INK4, pRB, cyclin D1 and p53. The objective of this study was to assess cell cycle proteins as potential surrogate markers for HR-HPV DNA testing to identify OPSCC with favorable prognosis after resection.Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) has an improved outcome and may allow for treatment de-escalation. High-risk HPV (HR-HPV) infection is associated with deregulated expression of the cell cycle-associated proteins p16INK4a, pRB, cyclin D1 and p53 using immunohistochemistry. Protein expression was scored as high or low based on the proportion of positive carcinoma cells. Tumor samples were analysed for HR-HPV DNA with polymerase chain reaction-based testing. Associations between cell cycle protein expression and HR-HPV DNA status were evaluated by calculating sensitivity, specificity, predictive values, and diagnostic odds ratios (DOR). Kaplan-Meier and Cox regression analysis were applied to evaluate associations between cell cycle protein expression and patient outcome.Tissue microarray cores of 313 surgically treated OPSCC were stained for p16INK4a, cyclin D1, pRB and p53 in tumor cells were observed in 51.8%, 51.4%, 41.9% and 33.5% of OPSCC, respectively. HR-HPV DNA positive were 158/313 (50.5%) tumor samples . P16INK4a showed a higher DOR to predict HR-HPV DNA positivity than pRB, cyclin D1 and p53. Both the p16INK4a/pRB and the p16INK4a/pRB/cyclin D1/p53 signatures had lower DOR than p16INK4a alone. Improved 5-year overall and disease-specific survival were associated with HR-HPV DNA positivity, high p16INK4a, low pRB, low cyclin D1, and low p53 expression. Associations with improved outcome were also observed for the marker combinations high p16INK4a/positive HR-HPV DNA, high p16INK4a/low pRB and high p16INK4a/low pRB/low cyclin D1/low p53. In a multivariate analysis adjusted for age, smoking history, pT and pN category, high p16INK4a expression showed the lowest hazard ratio for death.High expression of p16INK4a expression is a reliable marker for survival prognostication in surgically treated OPSCC patients. Protein signatures including the pRB, cyclin D1 and p53 proteins do not further increase the prognostic performance of p16INK4a as a single marker.High p16 High-risk human papillomavirus (HR-HPV) infection is a major cause of oropharyngeal squamous cell carcinoma (OPSCC) . HPV-relINK4a and down-regulation of cyclin D1. Disruption of both the p53 and pRB pathways promotes S-phase entry. The tumor cell signature of high p16INK4a expression and low/absent expression of cyclin D1, pRB and p53 correlates with HR-HPV infection [INK4a expression of tumor cells was associated with favorable prognosis and high cyclin D1 and p53 expression of tumor cells was linked to poor outcome [HR-HPV infection of tumor cells is associated with the expression of the viral E6 and E7 oncoproteins [ outcome –19. HoweINK4a as a prognostic marker in a large cohort of surgically treated OPSCC, and to directly compare the performance of p16INK4a with pRB, cyclin D1 and p53 to predict patient outcome. The second objective was to evaluate the cell cycle proteins p16INK4a, pRB, cyclin D1 and p53 as surrogate markers for HR-HPV DNA positivity.The objectives of our study were to validate p16n = 104; 33.2%), group 2 with resection and adjuvant radio(chemo)therapy . Pathological staging was performed using the 7th edition of the AJCC/UICC TNM classification system (2010).The cohort of this study represents a subgroup of a previous reported cohort . DemograOne representative formalin-fixed paraffin-embedded tissue block of each OPSCC resection specimen was retrieved. Hematoxylin and eosin (H&E) staining of sections was performed using standard techniques. Stained sections underwent histological review to confirm the presence of carcinoma.Formalin-fixed paraffin-embedded OPSCC tissue samples were used to construct tissue microarrays (TMA). Three cores (diameter 2 mm) with tumor tissue were punched out of each donor block and transferred to the recipient block. Tissue cores from tonsils of non-tumor patients were included as controls. H&E stained TMA sections were microscopically evaluated for the presence of tumor cells.INK4a , pRB , cyclin D1 , and p53 . Marker expression was scored as high or low based on the proportion of positive carcinoma cells  [Three-μm thick tissue microarray sections were deparaffinized followed by antigen retrieval . Stainings were performed on a Leica BOND MAX instrument (Leica) using the Bond Polymer Refine detection kit (Leica) and the following primary antibodies: p16 nuclei) , 21.Tumor cells were located and marked on H&E stained tissue sections obtained from paraffin blocks of primary OPSCC. Selective dissection of tumor cells was performed from three to five 4 μm thick serial tissue sections of the same paraffin block and genomic DNA was prepared. HPV DNA was detected by polymerase chain reaction (PCR)-based amplification using the L1C1/L1C2 consensus primer set as previously described . PCR proP values of < 0.05 were considered statistically significant.All analyses were performed with the SPSS Statistics 22 software . Demographic and clinicopathological patient characteristics were investigated with descriptive statistics. Associations between cell cycle protein expression and HR-HPV DNA status were analyzed using the chi-square test. Specificity, sensitivity, positive predictive values (PPV), negative predictive values (NPV), and diagnostic odds ratios (DOR) were calculated to evaluate cell cycle proteins and expression signatures as potential surrogate markers for HR-HPV DNA positivity. Overall survival (OS) was defined as time from the date of cancer diagnosis to the date of death, disease-specific survival (DSS) as the time from the date of cancer diagnosis to the date of death from OPSCC. Five-year OS and DSS rates were calculated using the Kaplan-Meier method. Univariate Cox proportional hazard models were used to explore associations between patient characteristics, biomarker results, OS and DSS. Hazard ratios (HR) with 95% confidence intervals were calculated. The Cox proportional hazard model was used for multivariate analysis. Three hundred thirteen patients with surgically treated and histologically confirmed OPSCC were included in the analysis. The clinicopathological characteristics of the patient cohort are shown in Table INK4a reactivity. pRB was strongly expressed in the nuclei of basal and parabasal epithelial cells, which also showed weak to moderate nuclear cyclin D1 and p53 staining (data not shown).In non-neoplastic tonsils, scattered epithelial cells showed weak to moderate p16INK4a staining in more than 70% of carcinoma cells was observed in 52.1% of OPSCC. pRB and cyclin D1 reactivity was low (< 25% of tumor cells) or absent in 48.6% and 57.8% of OPSCC, respectively. Nuclear p53 positivity stronger than in tonsillar epithelial cells was observed in 33.5% of tumors. A high p16INK4a/low pRB signature of tumor cells was present in 45.4% of OPSCC. Thirty-eight percent of the OPSCC displayed a high p16INK4a/low pRB/low cyclin D1/ low p53 signature.We next evaluated cell cycle protein expression in tissue cores of 313 OPSCC Fig. . Strong INK4a, pRB, cyclin D1and p53 in 91.8%, 17.1%, 15.8%, and 14.6%, respectively  were calculated. The value of a DOR ranges from 0 to infinity, with higher values indicating better discriminatory test performance. The largest DOR value was observed for high p16INK4a expression alone  tumors were HR-HPV DNA positive . HR-HPV DNA positive OPSCC were positive for p16INK4a, low pRB, low cyclin D1 and low p53 expression of tumor cells . The 5-year OS and DSS rates were 76.1% and 85.2%, respectively. Kaplan-Meier analysis demonstrated that both improved OS and DSS were associated with HR-HPV DNA positivity, and high p16INK4a expression .INK4a protein overexpression and suppress the expression of the cell cycle proteins pRB, cyclin D1 and p53 in infected cells [INK4a protein overexpression identifies transcriptionally active HR-HPV infection with specificity and sensitivity comparable to those of E6/E7 mRNA expression [INK4a immunostaining for the identification of tumors with positive HPV status, based on the conclusion that p16INK4a is a reliable surrogate marker for oncologically relevant HPV infection [INK4a expression is a direct consequence of pRB inactivation by the HPV oncoprotein E7, which induces degradation through the ubiquitin-proteasome pathway [INK4a expression alone as a biomarker for positive HR-HPV status may have limitations due to HR-HPV-independent mechanisms of up-regulation.Among patients with OPSCC, HR-HPV infection of tumor cells is associated with favorable prognosis, and therefore may identify patients who benefit from treatment de-escalation. Various methods are available for HR-HPV testing. HR-HPV E6/E7 messenger RNA (mRNA) expression demonstrated by reverse transcriptase-PCR or RNA in situ hybridization (ISH) is considered the most specific marker for biologically relevant HPV infection because it identifies transcriptionally active HPV infection and indicates the translation of the viral E6 and E7 oncoproteins . In addipression , 23, 25.nfection . In HR-H pathway . HoweverINK4a alone. In accordance with other studies, we observed that HPV-associated OPSCC are characterized by strong p16INK4a expression and low or absent expression of pRB, cyclin D1, und p53 [INK4a staining showed the strongest association with HR-HPV DNA positivity when evaluated as a single marker . The associations between low/absent expression of pRB, cyclin D1, or p53 alone and HR-HPV DNA positivity were significantly lower. In comparison to high p16INK4a expression alone, we observed higher specificity values for the 2-marker signature high p16INK4a/low pRB and the 4-marker signature p16INK4a/pRB/cyclin D1/p53, but reduced sensitivity values. Using the diagnostic odds ratio as a performance parameter, high p16INK4a expression alone was the best immunohistochemical marker for HR-HPV DNA positivity. Our findings confirm p16INK4a as a reliable surrogate marker for HR-HPV infection in OPSCC. They also indicate that signatures including the pRB, cyclin D1 and p53 proteins do not significantly increase the diagnostic performance of p16INK4a as a single marker.In our study, we aimed to evaluate pRB, cyclin D1 and p53 as alternative marker for HR-HPV DNA positivity, and to address the question whether protein signatures including pRB, cyclin D1 and p53 may increase the diagnostic performance of p16 und p53 , 18, 19.INK4a protein, p16INK4aprotein expression alone, or HR-HPV E6/E7mRNA expression identify OPSCC patients with favorable prognosis [INK4a, low pRB, low cyclin D1, and low p53 expression. Furthermore, HR-HPV DNA positivity and the combined parameters high p16INK4a/positive HR-HPV DNA, high p16INK4a/low pRB, and high p16INK4a/low pRB/low cyclin D1/low p53 were also associated with reduced risks of death. Parameters retained prognostic significance in multivariate analyses except high cyclin D1 and p53 expression. Our results confirm the findings of previous studies and validate them specifically for surgically treated OPSCC patients [INK4a expression provides comparable prognostic information as HR-HPV DNA positivity in surgically treated OPSCC patients and may therefore serve as a reliable surrogate marker for improved prognosis, if HR-HPV DNA testing is not available.Treatment options for patients with early OPSCC include surgical resection with or without adjuvant radio(chemo)therapy, or primary radio(chemo)therapy. In addition to its role in tumorigenesis, HR-HPV infection identifies OPSCC patients with improved survival who may benefit from treatment de-escalation , 20, 28.rognosis , 14, 15.rognosis , 16–18. rognosis –31. In tpatients , 23. TheINK4a expression and HR-HPV E6/E7 mRNA detection in surgically treated OPSCC.Limitations of our study include that cell cycle protein expression analyses were performed on TMA cores that may not be representative of the whole tumor. However, various studies have shown excellent concordance between TMA spots and whole tissue sections in immunohistochemical analyses of multiple tumor types. Furthermore, each tumor was represented by three tissue cores in our study to increase concordance. Two individuals blinded for HR-HPV DNA results and clinical outcome information scored all immunohistochemical stainings. However, digital image analysis may have increased standardization of scoring. We used HR-HPV DNA positivity as the gold standard to evaluate cell cycle proteins as potential surrogate markers for HR-HPV infection. However, HR-HPV E6/E7 mRNA detection is considered the best marker for biologically relevant HR-HPV infection. Further studies are necessary to directly compare the performance of p16INK4a expression in tumor cells is a reliable tool for survival prognostication in surgically treated OPSCC patients. Protein signatures including the pRB, cyclin D1 and p53 proteins do not further increase the prognostic and diagnostic performance of p16INK4a as a single marker. Therefore, p16INK4a may be used to select OPSCC patients with improved prognosis for treatment de-escalation, especially in the absence of HR-HPV DNA and HR-HPV E6/E7 mRNA testing.In conclusion, our results show that immunohistochemical demonstration of high p16


What are efficient approaches for triage of human papillomavirus–positive women in cervical cancer screening?This cohort study of 3225 women found that p16/Ki-67 dual stain, alone or in combination with human papillomavirus 16/18 genotyping, provides better risk stratification than comparable cytologic-based strategies.Triage of human papillomavirus–positive women with dual stain may lead to lower referral to undergo colposcopy with similar detection of precancerous lesions compared with cytologic screening, making cervical cancer screening more efficient. As cervical cancer screening transitions from Papanicolaou cytologic screening to primary human papillomavirus (HPV) testing worldwide, effective triage tests are needed to decide who among the HPV-positive women should receive further diagnostic evaluation to avoid unnecessary colposcopies and biopsies.To evaluate the performance of the p16/Ki-67 dual stain (DS) and HPV16/18 genotyping for the triage of HPV-positive women.A prospective observational study was conducted within the cervical cancer screening program at Kaiser Permanente Northern California of 3225 HPV-positive women undergoing HPV and Papanicolaou cytologic testing with a valid DS result from September 16 to October 31, 2015, with follow-up through December 31, 2018.Human papillomavirus screening with partial genotyping and cytologic triage compared with DS triage.Cervical intraepithelial neoplasia grade 3 or more severe (CIN3+) and grade 2 or more severe (CIN2+), diagnosed within 3 years after sample collection.P = .005). Similarly, DS showed better risk stratification for CIN3+ compared with Papanicolaou cytologic testing in HPV-positive women, irrespective of genotyping. The greatest reassurance against CIN3+ was observed in HPV16/18-negative women with negative DS results, with a risk low enough to extend retesting intervals. Dual stain triage strategies required substantially fewer colposcopies per detection of CIN3+ compared with Papanicolaou cytologic testing, with a 32.1% (859 of 2677) reduction of colposcopies compared with the currently recommended triage strategy of HPV screening with Papanicolaou cytologic testing. Results for CIN2+ were very similar.A total of 3225 women  participated in the study. For triage of HPV-positive women with partial genotyping, DS showed better risk stratification for CIN3+ than did Papanicolaou cytologic testing, with women with positive DS results having a higher risk than women with positive Papanicolaou test results for CIN3+  16/18 genotyping for the triage of women with HPV infection. Three primary screening strategies are currently approved in the United States: Papanicolaou cytologic testing, HPV testing with Papanicolaou cytologic cotesting, and HPV testing with partial genotyping.3 For all screening approaches, triage tests are needed to decide which patients should be referred to undergo colposcopy for diagnostic evaluation.5 Per current HPV screening recommendations, HPV16/18-positive women are referred to undergo colposcopy, while women with positive results for other high-risk types of HPV undergo cytologic testing.7 Partial genotyping for HPV16 and HPV18 identifies the 2 genotypes with the highest risk of cancer,8 but cannot distinguish a transient infection from a prevalent precancerous lesion. Triage with Papanicolaou cytologic testing is subjective, and its sensitivity varies widely, requiring retesting of HPV-positive women with negative cytologic results.9Cervical cancer screening is shifting from primary Papanicolaou cytologic testing to primary human papillomavirus (HPV) testing worldwide.15Primary screening and triage strategies should be evaluated together, because the safety and efficiency of a screening approach and subsequent management depends on the combination of component test results. An ideal screening and triage approach should identify as many precancerous lesions as possible, while referring as few women as possible to undergo colposcopy. Detection of p16/Ki-67  in cytologic specimens is an accurate marker for cervical precancerous lesions.We conducted a large clinical evaluation study at Kaiser Permanente Northern California (KPNC) evaluating DS together with HPV16/18 genotyping compared with the approved primary HPV screening strategy. We studied the performance of DS cytologic testing for triage of HPV-positive women, focusing on tradeoffs between colposcopy referral and detection of precancerous lesions in clinical practice.The study was conducted within the routine cervical cancer screening program at KPNC. Per KPNC guidelines, women with negative HPV and Papanicolaou cytologic (cotest) results should return for regular screening after 3 years. Women with HPV-positive atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesions, or more severe cytologic results are referred to undergo immediate colposcopy. Women with positive HPV results and negative cytologic results should undergo repeat cotesting after 1 year and are referred to undergo colposcopy when either HPV or Papanicolaou cytologic results (at a threshold of ASC-US or worse [ASC-US+]) are positive. Women with negative results of a repeat cotest are not referred to undergo colposcopy. Per KPNC recommendations, all women undergoing colposcopy should receive at least 1 biopsy. Pathologists evaluating cervical biopsies routinely had access to patients’ full medical records. Clinical management was not based on results of DS or HPV16/18 testing. In total, 3416 HPV-positive women who underwent cotesting at KPNC between September 16 and October 31, 2015, were included in the study. Follow-up for cervical precancer and cancer end points was conducted using electronic medical records through December 31, 2018. We excluded 85 women without evaluable DS results and 106 women without colposcopy when indicated per the algorithms described above . We evalHybrid capture 2  was conducted on specimen transport medium specimens per the manufacturer’s instructions. Papanicolaou cytologic samples were collected in SurePath (Becton Dickinson) fixative. SurePath slides were prepared, stained, and processed on the BD FocalPoint Slide Profiler (Becton Dickinson). Medical history, HPV test results, and FocalPoint results were transmitted to the cytotechnologists reviewing slides on guided screening microscopes. All slides from HPV-positive women were evaluated by guided screening–assisted screening and full manual review. All abnormal slides were sent for pathology review. In addition, all negative Papanicolaou cytologic slides from HPV-positive women were rescreened manually.SurePath tubes containing the residual pellet after preparation of routine Papanicolaou cytologic slides from HPV-positive women with final Papanicolaou cytologic results were removed consecutively from storage. SurePath slides were prepared by an experienced laboratory assistant. The prepared slides underwent staining by a histology technician who successfully completed CINtec PLUS (Roche Diagnositics) DS training, using the CINtec PLUS kit following the manufacturer’s instructions. The DS cytologic slides were evaluated by cytologists after a 2-day training and certification to evaluate p16/Ki-67 DS cytologic slides. The cytotechnologists marked at least 1 positive cell on any DS-positive slides and documented their results electronically. The pathologists reviewed all slides and documented their results electronically. Slides with at least 1 DS-positive cell were considered positive.The residual matching specimen transport medium tubes were removed from frozen storage and processed on the cobas 4800 instrument (Roche) by licensed and trained personnel per the manufacturer’s protocol. Results from cobas testing are either HPV16/18 positive, other 12 high-risk HPV positive, or HPV negative.16: negative for intraepithelial lesions or malignant neoplasm (NILM); ASC-US; low-grade squamous intraepithelial lesions; atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion; and high-grade squamous intraepithelial lesions. Final diagnosis was established by histopathologic findings classified according to the cervical intraepithelial neoplasia (CIN) scale: no indication for biopsy, normal, other (including atypia), CIN grade 1 (CIN1), CIN grade 2 (CIN2), CIN grade 3 (CIN3) and adenocarcinoma in situ, or cancer. Women who did not have an indication for colposcopy  were considered free of disease. The primary end point included CIN3, adenocarcinoma in situ, and cancer (CIN3+). The secondary end point included CIN2, CIN3, adenocarcinoma in situ, and cancer (CIN2+). We calculated sensitivity, specificity, positive predictive value, and negative predictive value for detection of CIN2+ and CIN3+ for primary HPV screening with partial genotyping and DS triage, and for DS triage in HPV-positive women without partial genotyping. The strategy currently approved by the US Food and Drug Administration (FDA) of primary HPV screening with partial genotyping and cytologic triage was the reference for the comparisons.We evaluated the distribution of HPV16/18 and other high-risk HPV types (high-risk HPV12), and positive DS results by baseline cytologic findings and poorest histologic test results. Cytologic findings were classified by the Bethesda System2 and differences in predictive values were evaluated using the method developed by Leisenring et al17 using the R package, DTComPair . In a supplemental analysis, we stratified clinical performance measures by age groups . We evaluated absolute risks of CIN2+ and CIN3+ in strata by cytologic testing, HPV16/18 genotyping, and DS. Exact binomial 95% CIs were calculated for proportions. We calculated the absolute risk of CIN3+for the currently approved primary HPV screening strategy with partial genotyping and cytologic triage, for DS in combination with partial genotyping, and for DS alone without partial genotyping , while the sensitivity was equal for both strategies (P = .005)  .Among the 2066 women without previous abnormal cotest results, the results with respect to positive DS and HPV16/18 positivity  and overall referrals to undergo colposcopy (2677 [83.0%]). This strategy required 9.7 colposcopies to detect CIN3+ findings at immediate referral. All DS strategies required substantially fewer colposcopies, from 1818 for the combined partial genotyping and DS strategy in which all women with positive results for either of the tests are referred to undergo colposcopy (a 32.1% [859 of 2677] reduction), to 1610 for DS alone, requiring between 8.3 and 7.7 colposcopies per case of CIN3+ detected. The currently approved HPV screening strategy requires retesting of women with positive HPV results and negative HPV16/18 NILM cytologic results after 1 year. Of these women, half have positive HPV or cytologic results at the 1-year visit and require colposcopy referral per current recommendations. Among women undergoing primary HPV screening with cytologic triage who are referred to undergo colposcopy after a repeat cotest, only 17 additional cases of CIN3 were detected in 549 colposcopies, requiring 32.2 colposcopies for each case of CIN3+ detected. In contrast, 1-year retesting of women with positive HPV16/18 results and negative DS results required 14.2 colposcopies per case of CIN3+ detected.7 Within KPNC, this strategy would lead to two-thirds of HPV-positive women being referred to undergo colposcopy immediately, and more than 80% total of HPV-positive women being referred to undergo colposcopy immediately after 1 year. The strategy creates a substantial burden for women and has implications for health care infrastructure and cost, because most referrals to under colposcopy do not lead to detection of precancerous lesions.Cervical cancer screening is transitioning from cytologic testing to primary HPV testing. Although a negative HPV test result provides reassurance against prevalent precancerous lesions or cancer, most HPV-positive women have transient infections that are not associated with cervical precancerous lesions, highlighting the need for additional triage tests. The currently approved algorithm for primary HPV screening in the United States includes partial genotyping and cytologic testing for triage of HPV-positive women.13 In the current study, we evaluated the programmatic clinical performance of DS in conjunction with partial genotyping, allowing a direct comparison of a novel DS and HPV16/18 algorithm with the currently approved approach for triage of HPV-positive women. Using a risk-centered approach with internal benchmarks based on current management,18 we found that performing DS would accurately identify most women at very low risk of precancerous lesions (negative HPV16/18 results and negative DS results) who may safely undergo retesting at extended intervals, a small group of women with somewhat elevated risk that is not high enough for colposcopy referral (positive HPV16/18 results and negative DS results), and the remaining women whose risk is well above the threshold for referral to undergo colposcopy . Women with normal cytologic test results, but positive HPV16/18 results, have a risk above the threshold for referral to undergo colposcopy, much higher than the risk of women with negative DS results and positive HPV16/18 results. Dual stain alone, without genotyping, provides very similar risk stratification, indicating that DS is also effective for triage of HPV screening tests that do not provide genotyping. Furthermore, the risk stratification of DS among HPV16/18–negative women is better compared with Papanicolaou cytologic testing, suggesting that DS is a good triage option for vaccinated populations that have reduced prevalence of HPV16/18 infections.A previous study reported that DS has higher sensitivity and specificity compared with cytologic testing for triage of HPV-positive women.We evaluated the approved primary HPV screening strategy and novel DS-based triage strategies with respect to colposcopy referral and disease detection. We found that DS-based strategies were more efficient (indicated by lower numbers of colposcopies needed per case of CIN3+ detected) compared with cytologic testing–based strategies. Our data show the poor efficiency of 1-year repeat testing in currently approved strategies, with large numbers of colposcopies after repeat testing yielding only very few cases of CIN3+. Compared with a combined DS and HPV16/18 genotyping approach, the few additional cases of CIN3+ that are detected by these strategies involving 1-year repeat testing have negative HPV16/18 results and negative DS results at baseline, suggesting that they are small, low-risk precancerous lesions that will either resolve or be safely detected at the next 3-year screening visit.12 These data, combined with the low risk estimates in the current study for women with negative DS results, support the finding that retesting intervals in women with negative DS results can be safely extended to 3 years.We also found that primary HPV with DS cytologic testing can supplant a high-quality cotesting program and provide sensitive detection of cervical precancerous lesions while referring fewer women to undergo colposcopy. Dual stain cytologic testing showed improved performance compared with Papanicolaou cytologic testing in both the subset of women undergoing routine screening and the full population including women undergoing cotests for management of abnormal screening results, after colposcopy, or after treatment. Long-term follow-up from a previous study shows that women with negative DS results have a low risk of cervical precancerous lesions over 5 years.19 allowing us to directly compare DS results with established management thresholds. All study procedures were implemented at the KPNC regional laboratory and performed in parallel with routine clinical operations, showing the feasibility of an integrated HPV screening and DS triage program in a clinical laboratory. A previous study showed the high reproducibility of DS when read by experienced cytotechnologists.20 Currently, approaches for automated evaluation of DS slides are being developed based on digital imaging; these approaches may further improve reproducibility, accuracy, and workflow of DS cytologic testing.21Our study has several strengths. We evaluated a large population with uniform and well-organized screening and management procedures, good disease ascertainment, and limited loss to follow-up. Our large population provides precise risk estimates for various combinations of test results. Furthermore, the risk levels from KPNC for different combinations of cytologic testing and HPV testing were the basis of current US screening and management guidelines,Limitations of our study should be noted. Papanicolaou cytologic testing at KPNC may be more sensitive than Papanicolaou cytologic testing performed at other health facilities. Thus, the specific performance comparisons between DS and Papanicolaou may differ in other settings. In our observational study, clinical management was based on HPV cytologic cotesting, with differential follow-up of women with positive HPV test results and positive cytologic results and women with positive HPV test results and negative cytologic results (repeat testing after 1 year). However, we had high completion rates of follow-up procedures. Furthermore, management guidelines were not always followed exactly, reflecting real-life practice in routine cervical cancer screening programs rather than in a clinical trial. Follow-up is ongoing; it will be important to determine how long a negative DS test result in combination with HPV16/18 genotyping is associated with a low risk of cervical precancerous lesions.We found that, for primary HPV screening, DS has both higher sensitivity and specificity compared with cytologic testing for triage of HPV-positive women. Because of the greater reassurance of negative DS results, screening intervals can be extended compared with the screening intervals after negative cytologic results. Dual stain reduces unnecessary colposcopy referral and unnecessary cervical biopsies, and may reduce unnecessary treatment compared with Papanicolaou cytologic testing. Our estimates of sensitivity, absolute risk, and colposcopy referral for various triage strategies can guide implementation of primary HPV screening.


A change of cervical cancer screening algorithms to an HPV-based screening setting is discussed in many countries, due to higher sensitivity of HPV testing compared to cytology. Reliable triage methods are, however, an essential prerequisite in such a setting to avoid overtreatment and higher screening costs.n = 5), cervical intraepithelial neoplasia grade 1–3 (n = 74), and normal cytology  were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 using the GynTect assay and compared to cobas® HPV and CINtec Plus® biomarker results. All samples from women with cervical cancer, 61.2% of CIN3, 44.4% of CIN2 and 20.0% of CIN1 cases were scored positive for the GynTect methylation assay. In contrast, all CIN, irrespective of severity grade, and carcinomas were positive by both, CINtec Plus and cobas HPV. The specificity of GynTect for CIN3+ was 94.6% compared to 69.9% for CINtec Plus and 82.6% for cobas HPV  and 90.6% for cobas HPV 16/18. DNA methylation analysis of this methylation marker panel (GynTect assay) in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1/2 lesions. The detection rate among cytologically normal samples is extraordinarily low (1.5%).In this study, a series of cervical scrapes collected in PreservCyt liquid-based cytology (LBC) medium from women with cervical cancer (GynTect shows excellent performance when using cervical scrape material collected in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to the other test systems used in this work, GynTect showed higher specificity while still detecting all cancer cases. With the availability of screening programs, cervical cancer incidence and mortality have markedly decreased, especially in developed countries . The effTesting for high-risk human papillomaviruses (hrHPV), the sexually transmitted infectious agents that evoke cervical cancer, could substantially improve the sensitivity of screening . Thus, iCINtec Plus® , which detects the two biomarkers p16 and Ki-67 simultaneously by immunocytochemistry, has been assessed as a triage test for HPV-positive women in several studies [In this context, hypermethylation of certain DNA regions during the course of carcinogenesis may provide a promising tool for triage of a highly sensitive screening, which finds virtually all disease cases, but lacks specificity, as is the case if testing for HPV , 9. DNA In the present work we investigated the performance of GynTect in comparison to cobas HPV 16/18 genotyping and CINtec Plus. Especially the latter assay is also discussed for triaging abnormal screening results.n = 5), CIN2 (n = 19), CIN3 (n = 50), and cervical cancer (n = 5).Residual samples from routine cervical cancer screening  with normal cytology findings (NILM) as well as residual samples from patients with abnormal histopathology findings , all available in liquid-based cytology (LBC) media, were used for the study. This cohort based on PreservCyt®  samples ) consisted of 201 anonymized screening samples from women with normal cytology , and 79 anonymized screening and triage samples from women with histopathology diagnosis CIN1  from routine screening were tested using GynTect. All samples had normal cytology (NILM) and were collected in the commercial laboratory ZyDoLab, Institute for Cytology and Immune Cytochemistry, in Dortmund, Germany.For performing the GynTect assay, samples were processed as described in the instructions for use. Briefly, LBC samples were vortexed for a few seconds, and 1 ml of each sample to be used for the assay was immediately transferred into a 2 ml Eppendorf tube. Cells were centrifuged, the supernatant removed and the pelleted cells were resuspended using 40 μl of GynTect lysis buffer and incubated at 60 °C for 30 min at 1000 rpm in a thermoshaker . 40 μl of the incubated material was directly used for bisulfite treatment using the EpiTect® Fast Bisulfite Kit . After elution of the bisulfite-converted DNA with 20 μl water, 70 μl water was added and 10 μl was applied to each single reaction in the GynTect real-time methylation-specific PCR (qMSP) assay as described elsewhere . The qMSFor each marker the Ct-value was determined and a delta Ct was calculated between the Ct-value of the quality control marker ACHE and the Ct value for each marker. To be scored positive, the delta Ct has to be ≤9 for ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ≤ 10 for ZNF671. Each marker has a score if positive  and GynTect is considered to be positive if the total GynTect score is equal or higher than 0.5. To be scored valid, the Ct value for the control marker ACHE had to be ≤32 for the respective sample.To determine the cytology status, PreservCyt® samples were analysed using computer-assisted cytology at CytoMol. The 352 SureThin samples were analysed by two experts experienced in morphological diagnostic at the cytological laboratory ZyDoLab in Dortmund, Germany.n = 59).The cobas® HPV test (cobas z 4800 system) and CINtec Plus® were in most cases performed during routine testing at the molecular diagnostics laboratory CytoMol in Frankfurt, Germany. For samples with missing HPV and/or CINtec Plus results, testing was done subsequent to GynTect testing. HPV testing was done for all samples, CINtec Plus® was performed for all CIN and cancer samples and a proportion of the NILM samples  yielded a “not valid” result due to a too high Ct value for the ACHE control marker.All five carcinomas included in the study were scored GynTect-positive. Of the 49 valid samples with histopathology-confirmed CIN3, 30 = 61.2%) turned out to be GynTect-positive, whereas eight of the 18 valid CIN2 samples (= 44.4%) and one of the five CIN1 samples were GynTect-positive , one sample was tested positive for CINtec Plus and 3 samples were invalid. The cobas HPV test was positive for all CIN and cancer samples except two CIN3 samples, which were scored HPV-negative. In the NILM group, 15 (8.0%) of the 201 samples were positive for the cobas HPV test, one sample was invalid. Using HPV16/18 genotyping, the false-positive rate in the NILM group decreases to 3.0%. One cancer case, however, which was not HPV16- or HPV18-positive, would have been missed. All CIN lesions showed 16/18 positivity rates between 60.0% (CIN1) and 66.0% (CIN3).The detection rate of CINtec Plus was 100% for all valid CIN and cancer cases . In the NILM group tested with CINtec Plus , all other HPV-positive samples were GynTect- and CINtec Plus-negative.Comparing the three different test systems, GynTect shows an increase in the detection rate with severity of the CIN lesion. Not only the percentage of samples scored positive within, but also the average GynTect score itself between the histopathology groups, increased, respectively. In contrast, cobas HPV and CINtec Plus do not differentiate between CIN 1, 2, 3 lesions or cancer cases  findings Table . GynTectCINtec Plus showed a 100% sensitivity for both, CIN2+ and CIN3+ samples in this cohort, its specificity is not directly comparable to GynTect and cobas HPV due to less NILM samples tested with CINtec Plus.Looking at the predictive values, GynTect demonstrated a PPV of 74.5 and 91.5% for CIN3+ and CIN2+ and an NPV of 91.7 and 87.3%. Cobas HPV 16/18 showed a PPV of 63.8 and 84.5% and an NPV of 91.9 and 88.7% for CIN3+ and CIN2+, respectively. Cobas HPV for all types shows higher NPVs  along with lower PPVs  compared to genotyping. NPV and PPV for CINtec Plus are not directly comparable to GynTect and cobas HPV due to less NILM samples, as already mentioned above.To confirm the specificity of GynTect for NILM samples, 352 samples from routine screening collected in SureThin liquid-based cytology medium  were tested. Of the 352 samples, only 6 were invalid (1.7%), and of the valid 346 samples 3 were tested positive with the GynTect assay, achieving a specificity of 99.1% in this sample group.In previous studies we have shown that hypermethylation of CpG islands in proximity to the genes DLX1, ITGA4, RXFP3, SOX17, and ZNF671 correlated with the presence of cervical precancerous lesions and cervical cancer . The molIn this study we evaluated whether GynTect is suitable for using residual material from liquid-based cytology samples and thus fulfils this prerequisite. Furthermore, we compared the results with cobas HPV testing including genotyping for HPV 16/18, and CINtec Plus data obtained from the same samples. The latter test is also discussed as triage test for patients with abnormal cytology or positive HPV tests.GynTect might provide the possibility to test if a woman with an abnormal cytology finding in the Pap smear and/or a positive HPV test result, has a precancerous lesion that requires follow-up and treatment. For this purpose we used samples for which the cytology findings and, for all Pap-abnormal cases, the histopathology results were available.GynTect showed a very good technical performance, since among all 280 PreservCyt samples only 4 (1.4%) and among the 352 SureThin samples, only 6 (1.7%) were tested invalid with GynTect.The excellent performance of GynTect from LBC samples was directly visible in the Ct values obtained for the control DNA regions detected in the test system. In many samples less than 28 cycles were observed as Ct value for the control markers included in the test, due to very good DNA quality. Not too surprising, several of the samples with normal cytology showed Ct values also for the cancer marker regions, but in the range above 37 cycles. Therefore, in contrast to the GynTect analysis of STM samples , for usiAll five cervical cancer samples included in the study were GynTect-positive, each of them for at least four of the six GynTect markers. The high sensitivity for cancer cases was already shown previously analysing 123 (123/123 detected) and 5 (5/5 detected) cases, respectively . A detecIt is well-known that not all CIN3 lesions proceed to cervical cancer , 16. In Taking these observations into account with respect to the GynTect-positive results obtained for the three CIN stages, one may hypothesize that only lesions from women tested GynTect-positive may progress to higher grade or cancer while lesions from women tested negative for GynTect are likely to regress to normal. Further studies will show whether regression of CIN lesions correlates with a negative GynTect result.To compare specificity, sensitivity, PPV and NPV, CINtec Plus was performed for all cytology-abnormal samples plus 59 of the NILM samples. Within this subgroup of randomly chosen NILM cases the CINtec Plus specificity was comparable to GynTect. The cytologically and histopathologically abnormal samples, however, were all positive for the CINtec Plus test. Thus, the test would not allow a differentiation between lesions prone to progression and lesions which may persist or regress. This is well reflected by the PPV (CIN3+) for CINtec Plus with 69.2% compared to GynTect with 74.5%. Other studies using CINtec Plus as triage option showed similar results, even though not all had a 100% sensitivity for CIN1+ samples.False-positive rates for CINtec Plus reported in the group with normal cytology are in the range of 27–55% , 24 and HPV genotyping was performed for all samples, and stratification for HPV16/18 positivity leads to a better specificity than HPV in total or CINtec Plus, but still has a lower specificity compared to GynTect for both, CIN2+ and CIN3+. NPV is slightly better for CIN2+ and similar for CIN3+ compared to GynTect. It has to be noted, however, that HPV-positively tested patients who are negative for HPV16/18 – as is the case for one of the five cancer cases included in the study – nonetheless may be detected in the triage following the HPV test, e.g. another cytology.Cytology results of the 74 CIN lesions from this cohort are difficult to compare to GynTect, HPV or CINtec Plus results since all histologically confirmed CIN lesions have originally been found due to an abnormal cytology result. Therefore, we did only compare GynTect, CINtec Plus and cobas HPV results, because all these were second-line tests after the initial Pap testing in this study.Methylation markers are discussed as a tool for triage in cervical cancer screening programs, since they have the potential of being more specific than other biomarkers such as p16/Ki-67 (CINtec Plus) or HPV testing, with excellent sensitivity for cervical cancer cases. A recent work from Ochs and colleagues  came to Meijer et al. established four methylation markers  with FAM19A4 and mir124–2 being the most promising ones –28. CompRegarding the specificity, especially for mir124–2 and FAM19A4 a comparison is difficult because no distinction between CIN1 and “no CIN” was made , 28. TheOur results demonstrate that the molecular diagnostic test GynTect based on methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 has very good performance using liquid based cytology samples. On one hand, GynTect displayed superior specificity in inconspicuous samples, on the other hand, the test showed excellent sensitivity in detecting the relevant cancer cases. Nevertheless, sample size in this study was small and thus, the power of the study is limited. Further studies have to confirm the results shown in this article.In conclusion, in this small sample cohort GynTect shows excellent results if performed on cervical scrape material in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to CINtec Plus or genotyping for HPV 16 and 18, higher specificity is achieved while still having the option to find all cancer cases and two thirds of high-grade lesions. High-grade lesions tested GynTect-negative may constitute the benign lesions not developing to cancer but instead regressing to normal. The latter has to be shown in prospective trials, of which one is already ongoing, which aims at determining the regression rate of CIN2/3 lesions negative for GynTect in women < 25 years of age.


The aim of this study was to determine the HPV positivity rate in patients with laryngeal cancer, and to determine the effect of HPV positivity on survival. An additional aim was to determine if patients with HPV positive laryngeal cancer are more sensitive to chemotherapy and if such sensitivity differs according to chemotherapy protocol.The study included laryngeal specimens obtained from 82 laryngeal cancer patients and 11 laryngeal specimens with normal laryngeal mucosa that were obtained from our hospital’s paraffin block archives between 1995 and 2013. HPV was detected via chromogenic in situ hybridization (cISH) and confirmed via genotyping.HPV was not detected in any of the 82 laryngeal cancer patients’ laryngeal specimens, nor in any of the 11 archived laryngeal specimens with normal laryngeal mucosa via cISH. Genotyping confirmed these findings; none of the HPV types studied were detected in any of the specimens. As none of the study samples were HPV positive, it was not possible to compare survival, recurrence, or chemotherapy sensitivity.HPV infection is not a leading cause of laryngeal cancer; however, additional research on HPV positivity in patients with laryngeal cancer and its effect on recurrence, survival, and chemotherapy sensitivity is warranted. Laryngeal cancer is the 20th most common cancer in Europe, with around 39,900 new cases diagnosed in 2012  . LaryngeAlthough the reported incidence of HPV positivity in laryngeal cancer patients varies –12 fewerRetrospective data analysis was performed to include patients with laryngeal cancer between 1995 and 2013. The patients who had laryngeal squamous cell carcinoma, who had a biopsy spesimen available in the Pathology Paraffin Block Archives, who received chemoradiotherapy as the first line treatment were included. All patients underwent chemoradiotherapy post diagnosis, and all chemotherapy protocols included an alkylating agent. Patients that received any treatment before direct laryngoscopy and biopsy, had surgery as the primary treatment procedure, received only radiotherapy, and those with follow-up < 2 years were excluded. 82 patients met the criteria and the formalin-fixed paraffin-embedded tissue specimens (FFPE) and slides of tumors stained with H&E for each patient were retrieved. All patient slides were re-examined for confirmation of the diagnosis.The retrospective data analysis included demographic and clinical findings, as well as the histopathological characteristics of the tumor. Grade of differentiation, primary tumor extension, and nodal status, were recorded. Treatment regimens and the recurrence status were also assessed. Disease-free survival (DFS) was defined as the time from the date chemoradiotherapy was initiated to the time recurrence was detected.The control group comprised of 11 laryngeal specimens reported as normal laryngeal mucosa obtained from the Department of Pathology Paraffin Block Archives. These pathological specimens included 6 male and 5 female patients. The mean age of these subjects was 47.45 years, ranging from 35 to 62 years. These 11 patients had undergone endoscopic laryngeal surgery for benign lesions. These lesions were resected and biopsied during the direct laryngoscopy procedure. These biopsy specimens were reported to be normal laryngeal mucosa without evidence of a premalignant or a malignant lesion.HPV was detected via chromogenic in situ hybridization (cISH) and was confirmed via genotyping. The study was approved by the University Ethics Committee and was conducted in accordance with the Declaration of Helsinki.High-risk (HR)-HPV DNA in-situ hybridization (ISH) was done using proprietary reagents , which can detect high risk HPV genotypes  . HR-HPV in accordance to the manufacturer’s instructions. The HPV GenoArray helps the identification of 21 HPV genotypes  and CP8304. Of the 21 genotypes, 5 were classified as low risk  and the remaining HPV genotypes are classified as high-risk .HPV positivity and all clinicopathological data, including age, gender, tumor stage, differentiation, tumor localization, and tobacco and alcohol use, underwent descriptive statistical analysis using SPSS v.10.0 for Windows .n = 58 [70.7%]) and glottis (n = 24 [29.3%]). In all, 5 (6.1%) patients were clinically stage II, 38 (46.3%) were stage III, and 39 (47.6%) were stage IV. In total, 46 (56.1%) patients had no evidence of nodal involvement at the time of diagnosis. Tumors were histopathologically classified as well differentiated carcinoma (n = 33 [40.2%]), moderately differentiated carcinoma (n = 44 [53.7%]), and poorly differentiated carcinoma (n = 5 [6.1%]). Table The study included laryngeal specimens obtained from 82 patients with laryngeal cancer and 11 laryngeal specimens reported as normal laryngeal mucosa. Among the 82 laryngeal cancer patients, 78 were male and 4 were female; mean age was 56.6 years (range: 40–75 years). Tumor localization included the supraglottis (Chromogenic In-Situ Hybridization (cISH) was performed to assess HR-HPV positivity. cISH findings were evaluated by 2 independent observers. None of the 82 laryngeal specimens obtained from patients with squamous cell carcinoma and none of the 11 laryngeal specimens with normal laryngeal mucosa exhibited a positive cISH staining pattern. Genotyping confirmed the cISH findings; none of the HPV types studied were present in any of the specimens.HPV infection has been shown to cause oropharyngeal cancer. However, the clinical significance of HPV infection in head and neck squamous cell cancer other than oropharygeal cancer is yet to be determined. The prevalence of laryngeal and hypopharyngeal squamous cell carcinomas that are HPV positive varies from 0% , 15, 16 PCR and ISH are widely used HPV detection systems. ISH is commonly used to detect HPV in clinical biopsy specimens. Some studies report that PCR-based methods are more sensitive than ISH. Besides, PCR-based methods can yield false positive results, because they may not differentiate biologically irrelevant HPV from clinically significant HPV, whereas ISH correlates with biologically and transcriptionally active HPV, differentiating between clinically significant and non-tumorigenic HPV DNA –21. HPV In the current study we used HR-HPV chromogenic ISH for detection of HR-HPV; however, none of the 82 laryngeal specimens of squamous cell carcinoma and none of 11 laryngeal specimens of normal laryngeal mucosa exhibited a positive cISH staining pattern. Although the reported prevalence of HPV positive squamous cell cancer of the larynx and hypopharynx varies widely, the literature generally indicates that the prevalence is higher than that observed in the present study, but some studies have also reported no HPV positivity or a very low prevalence of HPV positivity , 23–25. The effect of tobacco use on laryngeal cancer is well-known, and we also reviewed the data and/or asked the patients about their tobacco use. Although the data were not very reliable because they were collected via self-report and the patients did not remember precisely how long they smoked or how much they smoked, all our patients had a history of tobacco use. Gheit et al.  reportedSome studies regarding head and neck cancer found out that HPV positivity was observed in younger patients compared to patients with HPV negative cancer , 4. The Earlier studies on HPV positivity in laryngeal cancer in Turkey reported that 7.4%  and 10.6The present study is among the few from Turkey to investigate HPV infection in patients with laryngeal cancer, and compare it to HPV positivity in normal laryngeal tissue. The cISH technique used in the present study is not widely used for studying laryngeal cancer. A strength of the present study is that HPV negativity based on cISH was confirmed via genotyping. As some earlier studies reported, HPV positivity was not observed in any of the present study’s laryngeal specimens  , 15, 16 The present findings suggest that HPV infection does not play a major role in laryngeal cancer; however, additional research is required to increase our understanding of the prevalence of HPV positivity in laryngeal cancer patients and the effect of HPV positivity on recurrence, survival, and chemotherapy sensitivity.


Cardiovascular diseases are leading causes of death worldwide. Recent studies suggest that infection by some viruses, including the human papillomavirus (HPV), may increase the risk of developing atheromatous lesions on coronary arteries. However, there is a lack of data regarding the possible association between HPV infection and coronary artery disease (CAD) in women. To investigate whether HPV infection is associated with the occurrence of CAD among climacteric women. The presence of CAD and cervical HPV DNA was investigated in 52 climacteric women. Social and demographic variables and metabolic profiles were also investigated. Among 27 women with CAD, 16 were positive for HPV, whereas 11 were negative. The presence of cervical HPV was strongly associated with CAD, after adjusting for demographic variables, health and sexual behaviors, comorbidities, and known cardiovascular risk factors. HPV-positive women showed a greater likelihood of having CAD  as compared with HPV-negative women, particularly those infected with high-risk HPV types . These results support the hypothesis that HPV infection might be associated with CAD among climacteric women, though further studies are needed to investigate the mechanisms involved. Cardiovascular diseases are the leading cause of death and constitute a major public health issue worldwide. Globally, they account for millions of deaths and, in Brazil, represent 29.4% of all deaths , 2. Inde Chlamydia pneumonia have been suggested as risk factors for CAD, possibly by promoting a chronic inflammatory status [Infections with several agents such as HIV, Epstein-Barr virus, cytomegalovirus, andy status , 7. In ty status . In the y status . In devey status –11. Overy status , anal [1y status , and cery status  cancers.Previous reports  have desA cross-sectional study was approved by the Federal University of Maranhão Ethics Committee . Written informed consent was obtained from all women. The sample size was calculated considering a prevalence of 65% of women with coronary artery disease positive for HPV , a powerVolunteers with and without CAD were included if they were over 35 years of age and climacteric. Individuals with cardiac catheterization following coronary angioplasty were considered for enrolment and referred to the Gynecology Service of the Clinical Research Center/UFMA, where they underwent clinical examination to confirm eligibility. Exclusion criteria were previous chemotherapy/ pelvic radiotherapy or having serum HIV positivity.Sociodemographic variables  were investigated using a semistructured questionnaire specifically developed for this study.During clinical examination, volunteers reported the presence or absence of comorbidities . Next, blood pressure (BP) and body mass index (BMI) were determined for each participant. For BP, volunteers were required to have an empty bladder and have not exercised or consumed alcohol, coffee or any food for at least 30 minutes prior to measurement. Systolic and diastolic BP were measured following the Brazilian Society of Cardiology guidelines, using a stethoscope and a mercury sphygmomanometer . BP was Venous blood samples were obtained from volunteers after a 12-hour fasting period. After collection into tubes with and without anticoagulant, plasma and serum were separated by centrifugation and stored in freezer -80°C until further analysis. The samples were used to perform hemograms, glucose profile , lipid profile , renal profile (creatinine and urea), and high-sensitivity C-reactive protein (hs-CRP).μL sample solution was added to 400 μL of AL buffer and 20 μL of proteinase. Then, 400μl of 100% ethanol was added to each tube; the samples were centrifuged four times and, after a brief incubation period, were again centrifuged at 8,000 rpm for 5 minutes to elute the DNA, which was stored at -20°C. The DNA concentration was determined using a NanoVue (GE) spectrophotometer. HPV DNA detection was performed by L1 gene amplification using a PCR-NESTED technique and the primer pairs PGMY 09/11 and GP 5 + / 6 +. Next, the product of the first reaction with the primers GP5 + / GP6 + was amplified.Cervical material was collected and submitted to nested polymerase chain reaction (PCR) for HPV genotyping. Extraction of genetic material was performed using the QIAamp DNA Mini and Blood Mini kit  following the manufacturer's instructions. Briefly, 400 μL of each sample was submitted to 1.5% agarose gel electrophoresis to verify amplification. The PCR products were purified and subsequently sequenced using a MegaBACE 1000 automated sequencer . Chromatograms of the sequences obtained were analyzed by Chromas software (Technelysium) and the sequences were submitted to online BLASTn software for the identification of HPV types. A total of 36 DNA-HPV samples were analyzed, and HPV types 16, 18, 33, 35, 39, 45, 51, 56, and 58 were defined as posing high oncogenic risk.Following the PCR reactions, 5 p < 0.05 was considered statistically significant.Data are presented as absolute and relative frequencies. A binary logistic regression model was used to obtain adjusted odds ratio (OR) and confidence intervals (CI). Data were analyzed using EpIInfo 7 software . A Fifty-two women participated in this study, who were divided into two groups, according to the presence or absence of HPV infection: HPV-positive (n = 23) and HPV-negative (n = 29) . The prop = 0.008) and elevated systemic blood pressure (p = 0.002) when compared with the HPV-negative group.Variables related to metabolic risk for developing CAD in the both groups are summarized in p = 0.0467) and the OR for CAD was 3.74 for HPV-positive volunteers compared with HPV-negative women.Regarding the presence of HPV, 44.2% of all women were positive for cervical HPV DNA, 69.6% of which with CAD, and 30.4 % without CAD . Among H p = 0.0384). The presence of other types of HPV was not associated with CAD in this study.Sixteen women (30.8%) had high-risk HPV types . These volunteers included 44.5% of the women with CAD and 16.0% of those without CAD . After aHPV infection is the cause of multiple benign and malignant lesions, but a possible association with cardiovascular disease has seldom been proposed and remains speculative. Still, some evidence  has repoThe prevailing view is that HPV remains confined to its epithelial lesions, but some authors report the detection of HPV DNA and proteins on endothelial cells  and circAmong climacteric women, we observed a marginally significant association between HPV infection and CAD. Importantly, the high-risk HPV genotypes associated with cancer were selectively associated with CAD, whereas no association with low-risk genotypes was observed. This is in line with previous studies  that detThe present results support the hypothesis that infection with HPV is associated with CAD, and that there is a specific association with high-risk (rather than low-risk) HPV types. If HPV does reach coronary arteries and is present in atheromatous plaques, it might promote their development through its classical cellular targets, p53, and the retinoblastoma protein (pRb). The loss of p53 function in macrophages was found to be strongly associated with the increase of atherosclerotic lesions –30. In aThe present results also show a lower HDL and higher systemic blood pressure levels in HPV-positive compared with the HPV-negative group. While this could represent a menopause-related change, the statistical correlations with HPV infection were highly significant and call for an explanation, which may involve the interference of HPV on lipid metabolism . HoweverOverall, in the current study, HPV infection was associated with a greater likelihood of CAD occurrence among climacteric women. Prospective cohort studies are hereby necessary to investigate a potential causal effect of HPV infection on CAD development, and the investigation of biological mechanisms underlying this association is warranted.


Pipes are one of the key elements in the construction of ships, which usually contain between 15,000 and 40,000 of them. This huge number, as well as the variety of processes that may be performed on a pipe, require rigorous identification, quality assessment and traceability. Traditionally, such tasks have been carried out by using manual procedures and following documentation on paper, which slows down the production processes and reduces the output of a pipe workshop. This article presents a system that allows for identifying and tracking the pipes of a ship through their construction cycle. For such a purpose, a fog computing architecture is proposed to extend cloud computing to the edge of the shipyard network. The system has been developed jointly by Navantia, one of the largest shipbuilders in the world, and the University of A Coruña (Spain), through a project that makes use of some of the latest Industry 4.0 technologies. Specifically, a Cyber-Physical System (CPS) is described, which uses active Radio Frequency Identification (RFID) tags to track pipes and detect relevant events. Furthermore, the CPS has been integrated and tested in conjunction with Siemens’ Manufacturing Execution System (MES) (Simatic IT). The experiments performed on the CPS show that, in the selected real-world scenarios, fog gateways respond faster than the tested cloud server, being such gateways are also able to process successfully more samples under high-load situations. In addition, under regular loads, fog gateways react between five and 481 times faster than the alternative cloud approach. Technology is evolving at a fast pace and companies have to adapt to such a constant evolution. In recent years, the industrial application of the paradigm of the Internet of Things (IoT) and the principles of Industry 4.0 have derived into the introduction of the latest technologies for monitoring, controlling and optimizing processes ,2,3,4,5.The Joint Research Unit deals with different parallel lines that work on heterogeneous topics like robotics, factory automation, process analysis or the application of new wireless communication systems on a ship. One of such research lines is called “Pipe Auto-ID” and, as its name implies, it deals with the problem of identifying pipes automatically through their life-cycle. Note that pipes are essential in shipbuilding: a ship usually contains between 15,000 and 40,000 pipes that differ greatly in their typology , although they all are built at the same workshop in a shipyard that Navantia owns in Ferrol (Spain). The workshop is divided into two wings shown in , where pIn the previous work to this article ,8, activTherefore, this paper presents a fog computing based Cyber-Physical System (CPS) that makes use of active RFID technology to track pipes, to show information on them and, eventually, to detect and automate certain life-cycle events related to the pipes built in a shipyard. Thus, the main contribution of this work is the description, implementation and practical evaluation of a fog-computing architecture of a novel CPS for an industrial environment like a shipyard.The remainder of this paper is organized as follows. The life-cycle of a ship pipe begins in the pipe workshop, whose floor map is represented in The second stage for most pipes is bending . The procedure is performed with computerized bending machines , pipes and some tools can be exposed to high temperatures.Presence of corrosive substances. In some areas of the pipe workshop  it is common to make use of different acids, caustic solutions or fuel, which may condition the selection of sensors, actuators and other electronic devices.Presence of communication interference sources. The CPS communication architecture should take into account that there are in the workshop, besides common sources of electromagnetic interference , other elements that generate electrical and electromagnetic noise. For instance, it is difficult to make use of Power-Line Communications (PLC) in the workshop due to the presence of mechanical saws and other AC-motor based tools that interfere remarkably with the communication through power lines. In addition, wireless communications can be interfered, for example, by the radar tests performed in the shipyard, whose power can reach several KW.Long communication distances. Most shipyard workshops are between 100 m and 250 m long, therefore, communications require the use of the proper technology. In the case of making use of wireless communications, it is almost certain that a network of devices or repeaters would be needed to cover a whole workshop. Moreover, network devices should be placed at spots with access to the data network and to electricity.Exposure to pressure sources. Like in other industries, in a shipyard workshop the products are moved in groups from one area to another, which usually leads to collisions and to the accumulation of weight on the products placed at the bottom. In the case of the pipe workshop, up to 35 pipes are commonly moved together in a pallet that withstands a weight of up to 2 T. Therefore, if sensors, actuators or other electronics are placed on the pipes or on the pallets, they should be protected with a proper encapsulation.A shipyard workshop is in different aspects a tough scenario for deploying a CPS. Specifically, the main issues that can arise, are:Ships are generally built using a construction method that divides them into several blocks ,18,19. TTraceability is a field that has evolved remarkably in the last years thanks to the progress associated with technology and standardization. In the case of shipbuilding, some authors proposed systems with the objective of keeping traceability of people and of diverse elements present in shipyards . For insWith respect to cyber-physical systems, several challenges arise when they are applied to a shipyard or to other mission-critical infrastructures, since security, safety, trustworthiness, robustness, and interoperability are key aspects to be fulfilled for their broad adoption ,26,27. DShipbuilders usually do not develop their own cyber-physical systems, but rely on different commercial software that, in some cases, is integrated into a common system. The latest trend consists in incorporating a Manufacturing Execution System (MES), whose aim is very similar to the one of an industrial CPS: to track, collect and show information on the products during the manufacturing processes. Companies like Siemens or SAP sell their own MES, which is adapted to the specific necessities of a field, factory or workshop. A MES usually has to be integrated with two additional pieces of software: the Enterprise Resource Planning (ERP) and the Product Life-cycle Management (PLM) software. This integration eases data sharing between the functional areas of engineering, workshops and the front office.Finally, regarding the use of fog computing for cyber-physical systems, some examples can be found in the literature for smart manufacturing environments ,31,32 anThe proposed system architecture is shown in The internal cloud is in the top layer, where Navantia runs its own compute-intensive services and the ones offered through third-party software .At the workshop, active RFID devices, IAR interfaces and transducers from different sensor networks constitute the node layer. Regarding the RFID devices, there is a network of RFID readers that continuously collect data  readings) about the location of the pipes, which are controlled by a software system managed by the fog layer. Active RFID fixed readers  were selThe indoor positioning system is managed by a fog service that is divided into different modules, the Location module and the Business Intelligence (BI) module being the most important  and by the users that interact with the system. In the case of RFID readers, they constantly report tag SSI levels to the indoor positioning fog service. From the service perspective, the readers are just like clients that generate data through web requests at a constant rate. Note that, as the traceability system grows, the number of tags and readers increases, which implies that the number of requests to be handled also increases. In a cloud-centric approach, these increases might derive into traffic bottlenecks. Fortunately, the designed fog computing based architecture eases scalability as the indoor positioning system grows.In The samples obtained from all the readers need to be transmitted as fast as possible. When deploying in the same machine, both the process in charge of querying the readers and the location service, they communicate with each other by using Redis , an openPossible alternatives to the use of Redis would consist in sending the collected samples through standard HTTP POST requests or by using HTTP long polling, but since the number of requests per second is very high, they would introduce latency and overhead in both the network and the machines when sending and receiving the data. Finally, the chosen technology was web sockets , which aArea change events. When a pipe moves from one area to another  and it remains on the latter for a certain amount of time (configurable by the system), the event is captured by the system and shown to the operators that might be interested in it. This is especially useful for warning the operators about incoming work.Pipes leave the workshop towards an auxiliary company. When the workshop is overloaded, part of the procedures performed on pipes are outsourced to auxiliary companies. Such pipes are stacked on the outbound area of the workshop and leave it through a specific door that the trucks of the auxiliary companies go through to collect the pipes. In this situation, three events have to be detected. First, that some pipes have remained in the outbound stack area for a certain amount of time. Second, that the pipes include in their life-cycle a task that requires outsourcing. Third, that the pipes are no longer detected by the system readers either inside the workshop or in the neighboring dock.Pipes leave the workshop to go to another workshop or to be mounted on a ship. Similar to the previously described event, pipes are monitored by the system readers until they leave the workshop and then are detected again in a dock or in another workshop of the shipyard.Accessories are required for pipe manufacturing. When a pipe reaches the manufacturing area, in the case that it requires certain accessories to be added, it is automatically notified to the main warehouse, where the operators collect the accessories and carry them to the manufacturing area.As it can also be observed in For every tag, its position is associated with the RFID reader that receives the maximum power levels from the tag . Therefore, the system compares in real time the power levels reported by all the readers and chooses the highest one. Then, since only one reader is associated to one logic area, it is possible to indicate in which logic area the pipe is most probably located.t from reader i, the new i and The previous locations of a tag are considered when calculating its new position. Such a list of locations is called “tag trend” and consists in a set of data structures that store past SSI readings. Thus, the SSIs used for selecting the closest reader are conditioned by past readings. Therefore, if a tag has a The way logic areas are defined involves a pair of SSI thresholds that limit the size of an area, letting the system adjust each area size independently. Thus, one threshold  limits the total size of the area, while the other one (inner threshold) is set to indicate that the tag is really close to the reader. It is important to point out that, since logic areas are limited in size, blind spots exist . While this fact may seem a limitation, in practice, it adds a nice feature to the system, because, when all the possible pipe routes are covered by logic areas, it is straightforward to discover pipes that are located out of them  and then warn operators about such inconsistencies.The coarse estimations given by the location algorithm are refined in two ways to avoid false logic area detections and to correct ambiguous readings in the limits of the areas:In addition to the previous refinements, in order to avoid positioning the pipes in wrong areas or showing the operator non-continuous positions , the life-cycle of every pipe is modeled according to the state machine diagram shown in A pipe begins its life-cycle in the workshop when it is first detected by the RFID system. In that moment the pipe is considered to be in transit. Note that every pipe has to stop at every production area of the workshop, but the pipe can be processed or not in such an area. If there is no need to process the pipe , it is palletized . In the case that it needs further processing, then it first would have to be moved to the processing area, cross the outer area, be processed, and then leave the processing station towards a pallet where a quality control would be performed.This capacity for being able to determine when a pipe is in a specific area or next to a production machine enables the automatic detection of pipe-related events. Specific events are notified when a pipe moves from a processing area to another, or when it crosses from an outer area to an inner area. Thus, the BI module receives periodic updates of pipe locations, processing them in conjunction with past locations and pipe production orders in order to determine the next state for the pipe, which is notified to the corresponding operator to indicate that a task will be added soon to his/her pool.A generic notification process is presented in the sequence diagram shown in The display module in  has beenRegarding the interaction, operators can zoom in and out of the map by using the control panel in the top-left corner of the canvas. In addition, logic areas (represented as blue circles) can be clicked or tapped, opening a new window that shows details on the pipes located in such an area in . The detNote that the details shown about a pipe in an area depend on the role of the operator using the application. For example, only supervisors can make use of the sign button to approve a pipe quality control  and displays the relevant data to the shipyard operators in a user-friendly way.Estado), the FO number (N° de OdF), the pipe ID (Marca tubo) and the pipe material ID . On the right of the screen, the details on the different sub-operations to be carried out on the selected task are shown. At the bottom, diverse icons allow operators to begin, pause and stop the selected task/sub-operation. Other icons are related to show the quality characteristics of the task , to transmit to the engineering team the detected errors  or to show the position of the materials used in the task/sub-operation (by clicking on the Loc icon).The MES back-end was developed on top of Siemens’ Simatic IT . The froTag assignment to pipes. An RFID tag can be assigned to a pipe or to a pipe accessory by using the MES. For such a purpose, first, an operator starts a cutting operation related to a FO in the MES. After the pipe is cut and ready to be palletized, an RFID tag is attached to the pipe. Then, the operator reads the tag ID with a reader, which is sent to the MES. Next, the MES notifies the event to the CPS, which stores the relationship pipe-RFID tag in its internal database and starts tracking the pipe.Detection of a tag when it enters or leaves a logic area. For instance, after completing a pipe cutting operation, the next operation could consist in welding the pipe to an accessory. In such a situation, when the fog service detects the pipe entering the welding area, it notifies the event to the MES, which updates the welding task pool and sends a warning to the welder to let him/her know that a new pipe is coming to the welding area.An operator requests the location of a pipe using the MES. In this case the MES sends the ID of the pipe to be located to the indoor positioning fog service. Then, the service queries its local database of active RFID tags and looks for the one with the required ID. In the case of finding it, the RFID tag location is returned to the MES, which presents a map to the operator that highlights the area where the RFID tag is located.The ERP and MES group the individual tasks to be performed in FOs. A FO is composed by some materials  and a series of consecutive fabrication processes . Several actions regarding the FOs can be performed through the MES, such as starting or finishing FOs, starting or stopping fabrication processes, or outsourcing certain processes to auxiliary companies. Among the many possible actions, the following are the most relevant when integrating the MES and the pipe CPS:In order to evaluate the performance of the proposed architecture, it was tested when deploying the indoor positioning service either in the fog or in the cloud layer. In the case of the fog layer, the service was executed on an SBC that acted as gateway. Among the different SBCs that may be used . The goal of the second test was to find out the maximum number of supported tags. For such a purpose, both the cloud and the fog-based architectures were tested under abnormal high loads, when more than 10,000 tags sent information concurrently.In the case of the cloud-based system, it is important to note that the virtual machine that executed the positioning service had no privileges over other virtual machines and it was not possible to fiddle with advanced high availability features to optimize response latency. Specifically, the cloud-based system shared its public network interface with another 11 virtual machines, although, during the tests, only six of them were active . The pipe workshop is considered to be under a regular load when up to 1000 pipes are being processed or stacked in the different areas. Thus, in order to evaluate the performance of the CPS under such a regular load, 10 different tests were performed when varying the number of tags from 200 to 1000 and when using a cloud-based or a fog-based architecture. For each test, one million SSI samples were generated.The obtained latency results are presented in It can also be observed in Improvement of It is worth noting the field Despite the observed latencies, it can be concluded that, even in the worst case , the latency is small (around 3.3 s on average) and should not influence the regular operation of the positioning service, which is not a real-time service . Nonetheless, it is interesting to measure the sample processing rate of the service in every scenario . In such measurements it was obtained that, under regular loads, a processing rate of 99.99% was always achieved.Note that it is not 100% because of how the number of received SSI samples is computed. Since the location subsystem is asynchronous, the received SSI samples are processed in batch by several parallel co-routines that end up obtaining an approximation of the number of samples. Such a number reaches a maximum difference of 10,000 samples with respect to the real value. Therefore, due to the fact that one million samples are processed, a maximum of a 1% error in the sample processing rate is introduced with this measurement technique.The performance of the CPS can also be evaluated when it is under high loads, which would represent workshops with a remarkable density of monitored objects or a whole shipyard. Specifically, the performed high load tests made use of between 10,000 to 60,000 tags. Due to the large number of tags used, the total number of SSI samples was increased to 10 million to allow for receiving enough SSI samples from each tag.It is worth noting that, under regular network traffic conditions, the minimum/average/maximum round-trip times to the cloud are In the case of the cloud, for 20,000 and 50,000 tags, periodic high-load intervals occur because of sharing the system and network with other users, which impacted both the sample processing rates shown in Finally, it is important to emphasize that, like in the latency tests, several factors may lower the sample processing rate, but there exist different alternatives to tackle the traffic load, like the use of load balancing or distributed computing.When there is a regular number of monitored objects in a shipyard workshop , fog computing systems respond faster since they are closer to the data sources.For regular loads, the difference in response latency increases between the fog computing system and the cloud because of the amount of traffic exchanged, which collapses the cloud network progressively if no corrective measures are taken.In the shipyard, it is not usual to manage more than 1000 tags with only one computational device, but it is interesting to evaluate such a scenario to determine the performance of the implemented architectures. Thus, it can be observed that a single inexpensive fog gateway that runs the implemented positioning service can respond faster and with a higher sample processing success rate than a cloud-based system for up to 40,000 simultaneous tags. For 50,000 or more tags, the fog gateway gets saturated and the cloud system becomes a faster alternative, although its sample processing rate is lower than for the fog gateway.Response latency was the metric selected to measure the architecture performance in order to quantify user experience. Nevertheless, in certain scenarios, where it does not matter how fast the system updates its positions, other metrics could be used . Therefore, it is important to take the obtained results with caution since the system was designed explicitly according to Navantia’s requirements.Despite the observed under-performance of the cloud-based system, it is worth noting that it is possible to reduce the response latency by adjusting different high-availability parameters in VMWare ESXi . NeverthIt is also fair to indicate that, during the experiments, every fog gateway only executed the positioning service, while, as it is illustrated in It must be also noted that, during the tests, the behavior of the tags was emulated in order to evaluate the performance of the proposed architectures. In a real deployment, the results will differ due to the emulated tag behavior and because of the characteristics of the scenario . Note also that, when several hundreds of tags respond at the same time, two main issues would arise:-Real tags implement a medium-access technique that was not emulated for the tests. Therefore, in a real deployment, individual tag delays will be higher than the ones obtained for the experiments, since tags have to synchronize their transmission intervals to avoid collisions.-In a real deployment there would be interference from other devices  band) and from signals emitted asynchronously by other tags , which, in practice, will increase collisions and will decrease the sample processing rate .Finally, it is worth pointing out that the devised architecture, the technologies and the experiments discussed throughout this article were selected explicitly for a very hostile and specific industrial scenario, so the obtained results should not be generalized and each organization interested in deploying a similar system would have to adapt it to its own requirements.Since the different experiments performed are influenced by multiple factors , it is difficult to establish final conclusions, but the following are the most relevant key findings that would be preserved in similar industrial environments despite such deployment factors:Pipe traceability and tracking are essential in a shipyard due to their importance in shipbuilding. After analyzing the state of the art, this article presented the design and implementation of a CPS developed by Navantia and the University of A Coruña. The system is aimed at automating many of the tasks performed in a pipe workshop in order to accelerate production processes and increase the workshop output. Moreover, thanks to the use of a fog computing-based architecture, it is able to reduce latency, to provide location awareness and to cope with a large number of RFID tags.In addition, the proposed CPS was integrated and tested successfully with Siemens’ MES (Simatic IT). Furthermore, it was evaluated in terms of latency response and sample processing rate, showing that, in the test scenarios, fog gateways respond faster than the cloud alternative and are also able to process successfully more samples under high traffic loads and are between five and 481 times faster under regular loads.


High-risk human papillomavirus (HPV)-driven cancers represent a major health concern worldwide. Despite the constant effort to develop and promote vaccination against HPVs, there is still a high percentage of non-vaccinated population. Furthermore, secondary prevention programs are not ubiquitous worldwide and not widely followed. Metastatic disease is the cause of the great majority of cancer-associated deaths, making it essential to determine its underlying mechanisms and to identify actionable anti-metastatic targets. Within certain types of cancer , HPV-positive tumors show different dissemination patterns when compared with their HPV-negative counterparts, implicating HPV-related factors in the metastatic process. Among the many groups of biomolecules dysregulated by HPV, microRNAs have recently emerged as key regulators of carcinogenesis, able to control complex processes like cancer metastization. In this review, we present recent data on the role of microRNAs in the metastization of HPV-related cancers and on their possible clinical relevance as biomarkers of metastatic disease and/or as therapeutic targets. High-risk human papillomavirus (HPV) infection occurs at the basal cell layer of the stratified squamous epithelium  and these viruses are established carcinogens of the cervix, head and neck, anus, penis, vagina and vulva . ProphylMetastization is the process by which cancer spreads from a localized disease to a systemic disease . The metThe vast majority of tumors can promote angiogenesis and/or lymphangiogenesis, since these events are pivotal points in tumor progression ,10,12. NMicroRNAs (miRNAs/miRs) are small non-coding RNAs with approximately 22 nucleotides in length that play a pivotal role in most essential biological events . MiRNAs Importantly, the HPV oncoproteins can dysregulate the expression of several cellular miRNAs . In vivoMiRNAs that are able to promote or inhibit metastization have been termed metastamiRs . FurtherRASA1) expression, promoting migration [Cervical cancer is the fourth most common cancer among women worldwide and a major cause of cancer-related mortality ,30,31. Aigration . Rasa1 iigration . Taken tigration .SMAD2) and cyclin D1 (CCND1) and upregulation of tumor protein p53 (TP53) expression [The overexpression of miR-155 in cervical cancer tissue has also been correlated with lymph nodes metastases, as well as with International Federation of Gynecology and Obstetrics (FIGO) stage, vascular invasion and HPV status . Moreovepression . Thus, tLAMB3) which encodes a subunit of the protein Laminin-332, was identified as a direct target of miR-218 [SFMBT1) and defective in cullin neddylation 1 domain containing 1 (DCUN1D1) [SFMBT1 induced EMT, migration and invasion and DCUN1D1 also induced migration and invasion [DCUN1D1 is also a target gene of miR-195 and this miRNA is associated with reduced proliferation, migration and invasion of cervical cancer cell lines [MiR-218 is significantly reduced in cervical cancer tissue and its restoration inhibited cancer cell migration and invasion in both HPV-positive and HPV-negative cervical squamous cell carcinoma cell lines . Additio miR-218 . Other iDCUN1D1) . Overexpinvasion . Importainvasion ,41. Recell lines . Moreovell lines . Interesll lines .SP1) and consequently promotes cell proliferation, migration and invasion in vitro [GRB2) was identified as a target of miR-124 and its knockdown significantly reduced cell invasion and increased apoptosis [GRB2 and consequently to increased cell invasion [Downregulation of miR-375 in cervical cancer tissues is also correlated with pelvic lymph node metastases, FIGO stage, and other indicators of poor prognosis . Suppresin vitro . Downregin vitro ,44. Furtin vitro . Growth poptosis . Therefoinvasion .uPA) expression [uPA can also be promoted by miR-23b repression, resulting in increased migration of cervical cancer cell lines [MiR-34a and miR-23b seem to be downregulated by HPV E6 indirectly, since their expression is driven by p53 . Downregpression . Moreovell lines .PLXNB1) is upregulated in cervical cancer tissue and HeLa cells, promoting cell proliferation, migration and invasion [PLXNB1 upregulation may be caused by the downregulation of miR-214, since this miRNA was found to have a binding site within the 3’- untranslated region (UTR) of PLXNB1 [GALNT7) was also identified as a target of this miRNA [GALNT7 in cervical cancer cell lines inhibited cell proliferation, migration, and invasion [Plexin B1 (invasion . PLXNB1 f PLXNB1 . Ectopicf PLXNB1 . Anotheris miRNA . The knoinvasion .CYR61) and connective tissue growth factor (CTGF) were identified as potential targets of miR-205 and may play important roles in cervical carcinogenesis [MiR-205 expression is frequently increased in human cervical cancer, promoting cell proliferation and migration . Both cyogenesis .MST2), cell division control protein 42 homolog (CDC42) and ras homolog gene family member A (RHOA) and consequently phosphorylating Akt1 and Erk [Upregulation of miR-133b accompanies cervical carcinoma progression . In vitr and Erk . Mice tr and Erk .STAT3) [MiR-125a is downregulated in cervical cancer patients, and negatively correlated with tumor size, FIGO stage, and preoperative metastases . MoreoveSTAT3) . Thus, mSTAT3) .TIMP2) and thus induces cell morphological alterations and EMT, promoting migration and invasion [Increased expression of miR-20b is also promoted by HPV E6 . In vitrinvasion .KDM2B) [It was recently reported that the HPV E6 and E7 oncoproteins enhance the expression of c-Myc, which in turn mediates the downregulation of miR-146a-5p . In cervKDM2B) .PLK2) and promotes proliferation and invasion [PPARG) as a miR-27b target and its downregulation increases the expression of sodium-hydrogen exchanger isoform 1 (NHE1), inducing proliferation and invasion [HPV16 E7 also promotes miR-27b expression in cervical cancer cell lines by increasing DiGeorge syndrome critical region gene 8 (DGCR8), which in turn decreases the expression of miR-27b target polo like kinase 2 (invasion . Anotherinvasion .GSK3B), vascular endothelial growth factor A (VEGFA), and protein tyrosine kinase 2 (PTK2) genes, which are genes that play a crucial role in phosphatidylinositol 3-kinase (PI3K)-Akt signaling and focal adhesion [The expression of miR-106b-5p is upregulated in cervical cancer and significantly correlated with the number of metastatic lymph nodes . In siliadhesion .Besides cervical cancer, HPV infection has also been etiologically associated and implicated with the development of other anogenital cancers  ,59,60. AVulvar cancer is rare, representing about 5% to 6% off all gynecological malignancies ,63,64. GVaginal cancer is also considered a rare malignancy, accounting for about 2% of all gynecologic cancers . SeveralWith an estimated 27,000 new cases worldwide per year, anal cancer is considered relatively uncommon in the general population ,80. NeveEGFR) via miR-146a downregulation in penile cancer [Penile cancer is another rare disease ,85, occue cancer . The rese cancer ,97,98,99Scrotal squamous cell carcinoma has also been associated with HPV infection . High-riHead and neck cancers (HNC) are the fifth most common cancers in the world, with high rates of mortality . There aAs for cervical cancer, several metastamiRs have been identified in HPV-positive HNC. Specifically, a set of miRNAs, namely miR-151, miR-152, miR-324-5p, miR-361 and miR-492 is significantly associated with distant metastases in HPV-derived oropharyngeal carcinoma .MYO1B) is targeted by miR-363, and siRNA knockdown of MYO1B expression reduces migration in cell lines of HNC [MiR-363 is also upregulated in HPV16-positive HNC compared with HPV-negative cases . Myosin s of HNC . Thus, os of HNC .E2F3) and survivin [Transfection of HPV-positive or negative HNC stem cell lines with miR-34a mimics downregulates the expression of transcription factors such as Snail and Twist that regulate stem cell properties and EMT, suggesting that miR-34a may reduce cancer cell “stemness” and EMT, thereby reducing the invasions capacities of these cell lines, regardless of their HPV status . Kumar esurvivin . Additiosurvivin .The expression of miR-20a was also found to be significantly elevated in oral HNC , and thePDE2A), the gene harboring miR-139-3p [Sannigrahi et al. reported that miR-139-3p overexpression downregulates HPV16 mRNA/proteins and consequently decreases cell proliferation and migration . In factR-139-3p .Overall, dysregulated expression of miRNAs is associated with tumor cell migration, invasion and metastization in HNC, but most studies do not report HPV status, thereby limiting the conclusions that may be drawn concerning the relationship between HPV and miRNAs in this setting. Additional studies on this topic are required, given that HPV is clearly able to dysregulate the expression of several critical miRNAs and influence the prognosis of HNC patients ,111.High-risk HPVs have been found in cancers from several other locations than cervix, other anogenital and head and neck ,113. SevEsophageal cancer, in particular esophageal squamous cell carcinoma (ESCC), has long been proposed to be associated with HPV ,118,119.MTA-1) gene expression through inhibition of miR-30c-2* in NSCLC tissues. The same study correlated miR-30c-2* and MTA-1 expression with tumor stage and lymph node metastases [The involvement of HPV in lung cancer is still a matter for discussion ,116,121.tastases . High extastases .High-risk HPVs are oncogenic viruses associated with several malignancies, most prominently cervical cancer. In this review, we provide an overview of the metastamiRs found in HPV-driven cancers  and theiThere is a great lack of information about metastamiRs on less-frequent tumors, like the ones in vulva, vagina, anus and penis. Additionally, a great number of studies concerning miRNAs and metastization in cancers possibly related to HPV do not take into consideration the tumor HPV status. Further directions in this field clearly include the study of less-frequent cancers and the determination of common patterns of HPV-related miRNA dysregulation, as well as the validation of selected miRNAs for clinical applications.