The efficacy of cervical cancer screening programs is dependent on the participation rate. To increase participation among women not attending cervical cancer screening, self-collected samples for detection of high-risk human papillomavirus (hr-HPV) may be an option.The aims of this study were: to investigate the response rate to sending a self-collected vaginal sample for hr-HPV mRNA detection to long-term non-attendees; the compliance with follow-up among women positive for HPV in the self-sample; the prevalence of cervical dysplasia , atypical squamous cells that cannot exclude HSIL (ASC-H) or adenocarcinoma in situ (AIS)) or cancer among the responders; as well as to explore reasons for not returning a self-sample.A vaginal self-sampling kit was sent to 6023 women aged 30–70 years who had not provided a cervical screening sample for ≥7 years in the Region of Skåne, Sweden in November and December 2017. The self-sample was analyzed by Aptima HPV mRNA assay (Hologic). All vaginal self-samples returned no later than May 31, 2018 were included in the study. Follow-up of the results was registered until January 31, 2019 with a follow-up time varying between eight to 14 months. Women positive for hr-HPV mRNA were invited for a follow-up examination. This examination consisted of a cervical sample for cytological analysis and renewed Aptima HPV mRNA testing. Two hundred thirty-five women who had not returned the self-sample were randomly selected for telephone interviews, in order to explore their reasons.The response rate for the self-collected vaginal hr-HPV sample was 13.2%  and 9.9%  were positive for hr-HPV mRNA. The prevalence of severe dysplasia or cancer in the whole group of responders was 1.3% , with a cervical cancer prevalence of 0.4% . Only 27 women participated in the telephone interviews, no particular reason for not returning self-samples was observed.Self-collected vaginal hr-HPV samples increased participation in the cervical cancer screening among long-term non-attendees. The prevalence of cervical cancer was almost seven times higher for long-term non-attendees than in the organized screening population. The implementation of nationwide screening programs has caused a reduction in cervical cancer cases in many Western countries , 2. In SSeveral hr-HPV tests for cervical- and self-sampling are available on the market. The hr-HPV mRNA tests have shown similar sensitivity but improved specificity compared to hr-HPV DNA tests taken from the cervix , 12. ForPrevious studies have shown response rates for self-sampling among non-attendees to be between 15 and 58% –24. ReasThe first aim of this study was to investigate the response rate of a free of charge self-collected vaginal hr-HPV sample sent to women who had not attended organized cervical cancer screening for ≥7 years. The second aim was to study the compliance with follow-up among women positive for hr-HPV in the self-collected vaginal sample. The third aim was to analyze the prevalence of severe cervical dysplasia , atypical squamous cells that cannot exclude HSIL (ASC-H) or adenocarcinoma in situ (AIS)) or cancer among the responders. The fourth aim was to explore, by telephone interviews, the reasons for not returning a self-collected vaginal hr-HPV sample.Six thousand and twenty-three women in the county of Skåne, aged 30–70 years, who had not given a cervical smear for ≥7 years were identified through the southern regional cervical cancer screening registry. The registry contains information on all obtained smears, whether organized or spontaneously taken, in the region. Kits were sent out to randomly selected women who had not attended cervical cancer screening for seven years or more in the region of Skåne. The age range was chosen according to the ages that are tested with hr-HPV testing in the regular screening program in Sweden.In Sweden, all women 23–64 years old are invited to cervical cancer screening free of charge. Women aged 23–29 years old are invited every third year for primary screening with cytology. Women aged 30–49 years are invited every third year for primary screening with HPV testing and women aged 50–64 years are invited every fifth year (every seventh year in some regions in Sweden) for primary screening with HPV testing . Testing using HPV as the primary screening method has been recommended since 2015 in the Swedish national guidelines and was implemented in the region of Skåne in January 2017. As a strategy to reach the non-attending women, the national guidelines of Sweden recommend giving a telephone reminder to women with no cervical cancer screening > 3 years since the last regular screening invitation. The guidelines also recommend offering self-collected vaginal hr-HPV samples to women with no cervical cancer screening registered > 4 years since the last regular screening invitation .During November and December 2017, the identified women were offered a free of charge hr-HPV self-sampling test. The self-sampling parcel contained; 1) information about hr-HPV infection and written instructions in Swedish as well as descriptive illustrations showing how to perform the self-sampling, 2) one Aptima Multitest Swab and a tube prefilled with 2.9 ml Aptima Multitest Swab Transport Media , 3) one cylindrical container for transportation of the self-sample, 4) pre-printed labels with each woman’s social security number to mark the test, and 5) one prepaid padded return envelope. The self-sample was collected by placing a cotton swab 5 cm up into the vagina and rotating it, thereafter the cotton swab was put into the tube containing transport media. The women were asked to carefully check that the social security number was correct before affixing the pre-printed labels onto the test. The department of Laboratory Medicine, Region Skåne, Lund received the self-samples and conducted the human papillomavirus (HPV) analyses. All vaginal self-samples returned no later than May 31, 2018 were included in the study. No reminder was sent out if the kit was not returned. Follow-up of the results was registered until January 31, 2019 with a follow-up time varying between eight to 14 months. The self-samples were analyzed by Aptima HPV mRNA assay (Hologic Inc) on a Panther instrument, according to the manufacturer’s instructions. The assay detects HPV mRNA from 14 h-HPV types .Women with a negative HPV test result were informed that no hr-HPV types had been found and that no further testing was needed. This was done with an automatically generated letter from the department of Laboratory Medicine. Women with invalid test results were informed via a letter and were asked to make an appointment with a midwife to take a cervical HPV sample. Women with a positive HPV test result received a letter from the nearest midwife health station with information about the presence of hr-HPV and an invitation to attend a clinical follow-up examination with the midwife within three months. The follow-up examination included providing a cervical sample for cytological analysis and Aptima HPV mRNA testing. A reminder letter was sent if the woman did not attend her midwife appointment. One year after the self-sampling kit was sent out (November 2018), women with a positive hr-HPV test result that had still not attended their midwife appointment were reminded to do so by telephone. If the woman could not be reached by telephone a second reminder letter was sent informing the woman about the necessity for a follow-up examination. In case of abnormal test results at the follow-up examination, the women were managed according to regional guidelines . Since JTo investigate reasons for not returning a self-collected vaginal hr-HPV sample, telephone interviews were conducted in October 2018. Two hundred thirty-five women were randomly selected from the self-sample non-responders group and called on the number given to their care provider. If not reached, every woman was called a total of three times at different times of day. Women who were successfully reached were informed about the study, the voluntary participation, and that all answers were handled confidentially. If the woman agreed to participate the first question asked was “Have you received a vaginal hr-HPV self-sampling invitation?” If she had not, no further questions were asked. If the invitation had been received an open second question was asked “Why did you not perform the self-sampling?” The answers were classified into five different categories, namely: emotional/attitude, practical, physical, needless, or other.Statistical comparisons were based on the binomial distribution and 95% confidence intervals (CI) were calculated. Microsoft® Excel, Version 15.30 was used on a Mac computer for the statistical analyses.The study was approved by the Regional Ethical Review Board, Lund (DNR 2013/390). Returning the self-sample was defined as the woman’s consent to participate in the study.The response rate of the self-sample was 13.2%   stage IA1 and the other woman was diagnosed with adenocarcinoma FIGO stage IIB. Cytology of the third woman with cervical cancer showed she had HSIL, and she was diagnosed with clear cell adenosquamous carcinoma FIGO stage IIA1. According to our available registers in the Region of Skåne, none of the women diagnosed with cervical cancer had a registered cervical smear in the last 20 years and their last cervical smears had benign cytology.Among the 235 non-responding women randomly selected for telephone interview, 1.3%  were excluded due to death from unknown causes during the follow-up period. Out of the reminding 232 women, 30.6%  of the women were reached. Among these, 25.4%  were excluded due to former hysterectomy and 38.0%  agreed to participate in the telephone interview  when a self-sample was mailed to a woman’s home . In our Compliance with follow-up is essential if self-sampling is to be used. In this study, 83.5% of women with a HPV positive self-collected sample attended the midwife follow-up examination. This is similar to a recent meta-analysis by Arbyn et al. where the average participation rate for follow-up was 80.6% . CompareOur study is the first, as far as we know, using telephone interviews to explore reasons for not returning a vaginal hr-HPV self-sample among long-term non-attendees of the organized cervical cancer screening program. The participation rate of 12% in the telephone interviews was below our expectations, but the results should be interpreted with some caution because of the small sample size. Previous studies using questionnaires have reached participation rates of 3.4–38% , 20, 25.This study was performed in a setting of non-attendees of a current population-based cervical cancer screening program, which is a strength of the study. Furthermore, only one self-collected sample was invalid for analysis in the final compilation of results. Notably, during the analytical process we experienced problems with invalid samples (in one batch 25% (110/434) of the samples was invalid), but re-analysis of ¼ diluted samples (1 ml sample were transferred to 2.9 ml) rendered such samples as valid. However, for future studies of self-collected samples analyzed for HPV mRNA by the Aptima-system we will instead add a pre-heating step of samples, in order to reduce the proportion of invalid samples . One limIn conclusion, offering self-collected vaginal samples for hr-HPV mRNA testing was a promising strategy to increase participation in the cervical cancer screening among long-term non-attendees. The compliance with follow-up was 83.5%, which is a good level, comparable to other studies. The prevalence of cervical cancer was almost seven times higher among long-term non-attendees than in the organized screening population. No particular reason for not returning self-samples was noted from the conducted telephone interviews.


The aim of this study is to assess the projected incidence and prognostic indicators of gynecologic malignancies in the pediatric population. In this population-based retrospective cohort study, girls ≤18 years with ovarian, uterine, cervical, vaginal and vulvar malignancies diagnosed between 2000 and 2016 were identified from the Surveillance, Epidemiology and End Results (SEER)-18 registry. The Kaplan–Meier method was used to analyze overall survival (OS). The age-adjusted annual incidence of gynecologic malignancies was 6.7 per 1,000,000 females, with neoplasms of the ovary accounting for 87.5%, vagina 4.5%, cervix 3.9%, uterus 2.5% and vulva 1.6% of all gynecologic malignancies. Malignant germ-cell tumors represented the most common ovarian neoplasm, with an increased incidence in children from 5–18 years. Although certain subtypes were associated with advanced disease stages, the 10-year OS rate was 96.0%. Sarcomas accounted for the majority of vaginal, cervical, uterine and vulvar malignancies. The majority of vaginal neoplasms were observed in girls between 0–4 years, and the 10-year OS rate was 86.1%. Overall, gynecologic malignancies accounted for 4.2% of all malignancies in girls aged 0–18 years and the histologic subtypes and prognosis differed significantly from patients in older age groups. Malignant neoplasms are the fifth most common cause of death among children and adolescents aged 0–18 years and the third most common cause when excluding infants <1 year . GynecolThe aim of this study was to systematically describe the epidemiology, histologic types and prognosis of gynecologic malignancies in this age group.The Surveillance, Epidemiology and End Results (SEER) database of the National Cancer Institute was used to identify cases of gynecologic malignancies in children and adolescents. The SEER-18 population  from its November 2018 submission was used. This was limited to cases from 2000–2016 [Patients aged 0–18 years with a diagnosis of invasive cancer of the ovaries, uterus, cervix, vagina and vulva were identified in the SEER*Stat 8.3.6 database, and their clinical data was retrieved. In situ malignancies were excluded. Only patients with known age and listing in the research database were included. Patients’ age at diagnosis, year of diagnosis, ethnicity, site, histology, stage  and survival data were collected. Histologic subtypes were grouped into squamous cell carcinoma, adenocarcinoma, melanoma, sarcoma, germ-cell tumors and other subtypes/unspecified for vulvar, vaginal, cervical and uterine malignancies. Ovarian neoplasms were classified as epithelial, sex-cord-stromal germ-cell or mesenchymal tumors.Incidence rates were adjusted for age to the 2000 United States standard population and reported per 1,000,000 women 18 years or younger. Patients with secondary gynecologic cancers were excluded from the survival analysis. The SEER data is publicly available and deidentified and therefore excluded from Ethics Board approval.p-value ≤ 0.05 was considered statistically significant.Descriptive statistics were used to report demographic data. Continuous variables were compared using the Mann–Whitney-test or Wilcoxon test, as appropriate. More than two groups were compared using the Kruskal–Wallis ANOVA test. Cross-tables and Chi-square test were used to compare categorical data. The Kaplan–Meier method with log-rank test was used to analyze overall survival. Overall survival was calculated from the date of diagnosis to the date of death. Statistical analysis was performed using SPSS Version 26, IBM, Armonk, USA. A Gynecologic malignancies accounted for 4.2%  of all malignancies in girls ≤ 18 years and 0.3%  of all gynecologic malignancies in the SEER-18 population occurred in this age group: 1.1% of all ovarian, 1.0% of all vaginal, 0.1% of all cervical, 0.1% of all vulvar and <0.1% of all uterine malignancies were diagnosed in girls ≤18 years.The age-adjusted incidence of gynecologic malignancies and the relative frequency by organ are shown in Histologic subtypes identified were germ-cell tumors (71.6%), epithelial carcinomas (12.4%), sarcomas (7.9%), sex-cord stromal tumors (6.1%) and other/unspecified subtypes (2.1%). The majority of ovarian neoplasms were germ-cell tumors, whereas sarcomas represented the most common histologic type in uterine, cervical, vaginal and vulvar malignancies. The distribution of histologic subtypes is shown in Ovarian cancer accounts for 87.5% of gynecologic malignancies in children and adolescents ≤18 years, vaginal cancer 4.5%, cervical cancer 3.9%, uterine cancer 2.5% and vulvar cancer 1.6%. Abbreviations: n.s., not specified.p < 0.001), and girls with ovarian cancer were significantly younger than those with uterine malignancies (p = 0.011). Age at diagnosis differed between histologic groups (p < 0.001). Girls with sarcomas were significantly younger than all other histologic types (p < 0.001). Girls with germ-cell tumors were significantly younger than girls with epithelial (p < 0.001), sex-cord-stromal tumors (p = 0.026) and other/non-specified histologic types (p = 0.019). The annual incidence of gynecologic cancers by age is shown in p = 0.041) and among ovarian neoplasms  affect children between 0 and 4 years of age A. SarcomGerm-cell  tumors represent the second most common vaginal histology and have an excellent prognosis. A 10-year overall survival rate of 100% was observed in this cohort. This finding was similar to a recent single-center case series of 16 patients, where all patients, other than one who experienced a treatment-related death, had a complete response without recurrence at a median follow-up of 53 months  and is similar to the outcome in malignant germ cell tumors of the ovary, where the reported mortality rate was 1.9% over a period of 20 years ,28.In this study, sarcomas were also found to be the most common histologic subtype of cervical neoplasms. The majority of these were embryonal rhabdomyosarcomas with a similar prognosis D and treThe majority of vulvar malignancies were sarcomas, which had a very similar prognosis to vaginal and cervical sarcomas E,F and wUterine malignancies are rare in childhood compared to the general population, where they represent the most common gynecologic cancer . We obseThis study included a large series of gynecologic malignancies in children and adolescents using the SEER-18 population and is representative of the North American population. The data are population-based and therefore not limited to single-center experiences, avoiding publication bias. The study is, however, limited by its retrospective design and the use of registry data, which preclude a central pathology review. In addition, the medical treatment approaches used in this population are not reported here. The scope of this study was to investigate the epidemiology, characteristics and real-world prognosis, and is reflected in our results.Although gynecologic malignancies in children and adolescents are considered rare, they account for 4.2% of all malignancies in girls aged 0–18 years. Approximately 1% of all ovarian and vaginal cancers encountered in the general population affect this age group. The histologic subtypes of the reported pediatric malignancies differ from the commonly reported gynecologic cancers in the adult population. Treatment decisions should be based on a multi-disciplinary evaluation including pediatric and gynecologic oncologists. With regards to ovarian cancers, germ cell tumors represent the most common ovarian neoplasm. In vulvo-vaginal, uterine and cervical malignancies, sarcomas represent the most common subtype compared to squamous and adenocarcinomas in the adult populations. Overall survival in this study was found to be excellent; therefore, counseling for fertility preserving therapies is encouraged.


TP53 as the most mutated gene in locally advanced HNSCC. HPV-positive oropharyngeal tumours were less mutated than HPV-negative tumours in TP53 (p < 0.01). Mutational and HPV status influences patient survival, being mutated or HPV-negative tumours associated with poor overall survival (p < 0.05). No association was found between mutations and clinicopathological features. This study confirmed and expanded previously published genomic characterization data in HNSCC. Survival analysis showed that non-mutated HNSCC tumours associated with better prognosis and lack of mutations can be identified as an important biomarker in HNSCC. Frequent alterations in PI3K pathway in HPV-positive HNSCC could define a promising pathway for pharmacological intervention in this group of tumours.234 diagnostic formalin-fixed paraffin-embedded (FFPE) blocks from homogeneously treated patients with locally advanced head and neck squamous cell carcinoma (HNSCC) within a multicentre phase III clinical trial were characterised. The mutational spectrum was examined by next generation sequencing in the 26 most frequent oncogenic drivers in cancer and correlated with treatment response and survival. Human papillomavirus (HPV) status was measured by p16INK4a immunohistochemistry in oropharyngeal tumours. Clinicopathological features and response to treatment were measured and compared with the sequencing results. The results indicated  It constitutes a heterogeneous disease of tumours of the upper aerodigestive tract with different pathogenic origins and clinical prognosis. Tobacco smoking and alcohol consumption are still the most classical risk factors2 followed by viral infection4.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common neoplasia in the developed world5. With the aim of improving the clinical benefit, the addition of cetuximab, an IgG1 chimeric monoclonal antibody against epidermal growth factor receptor (EGFR), concomitant with RT was explored, resulting in longer progression-free survival (PFS) and overall survival (OS) compared to RT alone, although a direct comparison with CTRT has not been evaluated yet6.Most HNSCC are diagnosed as locally advanced disease (stage III or IV) and therefore multidisciplinary treatment strategies include surgery, radiotherapy (RT), chemotherapy (CT) and targeted therapy. However, treatment with chemoradiotherapy (CTRT) has become the standard of care after the publication of a large pool analysis9. Induction chemotherapy to improve organ preservation and survival may be an alternative to CTRT. The addition of cetuximab to radiation therapy in patients with laryngeal cancer stage III and IVA that respond to TPF could improve functional laryngeal preservation10, although randomized phase III trials did not find that induction chemotherapy provided benefit in time-to-treatment failure or OS14. On the other hand, a randomized phase II–III study suggested that adding TPF induction chemotherapy to CTRT resulted in a higher rate of radiological complete response compared with concurrent CTRT alone, improving PFS and OS by induction TPF15. The fact that patient populations in these trials were very heterogeneous, questions induction chemotherapy’s benefit thus, subgroups that will have a benefit from it need to be identified.The role of induction chemotherapy has remained a subject of controversy. The combination of docetaxel-cisplatin and 5-fluorouracil (TPF) has emerged as the most active regimen in locally advanced disease, showing better results than PF, although it did not show a convincing survival benefit in induction regimens compared with historical data of treatment with concomitant chemoradiotherapy alone17. For instance, the analysis of The Cancer Genome Atlas (TCGA) described the molecular landscape of HPV-positive and HPV-negative HNSCC as having molecular alterations not reported before18. Since the first description of the recurrently mutated genes in HNSCC19, additional studies have included other genes such as TP53, NOTCH1, PIK3CA, CDKN2A, CCDN1, HRAS, FAT1, FBXW7 and FGFR3, among others21. For this reason, targeted sequencing has become a flexible tool to study those genes previously reported as mutated in HNSCC21.Next-generation sequencing (NGS) has helped to identify genetic alterations that could be used as a molecular vulnerability for therapeutic discovery and target optimization. In addition, they could have a prognosis utility as biomarkers of response in different tumour types including head and neck squamous cell carcinomasTo contribute to the understanding of how somatic mutations influence the outcome of HNSCC treatment, we have studied a panel of 26 genes Table  by next-234 FFPE blocks with diagnostic biopsies from HNSCC patients within a multicentre phase III clinical trial were incorporated in this study Fig. . ClinicaTP53 wild-type (p = 0.009) tumours  followed by PIK3CA (10.3%), FBXW7 (1.7%), PTEN (1.3%) and CKIT and CTNNB1 (both with 0.43%) , followed by stop-gained (18.04%), frameshift (14.95%), splice-donor (8.76%) and in-frame deletions (2.58%) (Table Targeted panel sequencing in HNSCC FFPE blocks identified 162 samples (69.23%) with previously described pathogenic mutations whereas 46 (19.66%) did not carry any mutation and 26 (11.11%) showed variants of uncertain clinical significance (VUS). 194 pathogenic mutations and 72 VUS were found in the sequencing of the 234 FFPE blocks. All samples were sequenced > 5000 × . Globally, the most mutated gene was %) Table .Figure 1General comparison of the mutational status and tumour characteristics such as location, grade and histology, did not show any significant difference (p > 0.05) Table . HoweverTP53 was the most frequently mutated gene in both groups, these mutations were more recurrent in HPV-negative tumours (71.4% in HPV-negative and 30.8% in HPV-positive), difference statistically significant (p = 0.009). Conversely, the second most mutated gene, PIK3CA, although more represented in HPV-positive tumours (9.5% in HPV-negative versus 23.1% in HPV-positive), did not show any statistically significant difference (p = 0.178). While HPV-negative tumours did not present pathogenic mutations in other genes, PTEN was the third most commonly mutated in HPV-positive tumours (15.4%), followed by FBXW7 (7.7%).HPV mutational profile in oropharyngeal tumours is shown in Fig. 22. For that reason, both arms were evaluated within the same group as final response (or response after randomization). Evaluation of the two time-point responses according to the mutational profile did not show any statistical difference  patients were similarly randomized: 95 (50.53%) to conventional treatment and 93 (49.47%) to the experimental arm. Preliminary data indicated that the two regimens showed similar survival, response rates, toxicity and locoregional control% patientce Table .Table 4AConsidering only HPV profile in oropharyngeal tumours, there were no differences between HPV-positive and HPV-negative individuals either after induction chemotherapy (p = 0.396) or randomization (p = 0.914) Table .Table 5ATP53 and PIK3CA . By contrast, considering only PIK3CA mutations, a statistically positive association was found in the complete response group after induction chemotherapy (p = 0.024). However, this finding was not corroborated in final response group (p = 0.235)  Table  what cou23 and Neskey’s24 classification in patients harbouring TP53 mutations in relation with clinical response before and after randomization did not show any statistically significant association . This tendency was also shown in PFS, however, without statistically significant results ) Fig. A,B.FigurMoreover, OS was correlated with the mutational status. Patients without mutations in the selected genes had a better OS than patients with mutated tumours )  ) than non-mutated patients, with the exception of PFS ) . We observed a statistically significant lower percentage of mutated TP53 in HPV-positive oropharyngeal tumours (71.4%) than in HPV-negative (30.8%) as has been previously reported in HNSCC29. These results could be explained if TP53 sequestration by the viral oncoprotein E6 prevents gaining mutations in this gene under selective pressure of31. Comparing to other series, there was a higher percentage of TP53 mutations in HPV-positive tumours29. This fact could be explained by the coexistence of viral infection and other aetiological factors such as tobacco smoking and alcohol consumption during tumourigenesis32; these data were not collected in this study. TCGA data described 85% of TP53 mutation in HPV-negative tumours and only 3% in HPV-positive ones18. However, the sample population was very different with a high predominance of oral cavity tumours (62%) and mainly heavy smokers.Globally, the most mutated gene in our series was PI3K/AKT/mTOR has been reported as the most mutated pathway in HNSCC (13% to 56%), regardless of the HPV status18. PIK3CA gene, that encodes the catalytic subunit of the family, has been reported with an average mutational rate of 10.53% in HNSCC33, similar to the 10.25% found in this cohort, and with a higher frequency in laryngeal tumours34. Mutations in this gene have also been related to HPV-positive tumours4. Our results corroborate this fact, being PIK3CA more frequently mutated in HPV-positive tumours , similar to previously described data35. We did not, however, see an increased percentage in laryngeal carcinoma. 73% of the mutations in PIK3CA are commonly located in 3 hotspots 36, result also found in 76% of PIK3CA mutated samples in our study, emphasising the accuracy of using the targeted panel in HNSCC.FBXW7: an E3 ubiquitin ligase member of the F-box protein family, have been previously observed in HNSCC19. This tumour suppressor gene targets NOTCH1, being an important protein in cell proliferation control. Previous studies found FBXW7 mutated in 5% of HNSCC38 and a higher percentage of mutations was previously considered as a prevalent event in HPV-positive tumours39. Our cohort confirmed these results in FBXW7 with a similar percentage only found in HPV-positive tumours (7.7%).Mutations in PTEN was the third most mutated gene in 15.4% of the HPV-positive oropharyngeal tumours while not mutations were found in HPV-negative ones. Contrary to our results, TCGA study showed PTEN mutated in 12% of HPV-negative tumours and 6% of HPV-positive18. Apart from PTEN, there were other genes which mutated at a lower percentage in our series, such as CKIT or CTNNB1 , have been reported in HNSCC in varied percentages40. Together with PIK3CA, our result enhances the hypothesis of higher prevalence of PI3K pathway activated mutations in HPV-positive tumours41.TP53 mutations, recurrent alterations in PIK3CA, PTEN and FBXW7 genes, all belonging to the PI3K/AKT/mTOR pathway, could define a potential new target for pharmacological intervention in HNSCC, as it has been suggested in other publications42.Overall, excluding 47. Secondly, the presence of mutation in the targeted genes was associated with inferior outcome demonstrated by the presence of detrimental OS. These results could be an indirect measure of tumour aggressiveness, as has been reported in other series47. Moreover, the fact that carriers of tumours with more than one mutation have lower OS than those with non-mutated tumours reinforces this concept.In terms of survival, HPV-positive oropharyngeal tumours were associated with better prognosis, showing an increased OS and PFS compared to HPV-negative tumours as it was previously definedTP53 mutations stratified by Poeta’s23 and Neskey’s models24 and response to treatment or survival. These classification systems can serve as an important tool in individualizing and improving treatment for high TP53 mutated tumours, as it was previously identified in a subset of high-risk patients with a decreased response to platinum-based therapies48. Nevertheless, these classification models did not have any implication on outcome in our cohort.Lastly, there was a lack of association between mutational status and response after treatment. This can indicate that, excluding genetic-driven druggable targets, HNSCC mutational profile is not related to any clinical response but is a matter of mutational burden as is shown in the survival analyses. Similarly, there was no association between TP53 mutations, frequent alterations in PIK3CA, PTEN and FBXW7 genes, define possible pathways for pharmacological intervention. Finally, survival analysis showed that mutational status in the tumour could define patient prognosis, and may potentially be used as biomarkers to stratify patients for more intensive treatment. However, larger studies should be performed to confirm these results aiming at stratifying patients to different therapeutic interventions.Overall, our data strongly support and expand previously published studies exploring the presence and prognosis of mutations in this population. We have characterized the mutational profile of HPV-positive/HPV-negative oropharyngeal HNSCC in a representative cohort of patients. In this context apart from 22.234 FFPE blocks with diagnostic biopsies from HNSCC patients were included in this study. A consort diagram reporting the dropout is shown in Fig. It was a non-inferiority, randomized and controlled study with a parallel assignment intervention model and an endpoint of safety/efficacy, carried out between 2008 and 2013. The follow-up of the clinical trial finished in November 2016. According to protocol, written informed consent was obtained from living subjects and the protocol was approved by the University Hospital of Salamanca and the ethical committees of each hospital in accordance with the 1964 Helsinki declaration and its later amendments.2 d1, P 75 mg/m2 d1, F 750 mg/m2 CI d 1–5 q 21 d + G-CSF & ciprofloxacin, by 3 cycles; then, if objective response achieved, they were randomized to: conventional radiotherapy (RT) up to 70 Gy + P 100 mg/m2 d 1–22–43 vs conventional RT up to 70 Gy + cetuximab 400/250 mg/m2 weekly until the completion of RT, and they were stratified by primary tumour site. Surgery after RT (neck dissection) was allowed. The primary endpoint was non-inferiority of cetuximab-radiotherapy versus cisplatin-radiotherapy in terms of overall survival. Response rate, loco-regional control and toxicity in both arms were considered secondary objectives. Preliminary data of this trial did not show any difference in terms of survival or response rates, toxicity and loco-regional control as secondary end points in the two regimens22.Eligible patients: histologically or cytologically confirmed, previously untreated unresectable locally advanced (Stage III–IV) tumours , ECOG performance status 0–1. Unresectable disease was determined by Northern California Oncology Group in measurable disease. Treatment: docetaxel, cisplatin, 5-fluorouracil (TPF)-based induction chemotherapy  and DNA extraction was done using QIAamp DNA FFPE Tissue kit .Following TruSight Tumor 26 Reference Guide , DNA quality was measured by qPCR. Comparing FFPE-gDNA amplification potential with a reference non-FFPE gDNA (QCT), delta Cq value was used to predict the dilution required for each sample.TruSight Tumor 26 panel includes a set of 174 amplicons in complete exons of 26 cancer-associated genes Table . This pa49, and they were defined as likely pathogenic in the Catalogue Of Somatic Mutations in Cancer  or the National Center for Biotechnology Information  databases.Data were transformed in BaseSpace platform and the VCF file format were read in the Variant Studio Software . Following Illumina recommendations, somatic variants over 5% of frequency, with yields at least 1000 × cumulative coverage between the 2 strands and considered from the software of PASS filter were reported. Those variants of uncertain significance were considered pathogenic if at least two in silico prediction tools (SIFT and PolyPhen) classified them as deleterious/probably damaging50 as the gold-standard technique. FFPE sections were deparaffinized and exposed to 10 mM citrate buffer antigen retrieval at 92 °C for 30 min and then they were stained using a p16INK4a mouse monoclonal antibody . Percentage of p16 staining was measured and only those tumours > 70% nuclear and cytoplasmic p16+ were considered positive. 33 samples were considered HPV-positive following this methodology: 13 oropharyngeal, 4 hypopharyngeal, 2 laryngeal and 9 oral cavity tumours. However, after the publication of the guidelines from the college of American pathologists, p16 IHC is only recommended in oropharyngeal tumours but other locations, where DNA/RNA viral determination should be performed as a confirmatory test51. Since there was not more DNA from all the samples after the library preparation, only oropharyngeal tumours with > 70% p16 positive staining were considered HPV-positive.In the original study protocol, the assessment of HPV status was carried out by p16 immunohistochemistry (IHC), a surrogate marker for HPV infectionTP53 and PIK3CA (mutant or wild-type). Response was divided in two groups of treatment: after induction chemotherapy and after chemo/cetuximab plus radiotherapy  due to the similar outcome in both arms22. Response was classified in both groups as complete response versus partial response/stabilization. No progressions were shown in the cohort.Statistical analysis compared categorical parameters and mutational status by the Chi-square or Fisher’s exact tests; while in continuous nonparametric variables, the Mann–Whitney U or Kruskal–Wallis H tests were used. p-values were calculated excluding missing values and they were considered statistically significant when p < 0.05. Significant variables were included in the logistic regression analysis and size effects were indicated by odds ratio (OR) with their 95% confidence interval (95% CI). Mutational status was classified as presence or absence of mutations, number of mutations  and the status of Survival analysis was done according to the overall survival (OS) and progression-free survival (PFS) by Kaplan–Meier plots and log-rank test p-values were calculated in all the curves. Median was indicated in those plots in which it was achieved. Hazard-ratio was calculated to measure the risk of the event with its 95% confidence interval (95% CI) by Cox regression. Median follow-up in OS was 32.23 months while in PFS it was 15.31 months.TP53 mutations, we applied Poeta’s and Neskey’s classifications stratifying the mutations according to its change and functional effect, allowing a better comprehensive understanding on their relevance in clinical outcome. Following Poeta’s classification23, TP53 mutations were divided in two categories: disruptive and non-disruptive according to their functional effects on the p53 protein. Additionally, according to Neskey’s model24, also named as Evolutionary Action score of TP53-coding variants (EAp53), missense mutations were stratified into high-risk and low-risk through an in-silico scoring (https://mammoth.bcm.tmc.edu/EAp53/). Then, comparative analysis was performed in response to treatment, OS and PFS.Due to high prevalence in All these tests were conducted using SPSS software version 21.0  and GraphPad Prism software version 6.0 .Supplementary Information 1.Supplementary Information 2.


To investigate high-risk HPV (hr-HPV) genotype distributions and the association between hr-HPV infection with severity of the cervical lesions in women with normal cytology.In this cross-sectional study, the result of the hr-HPV test and biopsy of colposcopy of women with normal cytology from January 2012 to January 2019 were analyzed. The detection rate of high-grade squamous intraepithelial lesion (HSIL) and cervical cancer were calculated among different hr-HPV genotypes, viral load group, and age groups.P = 0.638), contrary to HPV18. In women without HPV16/18 infections, HSIL+ detection rates for single, double, and triple or more hr-HPV infections were 12.28, 20.31, and 37.50%, the risk of detection of HSIL+ significantly increasing. With the hr-HPV DNA load increases, the risk of detection of HSIL+  and invasive cervical cancer  increase. In age < 30, 31–40, 41–50, 51–60, > 60 group, HSIL+ detection rate were 24.80%、22.10%、19.59%、14.29, and 12.61%, respectively.Five thousand eight hundred eighty women were enrolled in this study. Overall, 59.97% had normal histological results, 19.32% had HSIL, and 1.07% had cervical cancer. The detection rate of HSIL or worse (HSIL+) in women with single HPV16(34.00%), HPV31(27.50%), HPV33(25.58%), and HPV52(20.88%) infection were higher significantly than single HPV18 (15.59%) infection, respectively. The HSIL+ detection rate between HPV16 single infection and multiple infections (excluding HPV18) was no significant difference (34% vs 35.47%, Women who have normal cytology with HPV 16/18/31/33/52/58 infections, multiple HPV infections and high viral load, have a higher detection rate of HSIL+. High-risk HPV (hr-HPV) infection is the main etiological factor for the development of cervical neoplasia , and rouTherefore, this study was designed to retrospectively analyze data for up to 7 years, to find the correlation between the severity of cervical lesions and hr-HPV genotypes, viral loads, and ages in HPV-positive/cytology-negative females in China.This study was cross-sectional investigation based on data from women who accepted Thinprep cytologic test (TCT) test, HPV test, and colposcopy at Qilu Hospital of Shandong University from January 2012 to January 2019, after approval of the Qilu Hospital of Shandong University【2018(054)】. Clinical information of women who met the inclusion criteria was collected retrospectively.Inclusion criteria: (1) women with normal cytology and hr-HPV infection(s); (2) women accepted colposcopic examination and underwent cervical biopsy under colposcopic guidance.Exclusion criteria: (1) women with history of treatments to cervical lesions, such as cervical surgery and medicine, et al.; (2) women with incomplete cervical cervix; (3) women with malignant tumors; (4) women with autoimmune diseases or receiving immunotherapy; (5) women with pregnancy; (6) women who had received HPV vaccine.Specimen collection, specimen preparations, and results of the tests are performed, according to the instructions of the manufacturers, respectively.Hybrid Capture 2(HC2) High-Risk HPV DNA Test : semi-quantitative detection of 13 h-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Samples were considered as positive if the ratio relative-light-unit (RLU)/cut-off(CO) was > 1.0  as described in package insert.the Cobas 4800 System : Qualitative detection of HPV 16, HPV 18 and 12 h-HPV .the HPV GenoArray test : qualitative detection of 15 kinds of hr-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68; 6 kinds of low risk HPV (lr- HPV) types: 6, 11, 42, 43, 44 and 81(CP8304).One of those HPV tests could be chosen:Specimen collection, specimen preparations, and results of the tests are performed, according to the instructions of the manufacturers, respectively. ThinPrep preparations was used, and the cytological diagnosis was performed by experienced cytopathologists, according to the Bethesda system TBS 2001.All women, enrolled in this study, underwent colposcopy and guided cervical biopsies according to the American Society for Colposcopy and Cervical Pathology Colposcopy Standards recommendations . The hisp-values < 0.05.Statistical analyses were performed using the SAS 9.4  for windows. The categorical variables were expressed by a percentage (%). The binary Logistic regression model was used to analyze the detection rate of HSIL+ and cervical cancer in different hr-HPV genotype groups. The relationships between the HPV viral load and the detection rate of HSIL+ were analyzed by the Cochran-Armitage Trend test. Results were considered statistically significant at A total of 5880 women with normal cytology and hr-HPV infection accepted colposcopic biopsy were enrolled in this study. 4332 women underwent HPV genotyping test and 1548 women were hr-HPV DNA positive by HC2 hr-HPV DNA test , followed by HPV3127.50%), HPV33(25.58%), HPV52(20.77%), HPV35(17.39%), HPV58(16.18%), HPV18(12.59%), HPV51(6.25%), HPV66(4.88%), HPV45(4.76%), HPV39(4.62%), HPV56(4.69%), HPV68(4.17%), HPV59(3.03%), HPV53(1.28%). Compared with single HPV18 infection, the detection rate of HSIL+ was higher in women with single HPV16/31/33/52 infection, with OR values of 3.576(95% CI:2.442–5.238), 2.633(95% CI:1.205–5.753), 2.386(95% CI:1.101–5.172), 1.819(95% CI:1.096–3.02), respectively. There was no significant difference in the detection rates of HSIL+ between single HPV35/39/45/51/56/58/59/66/68 and single HPV18 infection. The detection rate of HSIL+ with single HPV53 infection was lower than that in HPV18, while OR value was 0.090 (95% CI: 0.012–0.670). , not same as HPV18 (P = 0.022).There was no significant difference in the detection rate of HSIL+ between single infection of HPV16 and multiple infections with HPV16 (excluding HPV18) (P = 0.019). (Table In hr-HPV-positive/cytology-negative women without HPV16/18 infection(s), the HSIL+ detection rates in single hr-HPV infection, double infections and triple or more infections were 12.28, 20.31 and 37.50%, respectively. Compared with the detection rate of HSIL+ in single hr-HPV infection (excluding HPV16/18), OR of the detection rate of HSIL+ in double and triple or more HPV infection was 1.821(95% CI:1.212–2.738) and 4.287(95% CI:2.188–8.400), respectively. Moreover, there was a significant difference between double and triple or more HPV infections (excluding HPV16/18) (n = 516), medium viral load group , and high viral load group . The HSIL+ detection rates were 5.43, 17.33 and 27.15% in 3 groups, respectively; with the increase of viral load, the higher the risk was. The detection rates of invasive cancer in 3 groups were 0.19, 1.33 and 1.72%, respectively. As the same, viral load is proportional to the risk. was lowest , which means that more hr-HPV genotypes infected in women, the higher the HSIL detection rate.Multivariate analysis showed that the risk of cervical cancer was 19.9 times higher in women with single HPV infection, and 31.8 times higher in those with multiple HPV infection . This suP<0.0001), same as the detection rate of cervical cancer , which may be correlated to the fact that patients with low-load HPV infection are more likely to be eliminated, while those with high-load HPV infection are more difficult to eliminate [It is controversial whether the viral hr-DNA load can be used as a reliable marker to predict cervical lesions. Previous studies have reported that HPV viral load has been associated with persistence of HPV infection and subsequent cervical precancerous lesions , 36, 37.liminate .This study found that the absolute value of HSIL+ detection rate decreased with age, while the absolute value of cancer detection rate increased with age, in women with normal cytology and hr-HPV infection. The time of hr-HPV infection is short, and cervical cell morphology has not changed in young women, what’s more, the proportion of CIN2 that can regress is higher in young women . HoweverIn conclusion, about one in five hr-HPV-positive/cytology-negative women are diagnosed with HSIL+. Women with HPV 16/18/31/33/52/58 infections, multiple HPV infections whether combined with HPV16/18 infections, as well as high viral load, have a higher detection rate of HSIL+, although they have normal cytology.This study was a cross-sectional study without follow-up and it could not estimate the cumulative risk of cervical lesions within a period. The population enrolled in this study is the patients who underwent colposcopy in Qilu Hospital, Shandong University. So, the results of this study can not be directly applied to the general screening population, but it has certain reference significance for the diagnosis and treatment of patients coming to the hospital. Besides, we can not ignore the heterogeneity of 3 HPV assays and specifically analyze women infected with 12 h-HPV.


HPV causes specific cell-mediated immunity in the cervix. Mononuclear cells such as helper T cells (CD4+), cytotoxic T cells (CD8+), and dendritic cells play a critical role in the initiation of the HPV-specific immune response and destruction of virus-infected cervical epithelial cells. The programmed cell death ligand 1 (PD-L1) gene encodes an immune inhibitory receptor ligand and overexpression of PD-L1 inhibits T-cell activation and cytokine production. The aim of this study was to investigate the expression of PD-L1 in cervical tissue and its correlation with clinicopathological findings.n = 29), HR-HPV (−) CIN 0; Group II (n = 21), HR-HPV (+) CIN 0; Group III (n = 20), HR-HPV (+) CIN I; and Group IV (n = 24), HR-HPV (+) CIN II-III. A semi-quantitative scoring system was used to evaluate the degree of Ki-67, p16, and PD-L1 immunoreactivity in the cervical tissue samples.In this cross-sectional study, a total of 94 women who were referred for colposcopy due to abnormal Papanicolaou (PAP) test results and/or HPV positivity were evaluated. The presence of HR-HPV–DNA was analyzed using type- and gene-specific primers along with commercial real-time polymerase chain reaction. The cervical examination was done with a colposcope. Cervical biopsies were obtained from the areas that were evaluated as abnormal during the colposcopy. Histopathological result of cervical biopsies were defined as no intraepithelial neoplasia (CIN 0), mild CIN (CIN I), and moderate-to-high CIN (CIN II-III). All women were classified into four groups based on their HR-HPV positivity and cervical biopsy results: Group I  and mononuclear PD-L1 expression was correlated with HPV type, initial Pap test results, HPV persistence, and CIN persistence or recurrence . Moreover, mononuclear PD-L1 expression was also correlated with Ki-67 and p16 immunoreactivity . Epithelial PD-L1 expression was only correlated with HPV type and the presence of HPV persistence .We found that PD-L1 expression in both mononuclear cells and in cervical epithelial cells gradually increases from the HR-HPV (−), CIN 0 group to the HR-HPV (+), CIN II-III group (During the initial evaluation of the cervical histology results, the assessment of PD-L1 expression—especially in mononuclear cells in cervical tissue samples—may provide more information on the progression of HR-HPV infection and its persistence. Cervical cancer is one of the most common gynecological malignancies affecting women worldwide. The incidence and mortality rates vary depending on the use of effective screening strategy, sexual behavior, race, and socioeconomic status, and are approximately 7.8 and 2.3 per 100,000, respectively in the United States [HPV infection in the cervix causes HPV-specific cell-mediated immunity that plays an important role in the clearance or persistence of HPV infection , 5. DuriIt is also known that HPV is capable of immune evasion (immune escape) that prevents a robust immune response from the infected cells . HPV-medThe Programmed cell death ligand 1 (PD-L1 or CD274) gene encodes an immune inhibitory receptor ligand that is expressed by hematopoietic and non-hematopoietic cells, such as T cells, B cells, and various types of tumor cells . The intTherefore, we aimed to investigate the roles of PD-L1 expression in the pathogenesis of HR-HPV–related CIN. Thus, we examined the expression of PD-L1 on the mononuclear cells and cervical epithelial cells of cervical tissue samples from HR-HPV (−) CIN 0 and HR-HPV positive (+) women with different CIN grades and the correlation with clinicopathological findings.The investigation protocol of this study was in accordance with the Helsinki Committee requirements. All participant data were obtained from Balikesir University, School of Medicine, Department of Gynecology and Obstetrics between January 2015 and April 2020. Ethical approval was given by the institutional Ethical Committee of Balikesir University, School of Medicine (2020/64). Written informed consent was obtained from all participants.n = 29), HR-HPV (−) CIN 0; Group II (n = 21), HR-HPV (+) CIN 0; Group III (n = 20), HR-HPV (+) CIN I; and Group IV (n = 24), HR-HPV (+) CIN II-III  test results and/or HPV positivity in the co-test were included in the study population. All participants had an abnormal Papanicolaou (Pap) test results that included atypical squamous cells of undetermined significance (ASCUS), low-grade cervical intraepithelial lesion (LSIL), high-grade cervical intraepithelial lesion (HSIL), atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASC-H), and/or HPV infection. All women were examined by colposcopy at the next visit. Cervical biopsies were obtained during the colposcopic examinations. Cervical biopsy results were defined as no intraepithelial neoplasia (CIN 0), mild CIN (CIN I), and moderate-to-high CIN (CIN II-III) based on a consensus review by two experienced pathologists. All women were classified into four groups based on their HR-HPV and cervical biopsy results: Group I  was used. HPV-DNA was extracted from cervical samples using a DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions. Detection of HR-HPV–DNA was performed using type- and gene-specific primers along with commercial real-time polymerase chain reaction . The presence of the HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66, was analyzed by polymerase chain reaction (PCR) test as described elsewhere .Cervical cytological samples were taken with a cyto-brush and a conventional Pap test was used for the detection of cervical cytological abnormalities. Cytological diagnoses were obtained using the 2001 Bethesda System . For cytDuring the colposcopy, cervical samples were taken from the areas that were evaluated as abnormal with punch biopsy forceps. All cervical specimens were fixed with formalin and embedded in paraffin. Sections that were 4-μm thick were cut and stained with hematoxylin and eosin. For the histological examination, we evaluated the presence of cell dysplasia, cell irregularity, cell hyperchromatism, and increased nuclear-cytoplasmic ratio, loss of cell polarization, mitotic figure, koilocytosis, and infiltration of the immune cells.All tissue preparation and immunohistochemistry (IHC) procedures were performed as previously described . IHC anaIn all patients, the cervical tissue samples showed only moderate immunoreactivity for p16 and PD-L1, but showed strong immunoreactivity for Ki-67. The Ki-67 expression was predominantly localized in the nucleus of the cervical cells. The p16 expression was predominantly localized in the nucleus and cytoplasm of the cervical cells. Based on this observation, cytoplasmic p16 immunoreactivity alone was considered as negative. The PD-L1 expression was predominantly localized in the cell membrane of both cervical and mononuclear cells. Thus, we evaluated PD-L1 expression in these cells separately. All samples were stained with Ki-67, p16, and PD-L1 at the same time. During the evaluation, two experienced histopathologists were blinded to the clinical diagnosis and the cytological results of the patients Figs.  and 3.FA semi-quantitative scoring system was used to evaluate the degree of Ki-67, p16, and PD-L1 immunoreactivity. The degree of positive staining for Ki-67 was calculated by counting 100 endometrial epithelial cells: < 5 cells with nuclear staining were evaluated as negative; 6–25 cells as 1+; 26–50 cells as 2+; 51–75 as 3+; and ≥ 76 as 4+. The p16 immunoreactivity was evaluated based on four parameters: (1) intensity: strong (dark brown color similar to the positive control) versus weak (yellow color significantly lighter than the positive control); (2) extent: diffuse  versus focal (< 50% of the epithelium); (3) continuity: continuous  versus discontinuous ; and (4) location: positive cells reside in the lower third, two thirds, or full thickness of epithelium. Based on these four parameters, lesions were categorized as negative (0), ambiguous pattern (1+), and block-positive (2+). PD-L1 immunoreactivity was categorized based on the percentage of membranous positivity of the cells: < 5% positive cells per field as 0; 5–29% positive cells per field as 1+; 30–59% positive cells per field as 2+; and ≥ 60% positive cells per field as 3 + .https://www.medcalc.org; 2020). The distribution of all variables in both the CIN (+) and control groups were studied by describing the mean ± standard deviation (SD) or median (min-max), where applicable. Whether the distributions of continuous variables were normal or not was determined by Kolmogorov–Smirnov test. Also, the Levene’s test or F test was used for the evaluation of the variances. The Student’s t-test was used to compare normally distributed measurements for independent samples and the Mann–Whitney U test was applied for comparisons of the median values. The Chi-square test was used to compare categorical data. While the mean differences among more than two independent groups were analyzed by one-way analysis of variance (ANOVA), the Kruskal–Wallis test was applied for comparisons of the median values. When the p-value from one-way ANOVA or Kruskal–Wallis test statistics was statistically significant, the Scheffé test or post-hoc analysis nonparametric multiple comparison test was used to determine which group differed from which. A p-value of < .05 was considered statistically significant.All statistical analyses were performed using MedCalc Statistical Software version 19.2.1 . Clinical and pathological characteristics of the patients were summarized in Table In this cross-sectional study, the cervical sitology and histology results of 94 patients were evaluated. The mean ages and median parity of the patients were similar between the groups  groups frequently showed koilocytosis of the epidermal prickle cell layer and increased numbers of infiltrating immune cells Fig. a-d.Fig.According to control co-tests and subsequent colposcopy results of Group II-IV women, 2/21 in Group II, 3/20 in Group III, and 4/24 in Group IV had HPV persistence, and 1/2 in Group II, 0/3 in Group III, and 3/4 in Group IV women had CIN persistence or recurrence. Finally, a total 33 of 94 women underwent LEEP or cold-knife conization  Fig. .Fig. 5Fp = 0.0003 and p = 0.0394, respectively). Our subgroup analysis demonstrated that the HR-HPV (+), CIN II-III group revealed a significantly higher PD-L1 expression in mononuclear cells than the HR-HPV (+), CIN 0 (p = 0.0034) and HR-HPV (−), CIN 0 groups (p = 0.0016). Moreover, the PD-L1 expression of mononuclear cells was significantly higher in the HR-HPV (+), CIN 0 group than in the HR-HPV (−), CIN 0 group (p = 0.0477). However, there was no difference in mononuclear PD-L1 expression in the CIN I and CIN II-III (p = 0.3827) groups. We also found that epithelial PD-L1 expression in the HR-HPV (+), CIN II-III group was significantly higher than in the HR-HPV (−) CIN 0 (p = 0.0154) and HR-HPV (+) CIN 0 (p = 0.0130) groups , CIN 0 group to the HR-HPV (+), CIN II-III group and there was a statistically significant difference between the groups . Whereas, cervical epithelial PD-L1 expression was only correlated with HPV type and the presence of HPV persistence  . There were no differences between HR-HPV (−) women and HR-HPV (+) women with CIN 0 and I (p > 0.05). We also evaluated the correlation of mononuclear and cervical epithelial PD-L1 expression with Ki-67 and p16 staining and there was a positive correlation   CIN II-III women had significantly increased Ki-67 and p16 expression in their cervical tissue samples than HR-HPV (−) women and HR-HPV (+) women with CIN 0 or I (y) Figs. .In this cross-sectional study, we evaluated the expression of PD-L1 in the mononuclear and cervical epithelial cells of women who were HR-HPV (−) CIN 0 and HR-HPV (+) with CIN 0, I, and II-III. According to our results, HR-HPV (+) women with CIN 0, I, and II-III demonstrated significantly higher PD-L1 immunoreactivity in their mononuclear and cervical epithelial cells compared to HR-HPV (−) CIN 0 group. Moreover, PD-L1 expression was correlated with the presence of HR-HPV infection, HPV persistence, and CIN persistence or recurrence. Additionally, there was a positive correlation between mononuclear PD-L1 expression and Ki-67 and p16 staining in the cervical tissue samples. To our knowledge, this is the first study to investigate the expression of PD-L1 and its relationship with the histopathological changes of the cervical tissue of women with CIN.In the literature, a number of studies have demonstrated that the immunohistochemical expressions or circulating levels of PD-L1 are significantly higher in many types of cancers including lung, gastric, prostate, ureteral, and cervical –25. MoreOn the other hand, there are few studies evaluating PD-L1 expression in patients with CIN , 26. YanIt is known that Ki-67 is a proliferating cell marker, and previous studies have demonstrated that Ki-67 is a sensitive biological indicator of CIN progression . The p16In conclusion, PD-L1 expression in both mononuclear and cervical epithelial cells gradually increases with the presence of HR-HPV positivity and an increasing CIN grade during the initial evaluation of women with or without abnormal cervical histology results. Moreover, an overexpression of PD-L1 is associated with the persistence of HR-HPV and the persistence or recurrence of CIN. During the initial evaluation of the cervical histology results, the assessment of PD-L1 expression—especially in mononuclear cells in cervical tissue samples—may provide more information on the progression of HR-HPV infection and its persistence. We suggest that a new scoring system consisting of PD-L1 immunoreactivity and histopathological findings be developed to estimate the clearance of the HR-HPV infection and the management of CIN lesions.


We showed for the first time that the glycogen content in the cytoplasm decreased with the nucleus size of cervical cells in all studied groups apart from the cancer group. For the subpopulation of large-nucleus cells HPV infection resulted in considerable glycogen depletion compared to HPV negative cells in IIa, LSIL  and HSIL  groups. We hypothesize that accelerated glycogenolysis in large-nucleus cells may be associated with the increased protein metabolism for HPV positive cells. Our work underlines unique capabilities of Raman microscopy in single cell studies and demonstrate potential of Raman-based methods in HPV diagnostics.Using Raman microscopy, we investigated epithelial cervical cells collected from 96 women with squamous cell carcinoma (SCC) or belonging to groups I, IIa, IIID-1 and IIID-2 according to Munich III classification . All women were tested for human papillomavirus (HPV) infection using PCR. Subcellular resolution of Raman microscopy enabled to understand phenotypic differences in a heterogeneous population of cervical cells in the following groups: I/HPV E1–E8), two late genes  and long control region (LCR). Early genes code for non-structural proteins that regulate the maintenance and replication of the HPV genome in the infected cell, as well as the transition to the lytic cycle. The late genes in turn encode capsid proteins, which are also involved in the assembly of the virion [Cervical cancer, associated with the sexually transmitted human papillomavirus (HPV) was the fourth most common cancer among women in the world in 2018 [e virion . Integrae virion ,4Cytological examination, i.e., Pap smear, sometimes combined with an HPV test, is the basic method of cervical cancer screening . HoweverTests for detecting HPV are divided into three groups: nucleic acid hybridization assays, signal amplification assays and nucleic acid amplification assays . All tesAccording to the latest data, the HPV test is more effective at detecting cervical neoplasia vs. the Pap test. In fact, 2.3 and 5.5 of 1000 women developed CIN3 within 48 months of receiving a negative HPV and Pap test result, respectively . HoweverIn the case of cervical cancer, the nucleus to cytoplasm ratio is used as a cytological feature to identify abnormal cells . InteresTherefore, other spectroscopic techniques are proposed, including Raman spectroscopy, a label-free, unbiased technique suited to study cells in their natural environment . This meIn the case of cervical epithelial cells, glycogen levels vary in different phases of the menstrual cycle, and after menopause it decreases significantly . InteresChanges of the glycogen content in cancer cells are related to different signal transduction pathways in these cells . Akt, i.It is apparent that changes in the cell metabolism in the course of tumorigenesis affect the chemical content of various cell components. As HPV induces activation of various signal transduction pathways related to metabolic activity of cervical cells, in this work, Raman microscopy with subcellular resolution has been used to study distribution of chemical components in epithelial cervical cells collected from HPV positive or negative patients. Patients were classified according to the Munich III system ,52 to I,−, IIa/HPV−, IIa/HPV+, LSIL/HPV−, LSIL/HPV+, HSIL/HPV−, HSIL/HPV+ and cancer cells – squamous cell carcinoma  and glycogen can be separated  is rich in glycogen B,D as th−1 E. AdditiIn neoplastic cells, the diameter of nuclei is bigger than in healthy cells . This ph−1 both due to glycogen dispersed in the cytoplasm and glycogen in the form of glycogen-lipid-rich stores) occurs to be significantly decreased in cells with large cell nuclei in I/HPV−, IIa/HPV+ and LSIL/HPV+ .A decreased level of glycogen in cervical cells is usually attributed to increased glycogenolysis . Our res+ group suggests that pathological changes already may occur in cells in this group, which until now were considered dysplasia-free, although this hypothesis undoubtedly requires further studies. To investigate in more detail how a glycogen level is influenced by the dysplasia progress and HPV presence, the statistical analysis of the cytoplasm glycogen content in cells from all groups was performed without separation for cells according to the nucleus size  in all studied cells are presented in −, IIa/HPV−, IIa/HPV+, LSIL/HPV−, and HSIL/HPV− there are no statistically significant differences between the level of glycogen in the cytoplasm. The LSIL/HPV+ shows a difficult to rationalize increase in the glycogen level. Contrarily, the cells of patients with the cervical cancer have accelerated glycogen metabolism compared to all the above-mentioned groups, what agrees with previous findings showing that glycogenolysis is increased in cancer cells [+ group confirming their phenotypic similarity to SCC/HPV+ cells.As we have clearly identified small-diameter nuclei cells as metabolically different that the large-diameter nuclei cells, we compared separately these two groups of cells. For cells with small nuclei A for I/Her cells . This ef− groups I, IIa, LSIL and HSIL is similar. However, the results clearly show a decrease of the glycogen content for cells infected with HPV that is statistically significant for IIa and LSIL groups and shows a trend for the HSIL group. Reduction in the glycogen level equals to 45% for IIa/HPV+ vs. IIa/HPV−, 46% for LSIL/HPV+ vs. LSIL/HPV− and 37% for HSIL HPV+ vs. HSIL/HPV−, respectively. Moreover, these changes of the glycogen content are not dependent on age . For DNA isolation for PCR, cells were frozen in −20 °C until the assay was performed.Eight groups of patients have been analyzed in this study. The division into groups was based on the results of the cervical cytology test and classified according to Munich III and the PCR test for the presence of HPV infection. Research included patients whose epithelial cells were classified as group I, HPV negative; group IIa, HPV negative; group IIa, HPV positive; group IIID-1, HPV negative; group IIID-1, HPV positive; group IIID-2, HPV negative; group IIID-2, HPV positive ; and patients suffering from squamous cervical carcinoma (SCC), HPV positive. Due to only 1 and 2 samples in groups I, HPV positive and SCC, HPV negative, respectively, these groups were considered as statistically unreliable. Cells from every patient were divided into two parts and submitted for Raman imaging and PCR. For Raman imaging, freshly isolated cells were fixed using a 2.5% solution of glutaraldehyde for 4 min, then washed twice with PBS and stored in PBS in 4 °C until the measurement. Before Raman measurements, cells were placed on a Raman substrate . Written informed consents were obtained from all participants.−1 grating, spectral resolution of 3 cm−1) and a CCD detector . A laser power of ca. 28 mW on a sample provided by a solid state 532 nm laser source was used. A 63× water immersive objective  was applied to collect Raman spectra. Raman spectra were acquired with the integration time of 0.3 s for areas of 15 × 15 µm2 including cell nuclei with fragment of cytoplasm with the sampling density of 0.5 µm. For each patient at average 10 cells were measured  equipped with a UHTS 300 spectrograph  was carried out using the software WITec Project Plus 2.10. All imaging data were analyzed using the Cluster Analysis  and LSIL/HPV+ (big-diameter nuclei) and one cell from groups HSIL/HPV+ (big-diameter nuclei), LSIL/HPV+  and SCC/HPV+  was rejected from the analysis, as they were classified as outliers based on interquartile range.Statistical analysis of the data was performed using the Origin 9.1 software and the STATISTICA 13.3 software. The Shapiro–Wilk test was used to check whether the data met the assumption of normal distribution. Then, Kruskal–Wallis and Mann–Whitney U tests were performed . A Pearson’s correlation method was used to calculate the correlation between data. Two cells from groups IIa/HPVPCR reactions for the presence of genital types of HPV infection were carried out using a nested PCR method and 2 pairs of primers: external MY09/MY11 and internal GP5+/GP6+ using Mastercycler Nexus × 2 from Eppendorf . To visualize nPCR effects, an agarose gel electrophoresis with addition of bromodeoxyuridine (BrDU) was performed. The test used detected fourteen types of highly oncogenic HPV (HPVhr): HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. For confirmation that only highly oncogenic types of HPV were analyzed, Cobas HPV  test was used.PAS staining was performed according to a known procedure . Prior t−, IIa/HPV−, IIa/HPV+, LSIL/HPV−, LSIL/HPV+, HSIL/HPV−, HSIL/HPV+ and SCC/HPV+). In particular, due to the subcellular resolution of Raman imaging, we were able to separate a subgroup of cervical cells with large (over 10 µm) diameter of nuclei, showing unexpected chemical and metabolic characteristics.In this study, we assessed differences in the cytoplasm glycogen level of cervical epithelial cells, collected from 96 women, depending on the presence of HPV infection, dysplastic changes and nucleus diameter using Raman microscopy and chemometric data analysis. The applied methodology appeared to be crucial to account for a considerable heterogeneity of cells in studied groups (I/HPV+ group that is characterized by a decrease in the glycogen content in agreement with previous studies [+ group that is phenotypically similar to SCC/HPV+. Additionally, somewhat difficult to rationalize is an increase in the LSIL/HPV+ level. Importantly, there are no differences between the glycogen content for small-diameter cells in the HPV+ and HPV− respective groups. Contrarily, for the subpopulation of large-nucleus cells, the cytoplasm glycogen level is significantly reduced of about 37–46% for HPV+ cells compared to HPV− cells. It shows that for this subpopulation of cells, glycogen metabolism accelerates with HPV infection. The considerable depletion of the glycogen level in HPV infected cells may be associated with molecular pathways related with HPV E6 and E7 proteins [As our results demonstrate, in cervical cells glycogen is both dissolved in cytoplasm and aggregated with lipids in the form of glycogen-lipid-rich granules. For cells with small-diameter nuclei, the global level of glycogen in the cytoplasm is similar for considered groups apart from the SCC/HPV studies  and HSILproteins ,58 and iproteins . UnderstLast, but not least, lack of reagents and speed of Raman spectroscopy could be advantageous in HPV diagnostics compared to the DNA-HPV method. Raman spectroscopy may be in future a base for a simple, automatized test for HPV; however, it certainly requires further testing on a bigger cohort of patients and improvement of the methodology.


Preference-based care is required by the Centers for Medicare and Medicaid Services and is linked to improved quality of nursing home care. The federally mandated Minimum Data Set (3.0) Preference Assessment Tool (PAT) is a 16-item standardized measure used to facilitate preference-based care through rating how important residents’ daily and activity preferences are. Little work has explored how Veterans’ unique demographic backgrounds and functional/cognitive abilities may influence how they rate their preferences . Therefore, the purpose of this retrospective study was to explore the relationships between Veterans’ demographic/clinical characteristics and number of preference importance ratings. Our sample  consisted of Veterans admitted to community nursing homes after hospitalization at a Veterans Affairs facility for heart failure between 2010-2015. We used ordinal regression to explore predictors of preference importance ratings. Veterans were, on average, 78-years-old (SD=10.42) and mostly male (95%), white (81%), married (46%), cognitively intact (74%) with extensive functional impairment (60%) and minimal depressive symptoms (74%). Veterans rated an average of 12.47 preferences as important . Veterans living with cognitive impairment, depression, and extensive functional impairment who were not married or separated had a lower number of important preferences . Veterans that were female, under the age of 85, and any race but white had a higher number of important preferences . Discussion will include implications for planning and delivering preference-based care for Veterans as well as next steps in research and practice to better understand and fulfill Veterans’ everyday care preferences.


Numerical simulations of acoustic streaming flows can be used not only to explain the complex phenomena observed in acoustofluidic manipulation devices, but also to predict and optimise their performances. In this paper, two numerical methods based on perturbation theory are compared in order to demonstrate their viability and applicability for modelling boundary-driven streaming flows in acoustofluidic systems. It was found that the Reynolds stress method, which predicts the streaming fields from their driving terms, can effectively resolve both the inner and outer streaming fields and can be used to demonstrate the driving mechanisms of a broad range of boundary-driven streaming flows. However, computational efficiency typically limits its useful application to two-dimensional models. We highlight the close relationship between the classical boundary-driven streaming vortices and the rotationality of the Reynolds stress force field. The limiting velocity method, which ignores the acoustic boundary layer and solves the outer streaming fields by applying the ‘limiting velocities’ as boundary conditions, is more computationally efficient and can be used for predicting three-dimensional outer streaming fields and provide insight into their origins, provided that the radius of curvature of the channel surfaces is much greater than the acoustic boundary layer thickness ( Acoustic streaming comprises steady, time-averaged fluid flows driven by acoustic absorption in a fluid. Various acoustic streaming patterns have been analysed in acoustofluidic manipulation devices due to different mechanisms of attenuation, most notably Eckart  streaminMost bulk acoustic wave microfluidic manipulation devices utilise ultrasonic standing waves, where the scale of the fluid channel (in at least one dimension) is typically of the same order as the acoustic wavelength, meaning that the acoustic streaming fields are dominated by boundary-driven streaming as Eckart-type streaming generally needs longer distances to allow acoustic attenuation in the bulk of the fluid. Both types of streaming are typically found in surface acoustic wave (SAW) devices  into Eq. (1) and considering the equations to the first order, Eq. (1) for solving the first-order acoustic velocity takes the form,Repeating the above procedure, considering the equations to the second order and taking the time average of Eq. (1) using Eq. (2), the continuity and momentum equations for solving the second-order time-averaged acoustic streaming velocity can be turned into¯ Nyborg .Taking the curl of both sides of Eq. , we estaIt can be seen that the second-order streaming vortices are closely related to the rotationality of the RSFs. One advantage of this equation over Eq.  is that h Nyborg  points te of Eq.  to invesIn the results shown later, this relationship will be used to elucidate the driving mechanism of classical boundary-driven streaming patterns and to draw out the key causal factors.The numerical simulations were conducted in COMSOL 4.4 . In thismentclass2pt{minimThe required 1D standing wave fields in these rectangular channels were established by a harmonic excitation of the left boundaries at a frequency entclass1pt{miniman et al. , this diThermoacoustics, Frequency Domain’ interface was used to predict the first-order acoustic fields, which solves the acoustic pressure fields following . The supplied ‘Creeping flow’ interface was edited to reflect Eq. (4), which initially does not include the first term in (a) and the last term in (b). In 2D Cartesian coordinates shown in Fig. Computational fluid dynamics (CFD) step: the acoustic streaming fields were then solved. Here, a COMSOL ‘As illustrated, boundary-driven streaming originates from the dissipation of acoustic energy within the thin acoustic boundary layer region. The first method, the RSM, explores the origin of boundary-driven streaming flows, the net force on the fluid, and investigates how the boundary-driven streaming vortices inside and outside the acoustic boundary layer region are formed from the RSFs. The numerical process can be split into two steps:Pressure Acoustics, Frequency Domain’ interface was used to model the first-order acoustic fields, which solves the harmonic, linearised acoustic problem, as described in Eq.  can be approximated as a function of the first-order linear acoustic velocity field outside the acoustic boundary layer as long as the radius of curvature of the surface is much greater than the acoustic boundary layer thickness Nyborg . The strAs only outer streaming fields are solved in this method, with the assumption of low velocity, incompressible flow, the first term in the left-hand side of Eq.  is zero,In terms of channel dimensions, a series of channels with mentclass2pt{minimAs shown in Fig. The modelled first-order acoustic fields are shown in Fig. ers Fig. a with a The modelled acoustic streaming patterns are shown in Fig. In order to demonstrate the accuracy of these two methods, the modelled acoustic streaming velocity magnitudes were compared to Hamilton et al.’s analytical solution , care must be taken to accurately model the moving surface boundary condition, which is non-trivial in an Eulerian formulation Koster . In such