testing at the International HPV Reference Laboratory and the Scottish HPV Reference Laboratory (SHPVRL). Both entities were established in 2008, under the auspices of WHO (LabNet) and through the National Health Service of Scotland respectively. As such, we are committed to the evaluation and application of new technologies, including NGS technology, specifically to support the prevention and management of human cancers associated with HPV. In the present work we discuss the key stages of HPV-specific applications utilizing NGS and offer practical suggestions for quality-assurance procedures to support these stages, focusing on HPV whole-genome sequencing approaches. We believe that our experience may facilitate the implementation of NGS in laboratory settings so that it can play an increasing role in research, epidemiology, and importantly clinical testing. With respect to the latter, this is an important consideration, given the increased incorporation of NGS systems into routine departments of laboratory medicine, partly because of national strategies designed to harness the benefits of genomic medicine at the patient level in an agile but comprehensive way . NGS is All specimens used for the identification of HPV by existing molecular tests can in theory be used for NGS. However, various biospecimen types may present their own unique challenges. Liquid-based cytology samples, swab samples, or fresh frozen biopsies may typically be readily applied on an NGS platform. However, cells and tissue derived from formalin-fixed paraffin embedded (FFPE) material are more likely to be associated with fragmented nucleic acid and crosslinks between intracellular macromolecules such as proteins and DNA. Fragmentation can be a rate-limiting factor in approaches that demand longer amplicons, which is why it is useful to note that researchers have successfully used shorter amplicons when working with FFPE DNA using NGS technologies ,24,25,26Ideally, extraction methods should be assessed with a pilot panel of samples before embarking on a large project, in order to determine the quality and suitability of the specific extract for downstream NGS. Where possible, we recommend investigators evaluate at least two different extraction technologies to maximize nucleic acid yield. The quality, quantity, and fragment length prior to the library preparation must be determined, as different library preparations may require different nucleic acid input, quality, and length recommendations for library success. For Illumina  DNA libraries for example, most of the protocols are optimized for 1 ng of input. Assessing the DNA purity is needed to ensure that the extract does not contain possible contaminants  which can result in assay failure. UV absorbance is a common method used for assessing the purity of a DNA sample, and protocols generally define the “pure”/acceptable range as having an absorbance ratio values of 1.8–2.0 ,28. HighWhile HPV-positive clinical samples contain HPV nucleic acid, they are naturally dominated by nucleic acid from non-HPV sources . If NGS is performed directly on the extract, without a targeted approach, the HPV content is at a relatively low proportion vs. the total nucleic acid sample. As the human genome length is 3 billion base pairs vs. ~8000 base pairs for HPV, the relative proportion of human nucleic acids extracted is +200,000%.Sequencing studies reveal that viruses typically represent less than 1% of the total genomic material detected in a human specimen , and the(A).Depletion Protocols Using Saponin- or Lysis-Based MethodsSaponin is a non-ionic surfactant that depletes the human genome affecting the pathogen-human DNA ratio ,38. MolYA particular NGS application related to HPV is the detection of viral integration sites into the human genome. When performing WGS, authors have reported that tumors had either a small or a very large deletion in the viral genome and discovered that these deletions were the result of either HPV integration into the human genome or HPV–HPV sequence junctions . It has (B). Enrichment ProtocolsIn addition to depletion protocols, the two most common enrichment protocols for HPV are PCR enrichment in the absence of chemical components and using capture protocols. PCR enrichment involves performing a PCR reaction prior to library preparation. For WGS, this technique may include amplification of the whole genome of the target virus via overlapping primers covering the entire genome (other techniques may not require amplification of the whole genome). A disadvantage of this technique is that there is a need to know the target that is to be sequenced a priori—thus, the method is not valid for detection of novel or nontargeted HPVs. Additionally, in the case of fragmented material, the number of primers required can be high, which requires a structured a priori design approach, and the primers may vary in their affinity for the separate regions. Primer-dimer formation can also limit efficient target amplification, and therefore, pilot studies are needed to validate the protocol for each specific purpose ,53.To overcome these bottlenecks, unbiased amplification (not based on PCR) has been commonly used for viral enrichment, as it amplifies all DNA material present in the sample. Multiple displacement amplification (MDA) is the gold standard method for non-PCR-based amplification techniques, where the reaction is based on annealing random hexamer primers to the DNA template . MDA proAnother enrichment approach is to use a set of specific probes, “baits”, to recover HPV sequences from the entire genome of the virus. In brief, labelled biotinylated HPV specific probes are captured by streptavidin coated magnetic beads after hybridization, resulting in “pure” HPV-derived reads. Consistent with the overlapping PCR approach, these probes need to be designed in advance but can be modified as required if low read numbers are obtained for some regions of the viral genome. Although it can be expensive, the advantage of the probes/baits approach is the large number of different probes one may include, allowing thousands of probes in one design. This means that it is possible to load the analysis with probes for all known HPVs, depending on purpose. Furthermore, it is the current gold standard method for integration studies .Regardless of protocol applied, to validate the quality of host depletion or viral enrichment, positive and negative control material must be added at the nucleic acid extraction step and carried through the enrichment/capture/depletion step and all stages of subsequent analysis. As a positive control, cell line material infected with HPV is commonly used. However, we would recommend using specimens that contain both human DNA and HPV DNA in the typical concentrations that would correspond to real-life clinical specimens (99:1), as the sensitivity of the different approaches may vary. As a negative control, human DNA  or DNA-free water can be used. Note that if primer-based target enrichment is used, negative controls may contain noise such as primer-dimer bands when assessed by electrophoreses as well as unspecific amplification; these should be clearly discriminated from target sequences. Again, the introduction of homogeneous internal quality controls (IQCs) in the nucleic acid amplification should help in the identification of such issues. Internal positive control material demonstrates whether the depletion/lysis has removed/reduced the HPV target in large proportion during the sample preparation stage. For the enrichment step, positive control material should reflect if amplification of the target regions/genome occurs and negative controls help determine whether any nonexpected amplification/targeting occurred.Several operators have opted for performing WGS directly after nucleic acid extraction, without enrichment. As described above, reaching a high sequencing depth is required, due to the low proportion of viral sequences typically present in the human specimens. Direct sequencing has enabled an agnostic approach to DNA presence in clinical samples: while around 10% of cervical cancers are found to be negative for oncogenic HPVs by traditional PCR-based genotyping methods ,59,60,61At present, there are several different sequencing chemistries available. Each system has its own protocols and due to the diversity of platforms, and rapid pace of developments, we cannot recommend a specific one for all HPV applications. Akin to the assessment and introduction of any new technology in-house, we strongly recommend validation that includes confirmation of expected results from “known” quality materials and the evaluation of different kits with the specific analytical purpose in mind. For laboratories looking to embed NGS into the accredited scope of their clinical service, initial validation followed by yearly verification would likely be mandatory.Quality, quantity, and fragment length analysis is a must to confirm success of library preparation. Library preparation protocols usually inform the operator about the concentration and size expected for prepared libraries. If measurements do not reach the expected values,  optimization of fragmentation times, clean-up processes, or amplification steps should be performed. Larger fragments cluster less efficiently than smaller molecules, and a low concentration of prepared libraries translates into a low number of sequenced reads. Here, it is crucial to consider in which context libraries are analyzed; in a research study, suboptimal measurements can occasionally be acceptable but probably never in a clinical context where protocol adherence is paramount and actionable test results are needed.One also needs to perform accurate normalization to obtain homogeneous distribution (number of sequencing reads) of the samples and assure that the proper sequencing read length is used, depending on the insert size of the library. As an example, Illumina libraries prepared with dual-indexing that show a fragment length of 200 bp should not be sequenced with 2 × 150 bp, as part of the fragment length (around 130 bp) corresponds to adapter sequences, and the insert size, which is the actual query sequence, only comprises 70 bp (200 − 130 bp). Thus, 80 bp of the 150 bp sequenced (150 − 70 bp) does not contribute useful information. Libraries from positive and negative samples must be added into the final input dilution. Our recommendations for quality-control steps in the NGS workflow are summarized in The output data from a sequencing machine are often referred to as raw data. Raw data management generally includes filtering steps to remove poor-quality data and host-derived human reads and continues by mapping nonhuman high-quality reads directly to a known reference database or performing a de novo assembly approach, finishing with HPV taxonomy classification, phylogenetic analysis, and variant calling. There are several open-access tools that can be used to analyze “big data”. A set of bioinformatic algorithms, when executed in a predefined sequence, is collectively referred to as a bioinformatics “pipeline”. These pipelines can be designed in-house by teams with available bioinformatics expertise or obtained as ready-to-use applications from commercial suppliers. The premade pipelines are principally aimed to users with little bioinformatic experience and act as a “blackbox”  are used by the pipeline).Each bioinformatical tool deployed in the pipeline uses different algorithms and parameters when handling data. When the objective of a project is to resolve a 0.5–1% difference between sequences, these differences in parameter settings could mean a different interpretation on nucleotide/mutation level is reached depending on which pipeline is being used. Ergo, two studies on the same nucleotide position could reach two different conclusions as to whether a mutation is present.Errors due to poor sample handling and storage conditions, polymerase bias, PCR- or qPCR-induced errors, and incorporation errors within sequencing may be introduced during sample preparation, amplification, library preparation, and sequencing stages. While these errors might not interfere with the identification of an HPV type (where the sequence divergence is 10% relative to its most closely related type), they might compromise the identification of sublineages or variations in nucleotides that could have implications on accuracy, consistency, and, potentially, predicted phenotype. Careful sample handling, selection of a high-fidelity polymerase , and quaMost analysis applications use FASTQ files as input for analysis; however, different sequencing instruments may give different extensions for raw sequencing data , and the first step is the conversion of those files to a standard format (FASTQ). Platforms usually provide software for the desired conversion . Raw FASTQ files should be subjected to quality trimming and adaptor removal as a first step. Software for quality trimming and adaptor removal include Cutadapt, Trimmomatic, Trim Galore!, SeqTrim, and FastX among others . QualityTo obtain a dataset that contains only reads of interest, e.g., viral-related reads for HPV detection, nontarget sequences may be filtered out at the bioinformatics level to speed up downstream analysis and decrease the risk of misassemblies of genomic data. Most researchers applying WGS from sequenced extracted material opt for filtering out human genome sequences only (leaving HPV plus “other” microorganism reads). This is practical as human reads (according to our experience of multiple sequencing projects with a metagenomic perspective) account for approximately 90% of the total (with some variability depending on the sample origin) ,13,62. WNumerous aligners exist so far and are being developed in order to achieve greater accuracy pertaining to precision. Widely used tools include BWA-MEM  , SOAP2 , or BowtWhile some operators may, at this point, choose to filter out reads that are identical to the human reference genome , another approach is to employ looser parameters which accept reads as human if the identity and coverage across the human genome sequence of interest are at least 95% and 75%, respectively. This latter approach allows for the detection of possible mutations not anticipated in the reference sequence, although this flexibility should be tempered so as not to misclassify nonhuman reads as human. An important thing to consider after performing the aligning/mapping to the human reference genome is to select which “unmapped” reads are to be used in downstream analysis. Sequencing with paired-end reads may contain (1) paired-end reads where both reads are unmapped, (2) paired-end reads where one of the pair read maps to the genome and the other does not. Operators may decide to discard nonhuman single reads and continue only with nonhuman paired-end reads, or to include them all; such a choice is entirely dependent on coverage obtained and the aim of the project/resolution required.https://pave.niaid.nih.gov/).Once human (or host) sequences are filtered out from the high-quality data set, most operators align reads to a known and curated HPV database for HPV classification or to the reference HPV genome in question. Currently, there are 222 different HPV types officially established (data accessed on 18 March 2021 from Hpvcenter.se) and another 220 putative novel HPV types  whose complete sequence can be found in the public database from the papillomavirus Episteme  available at public databases, with GenBank having >33,500 hits retrieved when typing “human papillomavirus” (data accessed on 18 March 2021). A recent publication  detectedhttps://pave.niaid.nih.gov/; accessed 7 July 2021). The latter resource has a contemporary collection of internationally ratified sequences from the reference clones corrected for known sequencing mistakes in the original sequences.It is particularly key to note that, when aligning the sequencing reads to a specific HPV genotype, operators ascertain that the correct reference sequence is used. There are 3650 sequences belonging to HPV16 isolates in GenBank , showing differences that may reach up to 10% of the total genome. If each separate investigation were to use a different sequence as reference genome, then comparison between publications becomes challenging at best. The reference genomes that should be used for each HPV type are provided at the International HPV Reference Laboratory website (Hpvcenter.se); accessed 7 July 2021, as well as at the papillomavirus Episteme database  that infer phylogenetic trees after choosing the statistical method ,86.If the purpose is to detect HPV genotypes , once the reads are aligned to the HPV database, we recommended that cut-offs are applied on which HPV positivity is based .This could be, e.g., setting a minimum of 10 reads detected for a specific HPV type together with a coverage of at least 10% of the HPV genome (around 800 bp coverage). This approach would avoid false positivity generated by background noise . If phylogenetic analysis or variant calling is required, a FASTA file with the “query” sequence must be created. When creating a FASTA file from the obtained sequencing reads/contigs, investigators should be aware of the extent of genome coverage to see if there are missing regionsEvaluating the full coverage of the sequence is important, as several tools that convert the sequencing reads into a FASTA file use a reference sequence to account for the regions that are not covered. The use of “N”s is recommended for the positions that are not covered by the sequencing reads. For variant lineage assignment, exclusion must be considered for specimens with poor read depth (<200 median depth) and/or low genome coverage (<80% genome coverage) . For varWhen the correct reference genome is not known, the (re-)construction of the sequenced genome must be performed without a priori knowledge of either the correct original sequence (or the order of the DNA fragments), by assembling overlapping reads into one or more contigs. This process is known as de novo assembly. Subsequent post assembly assessment is mandatory to reduce the risk of chimeric sequences and possible miscalling of HPV positivity in samples and/or erroneous calling of new HPV variants/genotypes. HPV-Chimera scripts exist to help researchers determine the accuracy of their HPV contigs ,77.While positive and negative controls can be incorporated into laboratory experiments and several quality check-points are available during the whole laboratory process, we currently lack an agreed approach for the quality control of bioinformatical tools and pipelines. Digital IQCS can be prepared from confirmed and verified positive material and stored as FASTQ files (raw data). Interlaboratory exchange of data can provide reassurance by comparing results on sequences derived from the same specimens. However, this requires resources and collaboration which may not always be available in the short term. Therefore, it would be beneficial to have positive IQCS available in an online repository that could be used to verify the pipeline when setting up a new service/test or when a tool is updated.Recently, several journals have started to request that all authors who submit manuscripts containing NGS data provide a detailed summary of sequencing coverage and quality statistics. For example, the International Journal of Cancer requires a summary from submitting authors that must include all information about library preparation, sequencing technology information , as well as preprocessing, quality control, and filtering of the raw NGS data . FurtherApplication of NGS for clinical testing requires a level of quality assurance and monitoring likely to be even more stringent than systems set up in research laboratories. Verification and validation for each of the steps that make up the NGS process is the key to obtain and provide reliable results to clinicians and patients. Any minor change of the wet-lab protocol or any parameter in the data analysis requires a full verification with previously known samples/sequences. This in combination with the external quality assessment, and accreditation helps ensure the validity of the clinical results.Suppliers of EQA schemes have developed external quality materials to support NGS sequencing results for various pathogens and human genes ). At time of publication, we are not aware of any official HPV EQA scheme to support NGS for wet and in silico analysis or indeed data/dry analysis. This is arguably a current deficit, as while interlaboratory exchange of materials is undoubtedly helpful for quality assurance and validation of HPV NGS, such exchanges do not wholly “stand in” for consistent performance in a formal accredited EQA scheme(s). Should HPV NGS move to the diagnostic context, then this would increasingly require address.Data storage demands of NGS are often very large and need to be carefully considered before local implementation. Unfortunately, there is no  consensus on what and how data should be stored beyond the general recommendation that it should be stored in line with national and local capacity and governance policies. In high-level terms, we recommend storing the raw FASTQ files in a compressed mode (.fastq.gz), the final output, and the full-log file  making the whole analysis reproducible.If intermediate files are to be kept, they should be stored as standard open-file formats FASTQ, BAM, and VCF, facilitating the exchange with other laboratories, where governance permits. Cloud-based storage could be a very helpful tool; however, this may be challenging to reconcile with data protection. Currently, most journals ask for data availability and request that authors upload all nonhuman sequences detected in the study to different databases (such as the European Nucleotide Archive (ENA), Sequencing Read Archive (SRA), and Genbank) to make research publicly available and re-usable for other scientists without compromising confidentiality.Next-generation sequencing (NGS) has enabled researchers to detect human papillomavirus (HPV) infections with unprecedented sensitivity and accuracy while simultaneously providing the whole viral sequence to be analyzed for viral point mutations, variant lineages, and genome variations. Increasing incorporation of NGS into laboratories for research, epidemiology, and diagnostic purposes may be only a matter of time, particularly as costs reduce with increasing demand and competition.Certainly, the use of NGS for clinical workstreams generally requires accreditation/auditing from an independent regulator. As an example, the National Accreditation Body for the United Kingdom (UKAS) now has expertise to assess and accredit labs that have NGS in scope, working to ISO 15189:2012 standards. This process covers assessment of staff training, quality control of pipelines, initial validation of the whole process compared to a gold-standard approach, and reproducibility of results plus checks to ensure security of data and access is correct. The magnitude of the effort to achieve accreditation in a particular service laboratory is likely to depend on local support for the integration of NGS to support public health epidemiology and precision medicine in general terms. The SARS-CoV-2 pandemic has brought about an exponential increase in molecular testing and associated infrastructure, including that required to support sequence-level variant detection. This is likely to pay dividends for the establishment of “cross organism working” to a diagnostic standard .The bulk of HPV sequencing to date has been performed using the IonTorrent initially ,11,91, fTypically, published presequencing laboratory protocols appear well validated for providing detail on clear-quality assessment procedures and specifying the requirements needed for library preparation evaluation and success. Nevertheless, even though the inclusion of positive and negative controls should be a must, the presence and/or description of these controls is not commonly found within NGS publications. This may be in part due to the relatively high cost of NGS. Hornung et al. reviewed ~265 publications which had used NGS for microbiome research and found that only 30% of publications used any type of negative controls and that less than 10% reported positive controls. Additionally, they observed that some of the results reported were potentially indistinguishable from contaminants .Furthermore, to the best of our knowledge, quality assessment of bioinformatic analysis appears not to be as standardized, when compared to the analytical sample handling stages. However, we consider it key that the use of a curated and updated databases, use of standard reference genome sequences, and description of the parameters used for each step should be described fully in all publications.The present piece aims to describe and summarize the application of WGS for HPV detection and has mainly focused on DNA detection to detect the whole HPV sequence. Nevertheless, RNA sequencing should not be forgotten as an alternative method for HPV detection, as RNA transcription not only enables detection of HPVs but provides further information on the active HPV infection that drives viral oncogene expression. In the cancers that are known to be caused by HPV, transcription of viral genes is necessary for viral pathogenicity . StudyinInternational collaboration is essential to efficiently further knowledge, scientific development, and concerted efforts to combat globally prevalent viral infections. In the case of HPV, the International HPV Laboratory Network LabNet was created by WHO in 2006 to support global development of laboratory standardization and quality assurance of HPV detection methods. LabNet concentrated on evaluating and improving methods used for research and evaluation of HPV-based screening and vaccination . As partTogether. we now work between our two laboratories to exchange samples, know-how, and protocols for bioinformatical flow and sequence analyses. Hopefully, this will strengthen the quality of work produced by both settings and act as a catalyst/model for future international endeavors. We propose that it is time for NGS to be included in the global efforts on quality assurance and improvement in HPV-based testing and diagnostics. By establishing a set of quality standards and best-practice statements, the community could systematically develop and apply NGS guidelines suited for HPV research, epidemiology, and diagnostics to ensure this innovative and powerful technology is developed in an internationally comparable and robust manner.


Cytology and HPV genotype screening play an important role in cervical cancer detection. Whether multiple HPV genotyping can predict cytological lesions remains to be further studied.Two thousand two hundred twenty-four females were analyzed for cytology and HPV genotypes test. The possibility of predicting cytological lesions by HPV genotypes test was evaluated by multivariate logistic regression and area under the receiver operator characteristic curve (AUC).Abnormal cytological results were found in 479 participants. A total of 688 patients were detected with HPV infection, 619 with HR-HPV infection and 112 with LR-HRV infection. HPV-52 was found to be the most common type among these patients, and a relatively higher risk of cervical lesions was found in HPV positive females. HPV-16, 31, 33 and 58 were found to have significantly higher infection rates in patients with HSIL and higher lesions. The prediction model was developed based on age and HPV-specific genotypes, with the AUC of 0.73 for cytological abnormalities and 0.82 for HSIL and higher lesions.HPV-16, 31, 33 and 58 infection are significant risk factors for cervical lesions. Combined HPV genotypes test can effectively predict cytological abnormalities. Cervical cancer remains one of the most common cancers affecting women worldwide . Early dCytological lesion detection and HR-HPV detection are the two main methods to effectively screen cervical cancer and precancerous lesions . VariousFrom January 2004 to December 2010, the study was carried out in the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Cytology samples were collected from 26,455 women that came to our hospital’s outpatient clinic for genital tract disease or routine cytological screening. Two thousand two hundred twenty-four women who received the HPV genotype examination at the same time were enrolled in this study.Eligible patients received gynecological examinations performed by gynecological practitioners from Sun Yat-Sen Memorial Hospital. Samples of exfoliated cervical cells were collected using plastic cervical swabs during the examination. Insert a plastic cervical swab into the endocervical canal for 1–1.5 cm, and rotate it counterclockwise for 4–5 full circles. Immediately place the tip containing the cellular material into the conveying medium tube and store it at 4 °C. All store bottles with cellular materials were collected, mixed with 3 mL specimen stored liquid  and stored in 4 °C. Using the Bethesda system to evaluate the result. The evaluation system included: (1) negative (A0), (2) atypical squamous cells (ASC), (3) low-grade squamous intraepithelial lesion (LSIL), (4) high-grade squamous intraepithelial lesion (HSIL), (5) squamous cell carcinoma (SCC), (6) abnormal glandular lesions , (7) atypical glandular cells of undetermined significance (AGCUS) and (8) adenocarcinoma.Genotype detection   is a PCRP values were two-sided, and statistical significance was accepted if the P value was 0.05 or less.First, descriptive statistics were conducted, and then chi-square test  and t test (continuous data) were used to evaluate the correlation between Pap testing and covariates. In order to test the existence of collinearity and interaction, relationships between all covariates were evaluated through the cross-tabulations of categorical variables and the correlation matrices of continuous variables. Unconditional logistic regression was used to analyze the potential association between HPV infection and cervical lesions. Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated. Data management and statistical analysis were performed with SPSS software version 16. A total of 2224 females were included in this study, with a mean age of 37.6 years (±9.2 SD), ranging from 21 to 77 years. Patients were admitted for post-coital bleeding, postmenopausal bleeding, last abnormal LCT, abnormal menstruation, irregular bleeding, and uterine prolapse  was used to evaluate the prediction efficiency. The results show that the model has a good predictive effect on cell lesions, and the area under the ROC curve (AUC) is equal to 0.73 Fig. A. AdditiThis study analyzed the risk of HPV infection in different cervical lesions and the risk of different cervical lesions in the case of HPV infection. HPV infection is considered to be the main risk factor for cervical cancer pathologies, particularly for types 16 and 18, which are associated with approximately 70% of cervical cancers , but simIn order to investigate prediction value of specific genotype HPV infection for cervical lesions, the multivariate logistic-regression analysis was performed and suggests that HPV genotype infection is independently associated with cytological abnormalities. Using our multivariate logistic-regression model, combined HPV subtypes have a good predictive potential for predicting cytological abnormalities. Cytology combined with HR-HPV detection was recommended for cervical cancer screening by US Preventive Services Task Force . WhereasThe advantage of our research is that, as far as we know, there is no research on the use of HPV genotype testing to predict cervical cytology, and we are trying to fill the knowledge gap by using multivariable logistic regression analysis. However, there are some potential limitations to our research. First, the prediction model has not been externally verified, so its validity needs further discussion. On the other hand, it is not clear whether the retrospective analysis of the data from the study, which was carried out on the patients who came to the clinic or underwent a physical examination, had the same high predictive effect for all women. Finally, in the absence of corresponding histological results, the feasibility of substituting the results of human papillomavirus tests for cytological tests deserves further study.In summary, our study suggested that HPV-16,31,33 and 58 infections were significant risk factors for cervical lesions. Combined HPV genotypes test can effectively predict cytological abnormalities.


This observational study aimed to investigate the possible influence of sexual behavior and demographic characteristics in the expression of HPV-related biomarkers in a colposcopy population. Lifestyle factors that impacted HPV DNA positivity in a statistically significant manner were younger age at first sexual intercourse, a history of more than four sexual partners as well as a partner change during the last year before HPV DNA assessment. Although ambitious, the development and validation of lifestyle scoring systems that combine molecular and epidemiological patient data to effectively predict underlying cervical pathology will represent a milestone in the quest of cervical cancer prevention.p < 0.0001). Similarly, a reported partner change during the last year before HPV DNA assessment contributed to 2.5 times higher odds for DNA positivity (p = 0.0006). From this perspective, the further development and validation of scoring systems quantifying lifestyle factors that could reflect cervical precancer risk seems paramount.Despite the significant scientific evolution in primary and secondary cervical cancer prevention in the battle started by George Papanicolaou in the previous century, global cervical cancer mortality rates remain disappointing. The widespread implementation of HPV-related molecular markers has paved the way to tremendous developments in cervical cancer screening, with the transition from cytological approach to the more accurate and cost-effective HPV testing modalities. However, the academic audience and different health systems have not yet adopted a universal approach in screening strategies, and even artificial intelligence modalities have been utilized from the multidisciplinary scientific armamentarium. Combination algorithms, scoring systems as well as artificial intelligent models have been so far proposed for cervical screening and management. The impact of sexual lifestyle inherently possesses a key role in the prevalence of HPV-related biomarkers. This study aimed to investigate any possible influence of sexual behavior and demographic characteristics in the expression of HPV-related biomarkers in a colposcopy population from October 2016 to June 2017, and corroborated the determining role of age at fist intercourse; the older the age, the lower the probability for DNA positivity. Multivariate analysis illustrated additionally that a number of sexual partners exceeding 4.2 was crucial, with women with ≤5 partners being approximately four times less likely to harbor a positive HPV DNA test ( Cervical cancer remains the fourth most frequently diagnosed cancer and the fourth leading cause among cancer deaths, with an estimated 604,000 new cases and 342,000 deaths worldwide in 2020 . In spitThe deployment of cytology-based cervical cancer screening strategies with early detection and treatment of precancerous lesions has significantly reduced both the incidence and mortality from cervical cancer in the developed world ,6. HowevFurthermore, in Greece, despite several initiatives, no national cervical screening program is currently in place, and most women are being opportunistically screened, predominantly by cytology and lately by HPV DNA or mRNA co-testing . AccordiWe conducted a study in a colposcopy clinic population of women of reproductive age, studying the expression of HPV-related biomarkers (HPV DNA and mRNA E6 and E7) in relation to dependent variables such as cytology and colposcopy, using as gold standard cervical histology . We also sought to determine possible co-variations with demographic characteristics and lifestyle sexual factors .http://HPVGuard.org, accessed on 25 June 2021, Project Number: 11ΣΥΝ_10_250, Cooperation framework, Protocol Number: ΕΥΔΕ—ΕΤAΚ 1788/1-10-2012). The study included women of reproductive age who were referred in the Larisa Primary Health Centre Colposcopy clinic for evaluation of abnormal cytology. This clinic is affiliated with the University Hospital of Larisa, covering a large proportion of referrals from Central Greece. Experienced gynecologists and board-accredited colposcopists staff the aforementioned department.We conducted a prospective pragmatic observational study within the framework of a multidisciplinary research protocol in cervical pathology (Ministry of Education and Religious Affairs), under the frame of the HPVGuard research project ) or either positive or negative mRNA E6 and E7 test as tested by APTIMA® HPV Assay  at the initial visit. There were no prerequisites regarding the anti-HPV vaccination status in terms of the eligibility to participate in the study.We included all women of reproductive age who signed the informed consent form harboring any grade of cytological abnormalities and/or abnormal colposcopy (LGSIL/HGSIL). We also included individuals who tested positive for HPV DNA genotyping   were also recorded. ® )HPV DNA genotyping ).Detection of E6/E7 mRNA from the 14 high-risk HPV types  sample was obtained using a Rovers™ Cervex-brush just prior to the colposcopic evaluation. This was transferred in PreservCyt solution and subsequently underwent cytological and bio-molecular analysis for established HPV-related biomarkers, in particular:® HPV2 allows the simultaneous detection of 35 different HPV genotypes  by PCR amplification of a fragment within the highly conserved L1 region of the virus [® HPV Assay is a commercially distributed kit allowing the identification of E6 and E7 mRNA of the 14 high-risk HPV types  utilizing a transcription mediated amplification of viral mRNA, while testing and analysis were basically performed on an automated system  by using 1 mL from the ThinPrep sample transferred into an APTIMA Specimen Transfer tube. All specimens were analyzed for cytological and biomolecular evaluation, centrally at the University General Hospital “Attikon” of Athens.The cytological examination was expressed according to the Bethesda classification (TBS 2001 system) ,12. The he virus . The APTWomen with referral abnormal cytology of any grade as well as those who tested HPV DNA and/or mRNA E6 and E7 positive underwent colposcopic evaluation. Women with negative colposcopy had no biopsies taken and were referred for a repeat cytological and colposcopic assessment six months later. Women with low-grade colposcopy underwent colposcopically directed single or multiple punch biopsies. All women with high-grade colposcopy had either biopsies taken or loop excision of the transformation zone (LLETZ) of the cervix. LLETZ conization is considered the gold standard for histological diagnosis since it includes the entirety of the cervical transformation zone and was preferred over multiple punch biopsies in women who had completed childbearing . The subAs stated, based on the study design all women underwent a colposcopic evaluation in order to document any cytologic and/or biomolecular discrepancies. All colposcopic examinations were performed by expert board-accredited colposcopists. In all HPV unvaccinated individuals, a strong recommendation for HPV vaccination was given irrespectively to the study’s cytological and or colposcopical findings, since this represents universal standard clinical policy of the department.All women were informed about the scope of the study and were asked to undersign a consent form before entering the study. The study’s protocol has been approved by the Greek Central Government and subsequently received additional approval from the coordinating authority “Attikon” University Hospital Ethics Committee (Code: ΕΒΔ 623/14-5-13) .p < 0.05 and all tests were two sided.Statistical analysis was performed via the SAS for Windows 9.4 software platform . Descriptive values were expressed as mean ± standard deviation (SD) also reporting minimum and maximum values, as well as median and first and third quartiles  and for the categorical data using frequencies and percentages. Comparisons between groups for the qualitative parameters were made using the chi-square test and if required the Fisher exact test. We examined the relation between the number of sexual partners and the coitarche using the Spearman correlation coefficient since normality could not be ensured for these variables. We evaluated if these two characteristics can be used as single predictors of HPV DNA and mRNA positivity using the receiver operating characteristics (ROC) area under curve (AUC). The significance level for all statistical tests was set to The performances of cytology and colposcopy, as well as HPV DNA and mRNA detection in respect to the histological results, were set as primary outcomes of the study, and were assessed by counting the true positive and negative cases as well as the false positive and negative cases for various cytological and colposcopical cut-offs as well as for various histological thresholds. Subsequently, we calculated the sensitivity, specificity, positive and negative predictive value , and false positive and false negative rates . Moreover, as single number indicators of the overall performance we calculated the overall accuracy and the diagnostic odds ratios (DOR).In total 336 women participated in the study; both their demographic and medical baseline characteristics are depicted in At enrollment, about one in three (36.9%) were current smokers and similarly 34.5% were vaccinated, the vast majority (81%) with quadrivalent Gardasil. On average, the number of lifetime sexual partners was 6; 33.9% of the women had ≤3 lifetime partners and a similar percentage of 36.3% had more than 5. For the whole population, the average percentage of condom use during intercourse was 29.8%; 150 women were never users (44.6%) and 14 (4.2%) were consistent users (100% rate of condom utilization).In terms of medical conditions, this special colposcopy clinic population illustrated 53.6% aberrant cytology rates, while an even higher percentage (62.5%) harbored abnormal colposcopic findings. HPV DNA was found positive in 152 (45.24%) of the participating women, and of these 60 (39.5% of the HPV positive population) had co-infection with multiple HPV genotypes . In addition, 92 women (27.4%) were found to be HPV mRNA positive while no further mRNA typing was performed. Finally, history of warts (active or treated) was reported in 18 women (5.4%).Regarding the expression of HPV’s genotypes in our population, the results of the HPV DNA assay detected a distribution of particular HPV subtypes that concurs with reported geographical data ,17. Almop < 0.0001) related with negative cytology, . The results are presented in ogy, See  whereas p = 0.0108). Actually, if we use the number of lifetime sex partners as a predictor for HPV DNA positivity then the AUC of the ROC curve was 65.39% (95% CI: 59.39–71.39%) indicative that is a rather good predictor. Moreover, for the age of coitarche yielded an AUC of 44.88% (95% CI: 38.80–50.96%).In our study, the number of sexual partners gained importance for HPV DNA positivity when it exceeded the threshold of three partners (per lifetime)—actually for this cut-off the OR was 1.9 (95% CI: 1.2–3.0); in other words, women with more than three partners per lifetime had two times higher odds for a positive HPV DNA test. Notably, the odds were increasing with increasing numbers of sexual partners, for instance for five partners OR reached 3.8 and for 10 OR reached 4.2. In terms of sexual behavior, a recent (within the last 12 months) change of partner was also predictive of a positive HPV DNA outcome . Interestingly, vaccination status, history of warts and condom use did not yield statistical significance in our study (p = 0.0014), as 55% of HPV DNA negative women were smokers, in contrast to the higher percentage (72%) of HPV DNA positive women who were non-smokers, and this could be perhaps attributed to the relatively small study sample [Oddly in our patients smoking appeared to a play a “protective” role , indicative that the higher this age the lower the probability for DNA positivity. Interestingly the age of first sexual intercourse was found to be statistically important . Furthermore, a partner change during the last year before HPV DNA assessment conferred 2.5 times higher odds for DNA positivity . Finally, both smoking  and HPV vaccination  had a positive impact for a negative HPV DNA outcome. Among the various cut-offs for the number of sex partners, the multivariate analysis demonstrated that a cut-off of five sexual partners was crucial; specifically, women with ≤5 partners were about four times less likely to harbor a positive DNA test , indicative that these two characteristics were not related.Characteristic plots depicting graphically the role of age at first sexual intercourse and number of sexual partners vs. smoking and vaccination are depicted in p < 0.0001) compared to 5.4 for HPV DNA, a finding indicative that HPV mRNA might represent a better predictor of underlying cervical pathology. All mRNA positive cases also harbored colposcopical abnormalities . Moreover, despite the finding that positive HPV DNA and mRNA results were strongly interrelated, the study data analysis did not detect a role of multiple infections in HPV mRNA expression rates.In a similar manner, as for HPV DNA, we examined the role of all the studied variables in relation to HPV mRNA positivity (see results in p = 0.0019) and similarly there is a trend that the odds are increasing with rising number of sexual partners. The number of lifetime sex partners was also tested as a predictor for HPV mRNA positivity; the AUC of the ROC curve was 62.88% (95% CI: 56.11%–69.66%), indicative that it is a rather good predictor. Moreover, the age of coitarche yielded an AUC of 46.08% (95% CI: 39.20%–52.97%). A reverse role of smoking was also documented regarding mRNA positivity rates .In terms of the role of the number of previous sexual partners, data analysis illustrated that a history of more than one partner might affect mRNA expression . Finally, vaccine type did not affect HPV mRNA expression rates.p-value < 0.1 in the univariate analysis (see p < 0.0001), while as observed for DNA positivity, non-vaccinated women and oddly non-smokers illustrated higher rates of mRNA positivity .Similar to DNA positivity, we applied multivariate analysis for addressing any possible confounding factors influencing mRNA positivity. The factors entered in the multivariate models were those with a ysis see . As was Additionally, we calculated various performance indicators for the role of cytology, colposcopy, HPV DNA and HPV mRNA as tests which affect the corresponding cervical status —note that per protocol all women which simultaneously presented with negative cytology, HPV DNA and colposcopy were considered as being histologically negative. These results are summarized in Clearly, regarding cytological and histological cut-offs of ≥LSIL, sensitivity of cytology reached 72% while specificity was above 87% see .Similar to cytology, a colposcopical and histological threshold of ≥LSIL provided rather balanced results  [The low overall prevalence of HG disease in this cohort predictably had an impact on our findings; the main study’s results are in line with the existing epidemiological literature ,45. In a 0.0001) .p = 0.0006), a finding also consistent with the literature [In our study a partner change during the last year before HPV DNA assessment contributed to 2.5 times higher odds for DNA positivity (terature . In the terature .Particularly in recent times, the clinician should carefully balance between a too intrusive or disengaged approach during history taking in the colposcopy clinic; the presence of a midwife is helpful in this direction. After reassuring the patient on privacy concerns and that all data will be anonymized, women should be encouraged to provide a scheduled history covering basic aspects of reproductive and sexual demographic data. Epidemiological co-factors which augment hrHPVs in the development of severe precancerous lesions possess an established all-important role in cervical carcinogenesis which should not be overlooked or even dismissed whatsoever, in light of the elucidated role of hrHPV-mediated molecular mechanisms . AnotherFrom this perspective, the further development and validation of scoring systems quantifying lifestyle factors that could impact cervical precancer risk seems paramount . The recResearch in emerging HPV-related biomarkers should be coupled with the development, integration and validation of lifestyle scoring systems. These systems quantify lifestyle factors that could impact cervical precancer risk and together with artificial intelligence tools accurately predict underlying cervical pathology.


The paper develops the adaptive dynamic programming toolbox (ADPT), which is a MATLAB-based software package and computationally solves optimal control problems for continuous-time control-affine systems. The ADPT produces approximate optimal feedback controls by employing the adaptive dynamic programming technique and solving the Hamilton–Jacobi–Bellman equation approximately. A novel implementation method is derived to optimize the memory consumption by the ADPT throughout its execution. The ADPT supports two working modes: model-based mode and model-free mode. In the former mode, the ADPT computes optimal feedback controls provided the system dynamics. In the latter mode, optimal feedback controls are generated from the measurements of system trajectories, without the requirement of knowledge of the system model. Multiple setting options are provided in the ADPT, such that various customized circumstances can be accommodated. Compared to other popular software toolboxes for optimal control, the ADPT features computational precision and time efficiency, which is illustrated with its applications to a highly non-linear satellite attitude control problem. Optimal control is an important branch in control engineering. For continuous-time dynamical systems, finding an optimal feedback control involves solving the so-called Hamilton–Jacobi–Bellman (HJB) equation . For linIn the past few decades, reinforcement learning (RL)  has provTogether with the growth of optimal control theory and methods, several software tools for optimal control have been developed. Notable examples are non-linear systems toolbox , controlThe primary objective of this paper is to develop a MATLAB-based toolbox that solves optimal feedback control problems computationally for control-affine systems in the continuous-time domain. More specifically, employing the adaptive dynamic programming technique, we derive a computational methodology to compute approximate optimal feedback controls, based on which we develop the adaptive dynamic programming toolbox (ADPT). In the derivation, the Kronecker product used in ,14 is rehttps://github.com/Everglow0214/The_Adaptive_Dynamic_Programming_Toolbox, accessed on 10 August 2021.The remainder of the paper is organized as follows. We review the adaptive dynamic programming (ADP) technique to solve optimal control problems ,14. Consu that minimizes The objective is to find a control policy Algorithm 1. Policy iterationInput:  An initial admissible control Output:  The approximate optimal control 1: Set while do2:      3:   Policy evaluation: solve for the continuously differentiable cost function 4:   Policy improvement: update the control policy byif xthen5:   break6:       end if7:   8:   Set end while9: Moreover, the state feedback abilizes  at the oabilizes  over allshown in  that Vi+As proposed in ,14, consollowing ,14, chooAssume that solutions to  are welle system  with thee system  can be rCombined with  and 4),,4), the  x(t) of  is obtaix(t) of (V˙i(x)=−qLet e origin ,14. ThenEquation  is approK trajectories K time intervals K equations of the form . We provide two modes to generate approximate optimal feedback controls; one mode requires the knowledge of system model, but the other eliminates this requirement, giving rise to the ADPT’s unique capability of handling model-free cases.N is given byTo approximate +1(x) in  and 4),,Vi(x) anic order  asΦd(x)For example, if According to  and 10)10), the We take the highest degree of monomials to approximate of Vi in  and g(x)Theorem 1.Let a set of trajectories be defined as Then the coefficients whereand for wherefor with Proof. Combining , 14) an an14) anBy applying the property rewrite  and 18)〈A,BC〉=〈AThen, the system of linear equations in  readily K of trajectories, such that the minimization problem can be solved well numerically. Then the approximate optimal feedback control is generated by the algorithm as Algorithm 2. Computational adaptive dynamic programmingInput: An approximation degree Output: The approximate optimal control   1:  Apply   2:  Set while do  3:    4:  Generate   5:  Obtain if then  6:  break  7:        end if  8:    9:  Set end while10:  return 11:  We now give the computational adaptive dynamic programming algorithm in Algorithm 2 for practical implementation. To solve the least squares problem in line 5 in the algorithm, we need to have a sufficiently large number Remark 3.As in the statement of Theorem 1, several integral terms are included in 0. As in , u0 doesRemark 4.In Theorem 1, the Kronecker product that is used in ,14 for pRemark 5.In the situation where the system dynamic equations are known, the ADPT uses the Runge–Kutta method to simultaneously compute the trajectory points for a function https://github.com/Everglow0214/The_Adaptive_Dynamic_Programming_Toolbox, accessed on 10 August 2021.The codes of the ADPT are available at It is of great importance for an optimal control package that the user can describe functions, such as system equations, cost functions, etc., in a convenient manner. The idea of the ADPT is to use symbolic expressions. Consider an optimal control problem, where the system model is in the form  with, where more applications of the toolbox can be found.In this section, we apply the ADPT to the satellite attitude stabilizing problem because a stabilization problem can be formulated as an optimal control problem. In the first example, the system model is known and the controller is computed by the function Let given byq˙=12qΩ,echnique , the sysgiven byq˙=12qΩ,q˙=12qΩ,Ω˙=I−128) at thhttps://github.com/Everglow0214/The_Adaptive_Dynamic_Programming_Toolbox (accessed on 10 August 2021). For the initial control We set the option ‘xInit’ with three different initial states. For each initial state, the option ‘tSpan’ is set to ed ACADO  to numeropen-loop optimal control for each given initial state, which is a drawback of ACADO, while ADPT produces a feedback optimal control that is independent of initial states. Moreover, even for the given initial state ACADO takes a tremendous amount of time to compute the open-loop optimal controller. From these observations, we can say that ADPT in the model-based mode is superior to NST and ACADO in terms of optimality, speed, and usefulness all taken into account.We can see in Consider solving the same optimal problem as in e system  and 28)u0 and exFrom To see how the computed optimal controller works in terms of stabilization, the norm of the state error under the control with To compare with other toolboxes on ADP or RL, we investigate MATLAB reinforcement learning toolbox with the same control problem. Equations  and 28)28) are dThe adaptive dynamic programming toolbox, a MATLAB-based package for optimal control for continuous-time control-affine systems, has been presented. By employing the adaptive dynamic programming technique, we propose a computational methodology to approximately produce the optimal control and the optimal cost function, where the Kronecker product used in previous literature is replaced by Euclidean inner product for less memory consumption at runtime. The ADPT can work in the model-based mode or in the model-free mode. The model-based mode deals with the situation where the system model is given while the model-free mode handles the situation where the system dynamics are unknown but only system trajectory data are available. Multiple options are provided, such that the ADPT can be easily customized. The optimality, the running speed, and the utility of the ADPT are illustrated with a satellite attitude stabilizing problem.Currently control policies and cost functions are approximated by polynomials in the ADPT. As mathematical principles of neural networks are being revealed ,27, we p