Longitudinal data on the E6/E7 mRNA-based Aptima human papillomavirus (AHPV) assay exceeding three years in comparison to the gold standard Digene Hybrid Capture 2 (HC2) test are not available. We previously reported the cross-sectional data of the German AHPV Screening Trial (GAST) in which 10,040 women were recruited and tested by liquid-based cytology, the HC2 assay, and the AHPV assay. Longitudinal data on the E6/E7 mRNA-based Aptima human papillomavirus (AHPV) assay exceeding three years in comparison to the gold standard Digene Hybrid Capture 2 (HC2) test are not available. We previously reported the cross-sectional data of the German AHPV Screening Trial (GAST) in which 10,040 women were recruited and tested by liquid-based cytology, the HC2 assay, and the AHPV assay. Four hundred eleven test-positive women were followed for up to six years. In addition, 3,295 triple-negative women were screened after a median time of six years. Overall, 28 high-grade cervical intraepithelial neoplasia (CIN3) cases were detected. The absolute risk of developing high-risk HPV-positive CIN3+ over six years among those women that tested negative at baseline was 2.2  and 3.1  per 1,000 women screened by the HC2 and the AHPV tests; the additional risk for those with AHPV-negative compared with HC2-negative results was 0.9  per 1,000. In comparison, the absolute risk following a negative LBC test was 9.3 . The relative sensitivity of AHPV compared to HC2 was 91.5% for CIN3+, and the negative predictive values were 99.8%  for HC2 and 99.7%  for AHPV. Our data show that the longitudinal performance of the AHPV test over six years is comparable to the performance of the HC2 test and that the absolute risk of CIN3+ over six years following a negative AHPV result in a screening population is low.  Systematic screening has led to a significant decrease in cervical cancer cases worldwide. Since persistent human papillomavirus (HPV) infection with 13 high-risk HPV (HR HPV) types defined as class I or IIA carcinogenic for women –8. The l–9–Currently, more than 190 HPV assays are commercially available , and manHowever, cross-hybridization of the HC2 test with at least 26 additional HPV types of low carcinogenicity or undefined risk has been detected –19, whic–As stated by Meijer et al., new HPV tests should demonstrate noninferior sensitivity and specificity compared with the HC2 test in a representative set of samples from a routine screening population in a cross-sectional setting . HoweverThe Aptima human papillomavirus (AHPV) assay  is based on target-mediated amplification for the detection of viral mRNA. The test detects the mRNA of the two HPV oncogenes E6 and E7 of 14 HPV types, which include all high-risk HPV types targeted by the HC2 assay as well as the class 2B type HPV66 .The RNA-based AHPV test has been compared to the DNA-based HPV tests in a number of studies, several of which are typical screening populations , 24. In We recently reported the cross-sectional results of the German AHPV Screening Trial (GAST), where the clinical sensitivities and specificities of the AHPV and the HC2 HPV tests were determined and compared in cervical samples from 9,451 women age 30 to 60 years from a routine screening population . SamplesHowever, longitudinal data from a screening population cohort on the AHPV assay exceeding 3 years compared to the gold standard HC2 testing are important for reassurance, especially after the recent introduction of extended screening intervals (≥5 years) in some national cervical cancer screening programs. To address this lack of data, the GAST was continued by annually inviting all untreated women who remained positive by at least one of the three tests for follow-up screening. Furthermore, a randomly selected group of 4,000 women who were triple negative at baseline were invited for a second screening round after a mean of 6 years. We report here the first longitudinal data of more than 3 years regarding cumulative risk for CIN2/3+, clinical sensitivity, and negative predictive value (NPV) for the detection of histologically reviewed high-grade CIN by the RNA-based AHPV assay in comparison to the HC2 test .Women age 30 to 60 years from the routine cervical cancer screening population of three German centers in Tübingen, Saarbrücken, and Freiburg were invited to participate in GAST. The data of the baseline cross-sectional study have previously been published . Writtenn = 10,040) had single liquid-based cytology samples  taken during the annual routine gynecological examination. Liquid-based cytology , the Digene Hybrid Capture 2 (HC2) high-risk HPV DNA test, and the (AHPV) assay were performed on all samples. All women with a positive result in any of the three screening tests were referred for colposcopy within 8 weeks of receiving their test results.The design of the baseline cross-sectional study was described previously . In brieFor the positive follow-up arm of the GAST, all women who tested positive by any of these assays and who were not treated because of abnormal colposcopy and/or histology were retested annually for up to 5 years. After a mean interval of 6 years, 4,000 women who were triple negative at baseline were randomly selected and invited to be retested by all three tests when attending routine cervical screening . Those who tested positive by any of the three tests were referred to colposcopy. The rationale for the sample size among women who tested triple negative at baseline can be found in the Supplemental Methods in the supplemental material.As previously described for the cross-sectional trial , LBC resHPV testing was performed as previously detailed . ResiduaDigene Hybrid Capture 2 high-risk HPV DNA testing was performed as described previously , using tThe AHPV assay was performed following the manufacturer’s instructions. The earlier cutoff value of a signal/cutoff (S/CO) ratio of 1.0 instead of the current cutoff (0.5) was used throughout this study to provide continuity of the data. HPV genotyping was carried out using the INNO-LiPA HPV Extra genotyping test , as described previously , 28.Women who tested positive in either LBC, the AHPV assay, or the HC2 assay (HPV-positive women) were referred to colposcopy within 8 weeks. If lesions were detected after the application of acetic acid, a biopsy sample was taken from the suspicious tissue, and specimens were processed to produce hematoxylin and eosin (H&E)-stained slides. Current practice in Germany and some other European countries is to observe CIN2 lesions instead of treating them immediately, depending on the individual situation of the patient and her agreement. After local pathologist review, all slides were classified using the three-tiered CIN terminology. All slides with abnormal findings were reviewed by a second pathologist blinded to the first diagnosis, and slides with discordant review results were again reviewed by a third pathologist to reach a consensus diagnosis (two out of three agreement).Prior to analysis, data were checked for plausibility and monitored. This included violation of inclusion criteria (pregnancy or age below 30 years or above 60 years), and positive Pap test six months prior to baseline testing as well as HIV infection. Following this, the databases were sealed and sent to the statisticians for statistical analysis.Women with at least one positive test at baseline, who had at least one adequate screening test result on follow-up, and who were not treated nor diagnosed with CIN3 or worse at baseline were eligible for the follow-up analysis. In addition, women who tested negative at all three tests at baseline and had at least one adequate screening test result during follow-up were eligible for analysis.We present baseline demographic characteristics from all participants in the study and for those who attended follow-up. To assess whether there was a statistical difference between groups, we used a chi-square test to compare those attending follow-up with those who were eligible to attend but did not.The follow-up of women in whom all three baseline screening tests were negative was quite different from that in women who had one or more positive screening test results at baseline. Those with all negative results were rescreened once after approximately 6 years. Women who had a positive result at baseline were invited back at 12- to 18-month intervals until the results of all three tests were negative or until they were treated for high-grade CIN.At each visit, we estimated the hazard of having CIN3+ (or CIN2+) by multiplying the proportion of tested women who were eligible for colposcopy by the proportion of women attending colposcopy who were diagnosed with CIN3+ (or CIN2+). Further details can be found in the Supplemental Methods. Having estimated the hazard at each visit, we estimated the cumulative probability of disease after several visits using the Kaplan-Meier (product limit estimator) approach. The variance of the modified Kaplan-Meier estimator was derived in the same way as the Greenwood formula is derived for the usual Kaplan-Meier estimator. The formula is provided in the Supplemental Methods.We estimated the hazard at baseline separately for each of eight groups based on the result combinations (positive or negative) for each of the three screening test results. Subsequently, we estimated the hazards separately in just four groups based on the baseline result combinations of the two HPV tests. There was no evidence that the hazards differed depending on the LBC result within each of the four groups . Since we did not have 6-year follow-up data for women who were not triple negative at baseline, we assumed that the hazard observed at about 6 years in the baseline screen-negative group also applied to all other groups.We then estimated the number of cases of CIN3+ (and CIN2+) that would have been observed among women negative at baseline by each of the three tests (separately) had everyone been followed to 6 years by taking a weighted sum of the estimated cumulative risk in each group. The cumulative risk in each group was also estimated by dividing the number of cases by the number of women with that result at baseline.P values were estimated from the discordant pairs using the exact McNemar significance probability test.Confidence intervals were obtained by assuming that the logarithm of the cumulative risk is approximately normally distributed. The main analysis presents results that included all CIN3 cases. Since we were interested in comparing the performances of two HPV tests, both of which aim to detect the same 13 high-risk HPV types and HPV66, and since CIN3 caused by other HPV types is less likely to progress to cancer, a subanalysis excludes disease where the HC2 results are technically false positive due to cross-hybridization with noncarcinogenic HPV types .Analyses were carried out using STATA 15.0 (StataCorp).Out of the 4,000 women with negative screening test results at baseline who were invited, 3,295 women (82.4%) attended follow-up. Among those with at least one positive test at baseline, 606 women were eligible for follow-up, and 411 women (67.8%) attended . The basn = 606). Follow-up ceased when HPV infection and/or cervical abnormalities were cleared, if treated for cervical disease, or if they refused to participate in the follow-up study. Of the eligible women, 411 (67.8%) attended at least one follow-up examination and were eligible for analysis. The median time to the first follow-up visit was 14 months  , and the average number of follow-up visits per participant was 1.7 . Three women were excluded because they were missing an HC2 test result and did not return for follow-up. Of the 408 women with at least one follow-up visit with adequate HC2 and AHPV results, 77.2% (315) were negative on both HPV tests at their final visit. In total, 200 women tested positive during follow-up and were referred to colposcopy; 165 (82.5%) of these women attended colposcopy. Ninety percent of those who attended follow-up did so within 2.5 years of baseline. A total of 32 women were diagnosed with CIN2 or worse during follow-up.Untreated women with at least one positive test result at baseline and no CIN3 or worse were invited to attend annual follow-up examinations over a 5 year-period  was substantial, with a kappa (κ) value of 74.7% .in situ (AIS) or invasive cervical cancer cases were detected during follow-up.During follow-up of those women who tested positive on at least one test at baseline, a total of 24 women were diagnosed with CIN3 and 8 women with CIN2 . The basIn the baseline cross-sectional arm of the German AHPV Screening Trial (GAST), 8,752 women had a negative result in all three tests . Of these, 4,000 participants were invited for follow-up testing approximately 6 years postenrollment. In total, 3,295 participants (82.4%) attended follow-up . The medAt the second round, 3,057 women tested negative on all three tests (92.8%). A total of 140 women (4.6%) had at least one positive test results at follow-up, 115 (82%) of these women underwent a colposcopic examination, and a total of 9 women were diagnosed with CIN2 or worse disease (5 CIN2 and 4 CIN3 lesions). A summary of the LBC and HPV test results at the second screening round is found in n = 4 of 9), but 100% tested HPV positive  CIN2 cases that regressed, while 3/23 (13%) progressed to CIN3.Passive clinical follow-up data were available from a registry on the complete Saarbrücken subcohort of 2,147 women who tested triple negative at baseline; 887 of those women attended follow-up as part of GAST. During a 6-year passive follow-up period, only one CIN1 and one CIN2 case were observed in women who did not attend the second-round screening in GAST. Among the Saarbrücken cohort attending the second screening round, one case of CIN2 and two cases of CIN3 were detected at the second screening appointment.All HPV-positive samples were genotyped by the INNO-LiPA Extra genotyping test. Baseline HPV test results among the 41 women who went on to be diagnosed with CIN2+ during follow-up show that 9 women (22%) were HPV negative, 2 women (5%) tested positive for HC2 non-high-risk HPV types (HPV66 and HPV82), and 24 (58%) single-type and 8 (20%) multiple-type HPV infections were detected (results not shown).HPV genotyping results at the time of diagnosis (during follow-up) are presented in Table S2. At the time of diagnosis, 1/41 (2%) CIN2 case was HPV negative on both tests, one CIN2 case and one CIN3 case (5%) tested positive to non-high-risk HPV types , and 28/41 (68%) single-type and 12/41 (29%) multiple-type HPV infections were detected.HPV16 was the most frequent HPV type detected in patients with CIN3 in the cross-sectional part of the study and among those attending follow-up.The main analysis presents results that include all diagnosed disease. We present a subanalysis excluding two CIN3 cases whose HPV types were HPV82 and HPV67 and hence were considered technically false-positive HC2 test results. One case (HPV67) was diagnosed and treated at baseline, and the remaining case (HPV82) was diagnosed at follow-up.A summary of the 6-year cumulative risk per 1,000 women screened and negative predictive value among women testing negative at baseline can be found in P = 0.096). For comparison, the cumulative risk by 6 years among LBC-negative women was 1.66% . The relative sensitivity for CIN2+ of the AHPV test in comparison to the HC2 test was 91.4%. Among women testing negative by both HPV tests at baseline, the cumulative risk of CIN2 or worse was 0.38% .The cumulative risks of CIN2 or worse by the year 6 visit were 0.62%  and 0.47%  among those who tested AHPV and HC2 negative, respectively. The difference in AHPV-negative cases was 0.15%  and is not significant  of CIN3 that tested HPV82 positive and one (diagnosed at baseline) that tested HPV67 positive produced very similar results, with cumulative risks at 6 years of 0.59%  and 0.47%  among AHPV- and HC2-negative women, respectively. The relative sensitivity for CIN2+ of AHPV in comparison to HC2 was 93.0%.The cumulative risks of CIN3 disease by the year 6 visit were 0.31%  and 0.22%  for AHPV-negative and HC2-negative women , respectP = 0.1094). The cumulative risk by 6 years among those testing LBC negative was 0.90% . The relative sensitivity of the AHPV test to the HC2 test for CIN3 increased to 94.2%.The subanalysis excluding two CIN3 cases with technically false-positive HR HC2 HPV type results produced cumulative risks by 6 years of 0.28%  among those who tested AHPV negative at baseline and 0.22%  among those who tested HC2 negative  at baseline, we detected 8 CIN2 and 24 CIN3 cases. One CIN2 case was missed by both HPV tests and was positive only by cytology. One CIN3 case tested negative by the AHPV test at the time of diagnosis but was detected by the HC2 test, and it contained a non-HR type (HPV82). Results from a meta-analysis of type-specific HPV DNA prevalence in cervical cancer and women with normal cytology showed prevalences of HPV82 of 0.1%  and 0.1% , respectively. HPV82 is not targeted by the HC2 test but may yield positive results due to a cross-reaction. The known extensive cross-reactivity of the HC2 test may therefore explain the nonsignificantly higher sensitivity than that of the AHPV test in the baseline and follow-up results of this study. According to the Meijer et al. criteria , the canIn the second screening round of women who tested triple negative at baseline, a total of five CIN2 and four CIN3 cases were identified, of which one CIN3 was missed by the AHPV test at follow-up. This case was HPV16 positive and was repeatedly detected positive by the HC2 test at 1.86, 1.53 and 1.51 RLU, which is a borderline positive result according to the FDA approval but a negative test result in some countries , where an increased cutoff of 2.0 RLU is used for cervical cancer screening.The cumulative risks of CIN3 + 6 years after a negative screening test in this study are very similar to the ones observed earlier by Dillner et al. . In bothThe analysis including all CIN3 cases detected and the analysis excluding two CIN3 cases (one from baseline with HPV67 and one detected at follow up with HPV82) revealed highly comparable absolute risks for CIN3+ following a negative baseline HC2 or AHPV test and comparable longitudinal negative predictive values (NPV) for the tests. Note that women who tested positive (by any test) at baseline were not asked to return for testing 6 years after enrollment. Therefore, we assumed that the hazard at 6 years among those who tested positive but who did not develop disease was the same as that observed at about 6 years in the baseline screen negative group. This explains the sudden jump in the risk shown in Altogether, we found an absolute risk for developing CIN3+ after 6 years among those women who tested negative at baseline of 2.2 and 3.1 per 1,000 women screened by the HC2 and the AHPV test, respectively. This difference is not significant and is in line with one previous publication where very low 3-year risks for CIN3+ were detected after a negative baseline AHPV test .The fact that annual follow-up of all women from the routine screening population was unachievable complicated the data analysis and might be considered a weakness of this study. However, the CLEAR study found that annual follow-up of screen-negative women suffers from low compliance  and is nThe poor sensitivity of LBC in this study may be considered a weakness but simply reflects the low single-round sensitivity of cytology in Germany that has been noted in several studies previously . Since tIn summary, numerous studies from different populations  consiste


Post-hysterectomy collision tumors of the vulva has rarely been reported. Though long-term HPV infection may induce vulva tumor, but the relationship between HPV infection and collision vulva tumor is not clear. And there are no clear rules of the post-hysterectomy cancer surveillance for human papilloma virus (HPV) long-term infections. So here we first report a case of post-hysterectomy rare collision vulva tumor with long-term HPV infection composed of squamous cell carcinoma of the labia major and adenosquamous carcinoma of bartholin gland and hope to bring new direction to our future research.A 48-year-old woman with long-term HPV infection, 3 years after hysterectomy, gravida 3, para 2, was admitted to our hospital with complaints of a 4-month history of an itching vulva ulceration. An anabrosis was located on the surface of the solid mass of the bartholin gland at the posterior part of the right labium and the right inguinal lymph nodes were palpable. Result of the incisional biopsy of the ulcer area at local hospital was atypical squamous cells couldn’t exclude high-grade squamous intraepithelial lesion (ASC-H). Subsequently more authoritative pathological consultation results suggested squamous cell carcinoma of the vulva.Post-hysterectomy collision vulva tumor with long-term HPV infection composed of squamous cell carcinoma of the labia major and adenosquamous carcinoma of bartholin gland.The extensive excision of the vulva, bilateral inguinal lymph nodes dissection, and local skin flap transposition surgeon was done to this patient. The final certificate diagnosis was: vulvar tumor T1bM0N0 composed of squamous cell carcinoma of the labia major and adenosquamous carcinoma of bartholin gland; HPV infection; post hysterectomy, and bilateral salpingectomy.The patient recovered well after surgery, and consequently received 6 courses of TC (paclitaxel + carboplatin) chemotherapy, and 9 months and 13 days followed up. So far patient recorded as complete response (CR).Collision vulva tumor occurred post-hysterectomy is extremely rare. It is most likely related to long-term HPV infection, which suggests us should to modify the manner of the post-hysterectomy cancer surveillance for HPV long-term infections. For patients with high-risk HPV infection, even if the cytology results are negative, we may should perform colposcopy and vulva biopsy more positively to prevent the disease from progressing into cancer. And the pathogenesis of relationship between HPV infection and collision vulva tumor is still need further investigation. While adenosquamous carcinoma of bartholin gland is rare forms of vulvar malignancy, which are rarely reported and account for <2% of cases. Collision tumor are exceedingly rare in the vulva, with only two cases reported in the English literature, associated with squamous cell carcinoma or adenocarcinoma.Vulvar cancer accounts for 5% of all gynecologic cancers with an incidence of 2.5 per 100,000 women. It is found that in post-hysterectomy HPV prevalence was higher among vaginal than vulvar cases, and HPV16 accounted for most HPV-positive cases for both cancers. Colposcopy examination can detect most preneoplastic lesions, whereas HPV genotyping is suggested to be a more sensitive, inexpensive, and noninvasive method for diagnosis.Human papilloma virus (HPV) infection has been reported to have a role in the pathogenesis of post-hysterectomy vaginal and vulvar dysplasia.However, data of in post-hysterectomy on vulvar cancers are limited. We here first report a post-hysterectomy collision vulva tumor, who had long-term HPV infection, composed of squamous cell carcinoma of the labia major and adenosquamous carcinoma of bartholin gland.2A 48-year-old post-hysterectomy woman with long-term HPV infection, gravida 3, para 2 (G3P2), was referred to our hospital with a 4-month history of an itching vulva ulceration. The patient was diagnosed as grade III of cervical intraepithelial neoplasia with HPV-16 infection. In June 2015, and underwent laparoscopic hysterectomy and bilateral salpingectomy at their local hospital. After the surgery her follow-up revealed a long-term vaginally HPV-16 infection but negative vaginal cytology. At admission, the clinical examination demonstrated 2.0 cm × 1.5 cm sized anabrosis was located on the posterior part of the right labium on a 3.0 cm × 2.0 cm well-circumscribed solid mass of the bartholin gland  and the International Federation of Gynecology and Obstetrics (FIGO) staging systems.The patient underwent extensive excision of the vulva, bilateral inguinal lymph nodes dissection, and local skin flap transposition. Vulvar cancer of this case was staged as TThe postoperation histopathological results certificated the diagnosis as: collision vulva tumor with long-term HPV infection composed of squamous cell carcinoma of the labia major and adenosquamous carcinoma of bartholin gland. The gross postoperative pathology description illustrated, a 2.0 cm × 1.5 cm tumor was found with a well-defined, expansile margin on the posterior part of the right labium; meanwhile another solid mass of the bartholin gland was found which was a 3.0 cm × 2.0 cm well-circumscribed unilocular cyst filled with mucoid material and a hemorrhagic lining, without papillary or polypoid projections in. Subsequently on microscopic examination, it indicated, the tumor of the large labia was a 2.0 cm × 1.5 cm × 1.0 cm poor differentiated squamous cell carcinoma and appeared to be submucosal invasion with infiltration depth approximately reaching 1.0 cm. Intravascular tumor thrombus were found and extensive vaginal intraepithelial lesions II to III existed in peri carcinomatous tissue. Simultaneously, the solid mass of bartholin gland revealed to be completely another poorly differentiated adenosquamous carcinoma with partial undifferentiated element  = 4. As a follow-up, the patient underwent a monthly clinical evaluation, including testing of various blood biochemical and tumor marker levels; and the imaging examinations every 3 months, including vaginal ultrasound, magnetic resonance imaging (MRI) or positron emission tomography–computed tomography (PET-CT). From the end of the operation to June 30th, 2019, totally 9 months and 13 days were followed up. So far patient follow-up status has been recorded as complete response (CR) according both to response evaluation criteria in solid tumors (RECIST 1.1), and modified response evaluation criteria in solid tumors (mRECIST), and no recurrence was found.The patient was discharged after 12 days of hospitalization. Chemotherapy was initiated in our hospital tumor center as soon as her surgical incision achieved healing 4 weeks after the surgery. According to the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology of vulvar cancer, the patient received 6 courses of TC (paclitaxel + carboplatin) chemotherapy: paclitaxel 135 mg/m3In this case we first report a post-hysterectomy collision vulva tumor, who had long-term HPV infection, composed of squamous cell carcinoma of the labia major and adenosquamous carcinoma of bartholin gland. Though the squamous cell carcinoma of large labia and adenosquamous carcinoma of bartholin gland are 2 common histological types respectively in vulva, they are exceedingly rare to be found together. Therefore, we hypothesize that the presence of vulva collision tumors may not be purely coincidental, but because of that the vulva was stimulated by a same carcinogen, resulting in a simultaneous proliferation of different cancer cell lines, and the carcinogen may should be HPV infections. Of course, our assumptions are supported by some evidences. It has already been reported that there is some correlation between the occurrence of squamous cell carcinoma of vulva and long-term infection of HPV.,5 However, adenosquamous carcinoma of bartholin gland, as one of the rare forms of vulvar malignancy, its association with high-risk HPV infection is still unclear, although there have been some reports about the etiologic role of HPV in the pathogenesis of squamous cell carcinomas (SCC) of bartholin gland. All in all, there might be proposal relationship between long-term HPV infections of post-hysterectomy and the collision tumor of vulva, which requires our further investigations.Back to the patient's medical history, we found that long-term HPV infection was an obviously high-risk throughout the course of disease. According to our current knowledge, the patient suffered CINIII 3 years ago, infection of HPV-16 could be the cause of the disease; however, during the follow-up of the hysterectomy, the patient's HPV infection was not cleared but had persisted till now. Therefore, it is reasonable to infer if there is proposal relationship between the collision tumor of vulva and the long-term HPV infections of post-hysterectomy. It has been reported that a single carcinogenic agent may interact with 2 neighboring tissues, inducing the development of tumors of different histological types  in the same organ.–10 Guidelines clearly suggest that for patients with long-term high-risk type infection of HPV in cervical and vaginal secretions, such as HPV-16 or HPV-18, colposcopy should be added, and biopsy of cervical and vaginal tissues should be done if any suspicions, no matter what result of the cytology smear screening is. By further review of the literature on post-hysterectomy cancer surveillance, we found that cervical screening and the histology test of the cervix are recommended for women with a past history of high grade squamous intraepithelial lesions of the cervix (CINII-III). Colposcopy and further vulvar tissue biopsy should be done if having suspicion, especially for long-term HPV infections even without vaginal dysplasia. However, there is no literature mentioned that whether the vulvar tissue should be also focused, and if it is necessary for biopsy of vulvar tissue in the case of negative cytology? The current manner of cancer surveillance might be just the limitation of this case, by which long-term HPV infection has been neglected and eventually developed into a cause of vulvar tumors.Lessons that this case brings to us from another side of the coin is about the post-hysterectomy cancer surveillance. Although this patient has been undergoing vaginal cytology and HPV DNA monitoring every year after hysterectomy, the long-term HPV infection have been ignoring because her vaginal cytology results have been negative. Eventually biopsy confirmed the occurrence of vulvar cancer until the symptom of her vulvar ulceration occurred. Looking back at the entire course of the disease, the long-term high-risk HPV infections of patient did not receive enough attention and vigilance. We reviewed relevant guidelines and literatures on vulvar tumors and found that HPV infection is a high-risk factor for cervical, vaginal, and vulvar tumors.In conclusion, this was an extremely rare collision vulva tumor occurred after hysterectomy. Its occurrence is most likely related to long-term HPV infection. This tragedy suggests us should to modify the manner of the post-hysterectomy cancer surveillance for HPV long-term infections. For patients with high-risk HPV infection, even if the cytology results are negative, we should perform colposcopy and vulva biopsy to prevent the disease from progressing into cancer. At the same time, the pathogenesis of relationship between HPV infection and collision vulva tumor is still need further investigation.Funding acquisition: Xiaorong Qi.Investigation: Fan Yang, HongYi Li, Xiaorong Qi.Methodology: Ce Bian.Resources: Fan Yang, Ce Bian.Software: HongYi Li.Writing – original draft: Fan Yang.Writing – review & editing: Fan Yang.


Sci., 2017, DOI: ; 10.1039/c7sc00732a.Correction for ‘Tailored theranostic apolipoprotein E3 porphyrin-lipid nanoparticles target glioblastoma’ by M. A. Rajora  In The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Background: With the growing interest in treatment de-intensification trials for human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPC), prognostic models have become essential for patient selection. The aim of this paper is to validate and compare the prognostic ability of the TNM 8th edition and previous published risk group classifications of Ang et al. and Rietbergen et al. and to derive a patient selection classification for de-intensification trials.Materials: Patients with HPV+ OPC treated with curative (chemo)radiotherapy between 2004 and 2017 were classified according to the TNM 8th edition, the model of Ang et al. and of Rietbergen et al. HPV status was determined by p16 immunohistochemistry staining. Overall survival was estimated using the Kaplan-Meier method and groups were compared using the log-rank test. Harrell's C-index was used as measure of discriminative performance.Results: A total of 333 OPC were identified of whom 100 were HPV+. The median follow-up was 63.7 months. The 5-year overall survival (5Y-OS) of stage I, II and III were 91.6, 55.2, and 38.0%. There was a significant difference between stage I vs. II and III. The Harrell's C-index for TNM 8th edition stage was 0.67. Including only HPV+ OPC, the Harrell's C-index for the model of Ang and Rietbergen were both 0.62. We combined the main prognostic factors defining the low risk groups in the three models, stage I, low comorbidity and ≤ 10 pack years, into one new low risk group to identify patients who may benefit from de-intensification trials. Intermediate risk was defined as stage I with high comorbidity or >10 pack years, high risk as stage II-III. The 5Y-OS were 100, 85.7, and 51.3%. The Harrell's C-index for the new classification model was 0.67.Conclusion: Although TNM 8th edition provides better OS stratification than the 7th edition, it is not performant enough for patient selection, neither are the models from Ang et al. and Rietbergen et al. Therefore, we propose a patient selection classification for de-intensification trials based on the new TNM classification 8th edition, comorbidity and smoking pack years. In addition, this study emphasizes the importance of patient selection and personalized treatment for HPV+OPC. Due to a change in etiology, oropharyngeal squamous cell carcinoma (OPC) is nowadays the shared name of two distinct clinical entities. First, OPC can be related to tobacco and alcohol consumption, which is also a well-known cause of multiple other types of cancer such as lung cancer or head and neck squamous cell carcinoma (HNSCC) in general. In the western world, the incidence of this type of OPC decreased by changing tobacco and alcohol habits.  Second, The prognosis of these two types of OPC is completely different. HPV+ OPC have a much better prognosis than tobacco and alcohol related OPC  OPC). HPV+ OPC were often diagnosed as a stage III or IV [TNM 7th edition (7th Ed)] since they frequently present as a small primary tumor with multiple cervical lymph nodes. Such a stage IV (7th Ed) OPC had a 5-year overall survival (OS) around 70% in contrast to a 5-year OS of 30% for stage IV HPV− OPC  has developed new staging rules for better discrimination of stages for HPV+ OPC . Their pHowever, the outcome has not only been determined by TNM classification and HPV status but has additionally been influenced by patient related factors for example, smoking and comorbidities. In the last decade several different prognostic models have been developed and validated to take these influencing factors into account. These prognostic models have been either nomograms, calculating a specific percentage of probable survival, or risk group classifications. Ang et al. was one of the first reporting on a risk group classification with HPV status as independent prognostic factor for OS and progression-free survival. They classified patients with OPC in three risk groups of death based on four factors: HPV status, smoking pack years, T-stage and N-stage 7th Ed) . This clth Ed . TThe aim of this project was first to validate the new cTNM 8th Ed in a Belgian HPV+ OPC population, second to investigate if there is still an added value of the previously published prognostic models from Ang et al. and Rietbergen et al. Third we combined these three different models in one new risk group classification to select patients for de-intensification trials.A retrospective chart review was performed of all non-metastatic oropharyngeal squamous cell carcinoma patients treated with primary radiotherapy (RT) in a single institution between January 2004 and July 2017. Patients with a synchronous second primary or with positive cervical lymph nodes without known primary tumor despite p16 positive status of the involved nodes were excluded from analysis. Local ethics committee approval was obtained before the start of the chart review.Patients were scored for comorbidity, assessed with the adult comorbidity evaluation 27 (ACE27) , and for2 3-weekly, or RT concomitant with weekly cetuximab, an EGFR-inhibitor, if no contra-indications were present. Patients treated with primary surgery were excluded from analysis. Treatment was decided by the institutional multidisciplinary tumor board. After RT patients were followed every 2 months during the first 2 years, every 3 months in the third year, every 4 months in the fourth year, every 6 months in the fifth year and yearly thereafter.All patients were treated with curative RT to an EQD2 of 70Gy. Locally advanced tumors (stage III and IV 7th Ed) were treated with concomitant chemoradiotherapy (CRT), cisplatin 100 mg/mHPV status was evaluated by p16 immunohistochemistry (IHC) staining on formalin-fixed, paraffin-embedded tumor tissue. The biopsies were obtained during the diagnostic work-up of the patients. More than 70% diffuse nuclear and cytoplasmic staining was considered as HPV positive, less than 70% p16 staining was considered as HPV negative. P16 staining could not be performed if only fine needle aspiration cytology was obtained or if tissue blocks were not available. Patients with unknown HPV status were not eligible for analysis.All HPV+ OPC were classified according to the TNM 7th Ed and to the cTNM 8th Ed. In the new cTNM 8th Ed, the T-stage remained practically unchanged apart from T4a and T4b (7th Ed.) combined to one T4-stage. N-stage, on the other hand, has changed extensively except for N0 and N3. Ipsilateral lymph nodes smaller than 6 cm independent of the number of suspicious lymph nodes, were all classified as N1. Bilateral or contralateral lymph nodes smaller than 6 cm were classified as N2 instead of N2c. Stage groups were stage I for T1T2 N0N1 tumors, stage II for T1-T3 N2 and T3 N0N1 tumors and stage III for T4 or N3 tumors.  were at low risk. Intermediate risk was reserved for HPV+ OPC with more than 10 smoking pack years and N2b-N3-stage (7th Ed), together with the HPV− OPC with less than 10 pack years and T1-T3-stage. HPV− OPC with more than 10 smoking pack years or with T4-stage were at high risk  , comorbidity index and T-stage (7th Ed)  , locoregional control (LRC) and distant metastatic control (DMC) were calculated from the date of histological diagnosis to the date of death from any cause, to the date of locoregional relapse  or to the date of distant metastases. OS rates were estimated by the Kaplan-Meier method and comparison between the curves was carried out using the log-rank test. The Cox regression model was used for analyzing the association between prognostic factors and OS. Given the limited number of events in the HPV+ OPC, a backward selection procedure was applied to construct a multivariable model. LRC and DMC were estimated by the cumulative incidence function using the Fine and Gray model, accounting for death as competing risk and were compared by Pepe and Mori test. The Harrell's concordance index (Harrell's C-index) was used as a measure of discriminative performance. The maximum value of the Harrell's C-index, 1, indicates a perfectly discriminating model while a value of 0.5 indicates that discrimination is not better than chance . All tesPatients charts of 333 patients with OPC were reviewed. P16 status could be determined of 260 patients of whom 100 (38.5%) were p16 positive and as a result considered HPV+. Patient characteristics of the 260 patients included in this study are listed in Table There were significantly more smokers and smokers with more than ten pack years in the HPV− group than in the HPV+ group. The mean number of pack years was also significantly higher in the HPV− group. Patients with HPV− OPC had significantly more comorbidity, measured with the ACE27 comorbidity index. The predominant TNM 7th Ed stage was stage IVa for HPV− as well as for HPV+ OPC. T-stage, N-stage and TNM group staging distribution (7th and 8th Ed) are shown in Table p < 0.002). Locoregional control (LRC) was also significantly better in the HPV+ OPC with a 5Y-LRC of 85.8%  compared to the HPV− OPC [68.0% ] (p < 0.001). Distant metastatic control (DMC) of HPV+ and HPV− OPC was comparable .  overall survival (OS) was significantly better in the HPV+ OPC than in the HPV− OPC  vs. 52.5% ; p = 0.03), tumor volume (p = 0.003) and comorbidity (p = 0.009) were significant determinants of OS for HPV+ OPC but not for HPV− OPC. The use of concomitant cisplatin or cetuximab and age were not significant for neither HPV+ nor HPV− OPC. In multivariable analysis, tumor volume (p = 0.001) and comorbidity (p = 0.004) were independent predictors of OS for HPV+ OPC, while smoking pack years was borderline non-significant    = 4.24 ] and for stage III compared to stage I [p = 0.004 HR = 5.40 ]. Nevertheless, there was no significant difference between stage II and III . The Harrell's C-index of the TNM 8th Ed was 0.67 .The 5Y-OS for TNM 7th Ed stage II, III, IVa and IVb were 88.9% ; 70.0% ; 71.4%  and 29.8% , respectively , was 82.9%  vs. 52.2%  for the intermediate risk group, HPV+ OPC with more than 10 pack years and N2b-N3-stage, resulting in a Harrell's C-index of 0.62 . Applying the model of Rietbergen et al. the 5Y-OS for the low risk group, HPV+ OPC with ACE 27 score 0 or 1, was 81.1%  and for the intermediate risk group, HPV+ OPC with ACE 27 score 2 or 3, 44.9% , with a Harrell's C-index of 0.62 .p = 0.005)   and smoking (≤ of > than 10 pack years)  Figure . The HarAlthough the model of Ang et al. also stratifies according to N-stage (7th Ed), we did not incorporate this in our proposed risk groups as the N-stage division into N2a vs. N2b does not exist anymore in the new cTNM 8th Ed and we could not observe a significant difference either in OS between the different N-stages RT and shows a clear survival difference between stage I vs. stage II and III. As expected, most of the HPV+ OPC were diagnosed as stage IV according to the TNM 7th Ed, while most HPV+ OPC were diagnosed as stage I according to the 8th Ed. Although, currently, this switch in stage classification does not yet lead to a shift in treatment, the psychological impact for the patients being diagnosed with a stage I instead of a stage IV disease and the opportunity to give accurate prognostic information are crucial for good patient care.To our knowledge, this is the first European publication showing the improved prognostic and differentiating ability of the clinical TNM 8th Ed for patients treated exclusively with primary (C)RT. Although two of the seven centers in the original O'Sullivan et al. study were European, the ICON-S population mainly existed of a northern American population. As previous research has shown that the European and American population differ in terms of risk profile, it is valuable to validate the TNM in a European population , 8. ReceOur results are in keeping with the findings of previous work by Australian researchers showing a significant OS difference between stage I and II and between I and III but not between stage II and III . LookingAlbeit the prognostic ability of the 8th edition is better than the 7th edition, the TNM staging system alone is not performant enough for patient selection for de-intensifying studies. Previously, the risk group classification of Ang et al. and the European risk group classification of Rietbergen et al. have been proposed as patient selection models for de-intensification trials  all as N1-stage. Interestingly, not all patients within the N1-stage (8th Ed) can be treated in the same manner as O'Sullivan et al. showed that HPV+ OPC with more than 10 pack years and N2b-stage (7th Ed) have a reduced distant control when treated with RT alone compared to CRT . IncludiUnfortunately, the Harrell's C-index of the new model was not better than the cTNM 8th Ed and our patient population was too small to make firm conclusions. Therefore, our proposed risk group classification is purely hypothesis generating and must be validated in the future. In spite of this, it points out the importance of patient selection in a HPV+ OPC population.The strength of this paper is that all patients were treated with curative primary RT and all HPV+ OPC were classified according to the clinical TNM 8th Ed. Our study has a couple of limitations, particularly the retrospective nature of the data collection and the rather small fraction of HPV+ OPC influencing the strength of our analysis. The cTNM 8th Ed was additionally only validated in the HPV+ OPC and not for the HPV− OPC. Although, in practice, the TNM 8th Ed for HPV− OPC with the addition of N-stage N3b, will only change the group staging of a very restricted subgroup with a switch from stage III or IVa to IVb and will consequently only have minimal influence on the predicted prognosis.in situ hybridization (ISH) to reduce the number of false positives, nevertheless, the best method for HPV detection and their prognostic implications remain controversial. Lewis et al. found that all p16 positive OPC, whether HPV positive by ISH, HPV negative by ISH alone, or HPV negative by ISH and PCR, showed statistically significant better overall and disease free survival than p16 negative OPC radiotherapy. The prognosis of HPV+ OPC is additionally influenced by patient specific characteristics such as smoking pack years and comorbidity, demonstrated by the risk group classifications of Ang et al. and Rietbergen et al. We propose a new risk group classification combining the cTNM 8th Ed with these risk factors. The low risk group patients defined as stage I, never smokers or smokers with less than 10 pack years and low comorbidity have an excellent prognosis and may benefit from de-intensification trials. In addition, this study emphasizes the importance of patient selection and personalized treatment for HPV+ OPC.This study was carried out in accordance with the recommendations of the ethics committee of the University Hospitals of Leuven. This protocol was approved by the ethics committee of the University Hospitals of Leuven. This manuscript involves research with human subjects and was conducted in accordance with the World Medical Association's Declaration of Helsinki. This retrospective study was exempt from patient written informed consent by the ethics committee of the University Hospitals of Leuven.Each author has provided substantial contributions to warrant authorship. SD and SN contributed conception and design of the study; SD organized the database; RD and EH performed the immunohistochemistry staining; AL and SD performed the statistical analysis; SD and SN wrote the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oncorhynchus mykiss, aside from yellow-albino phenotypes, cobalt blue variants with autosomal, recessive inheritance have also been reported. In this study, we investigated the inheritance pattern and chromatophores distribution/abundance of cobalt blue trouts obtained from a local fish farm. Based on crosses with wild-type and dominant yellow-albino lines, we could infer that cobalt blue are dominant over wild-type and co-dominant in relation to yellow-albino phenotype, resulting in a fourth phenotype: the white-albino. Analysis of chromatophores revealed that cobalt blue trouts present melanophores, as the wild-type, and a reduced number of xanthophores. As regards to the white-albino phenotype, they were not only devoid of melanophores but also presented a reduced number of xanthophores. Cobalt blue and white-albino trouts also presented reduced body weight and a smaller pituitary gland compared to wild-type and yellow-albino phenotypes. The transcription levels of tshb and trh were up regulated in cobalt blue compared to wild type, suggesting the involvement of thyroid hormone in the expression of blue color. These phenotypes represent useful models for research on body pigmentation in salmonids and on the mechanisms behind endocrine control of color patterning.Albinism is the most common color variation described in fish and is characterized by a white or yellow phenotype according to the species. In rainbow trout  The integumental pigmentary system of fish shares several conserved features with other vertebrate classes . One of tyr-1, tyr-2, and slc45a2 were shown to be implicated in pigmentation [In rainbow trout, different color variations have been described such as the albino, palomino, and cobalt/metallic blue phenotypes ,12. Regaentation ,10. Howeentation , suggestThe inheritance of albino phenotypes is determined mainly by recessive genes , but theUnder this scenario, this study aimed to better characterize our cobalt blue trouts by performing crosses with wild-type and dominant yellow-albino strains of rainbow trout. We first investigated the inheritance of body color in these and in another phenotype, named as white-albino. Then, we also compared the density of chromatophores, body growth, and pituitary morphology of these four phenotypes.This study was conducted in accordance with animal rights of Colégio Brasileiro de Experimentação Animal (COBEA) and approved by Comitê de Ética em Experimentação Animal do Instituto de Pesca de São Paulo, Brazil (04/2017).A total of 137 rainbow trout juveniles with total body weight of about 100 g and cobalt blue phenotype were obtained from a local trout farm at Sapucaí-Mirim . According to the farmers, these cobalt blue rainbow trout juveniles were produced by crosses between cobalt blue and wild-type phenotypes. They were transferred to Salmonid Experimental Station at Campos do Jordão (SES-CJ) and reared for more 12 months until reaching sexual maturity for being used as broodstock in crosses experiments. Broodstock fish of wild-type and dominant yellow-albino strains from SES-CJ were used in crosses with cobalt blue females and males.loci. For those reasons, we have ascribed the letter B for the allele responsible for blue color due to its dominance over wild-type (see 3 solution (pH 8.0) for sperm activation. After two minutes, eggs were hydrated for 20 minutes and then washed with water. Egg incubation and larvae-juvenile rearing were performed separately in open flow through systems at temperatures ranging from 10 to 13 °C.To verify the inheritance of these phenotypes in relation to others, both females and males of cobalt blue rainbow trout were crossed between themselves and with the following strains: wild-type and dominant yellow-albino. According to a previous study on cobalt blue rainbow trout  and alsotype see , and A fDanio rerio) [Two-year-old fish with wild-type, yellow-albino, cobalt blue, and white-albino phenotypes were euthanized in an overdose of benzocaine solution. After complete removal of scales, skin tissues with about 2 cm x 2 cm from the dorsal region located between the lateral line and dorsal fin were stripped off. Fresh skin samples were mounted in slides and analyzed under a fluorescent microscope . Images were captured with a CCD Camera (DP73) and analyzed by CellSens software . The number of xanthophores from five to seven fields were counted for each color phenotype. The area of those cells (n = 15 cells per color phenotype) was measured using Image J software . This meo rerio) ,24.3.Body weight (BW) and standard length (SL) were measured in four color phenotypes (n = 14 to 25) obtained from the cross between blue female (F0) and white male (F1). All measurements were performed in 18 months-old adult females. Males were not included in the analysis due to the presence of precocious males. Condition factor (K) was calculated as BW (Kg)/SL (cm) The pituitary was carefully dissected from juveniles (about 6 months) and adult females and males (about 2 years) from the four color phenotypes . The rel® Green PCR Master Mix (ABI), 1 μl of first strand cDNA (approximately 25 ng) and 5 pmol of each primer in a Step One Real Time PCR System , using standard program. Transcript abundance was quantified using the Standard Curve Method with four dilution points and normalized against respective β-actin values.The mRNA expression of growth hormone, prolactin, deiodinase 1 and 2, thyroid stimulating hormone, and thyroid-releasing hormone was analysed in the pituitary of female adult trouts from cobalt blue  and wild type phenotypes . Samples were fixed in RNAlater and stored at -80 °C until processing. Total RNA was extracted from pituitary using 1 ml of TRIZOL Reagent (Invitrogen) following the manufacturer’s instructions. RNA samples (1 μg) were treated with Deoxyribonuclease I Amplification Grade (Invitrogen) and reverse-transcribed using SuperScript III RNase H- (Invitrogen) with oligo(dT)12-18, following the manufacturer’s instructions. Primer sequences, amplicon size, and GenBank accession numbers are described in p < 0.05.The significance of deviations from 1:1 and 3:1 in the proportion of color phenotypes in the progenies was analyzed by the Chi-square test, with Microsoft Excel 2013 . The difference between groups was analyzed by One-way ANOVA followed Bonferroni’s test using GraphPad Prism  or by Student’s T test. Differences were considered as significant for Crosses between blue female vs. wild-type male and wild-type female vs. blue male generated balanced proportions of both cobalt blue and wild-type phenotypes . For thrp ≥ 0.05); in the remaining six, the proportion of wild-type were higher than that of cobalt blue in four crosses. From these results we can surmise that those eight individuals are homozygous for the blue color and the other 12 heterozygous for the locus responsible for the blue phenotype.The F1 cobalt blue trout generated from crosses #4, #5, and #6 were used for progeny tests. Single-pair crosses between cobalt blue females (n = 10) and wild-type males, and between wild-type females and cobalt blue males (n = 10) generated eight all-cobalt blue progenies. The remaining 12 crosses yielded mixed-populations of blue and wild-type offspring , wherebyp < 0.05). In the intercross between F1 white-albino trouts, white-albino, yellow-albino, cobalt blue, and wild-type trouts were obtained (p < 0.05), respectively, as expected from a cross between double heterozygous, based on Mendelian segregation. The F1 yellow trouts were also intercrossed, yielding yellow and wild-type trouts (p < 0.05), respectively, as expected from a cross between trouts with both alleles recessive for the blue locus (bb) and those for yellow in heterozygous condition (Aa), based on Mendelian segregation.In a cross between F0 cobalt blue and F1 white-albino trouts, white-albino, cobalt blue, yellow-albino, and wild-type trouts were obtained  in a proobtained ; Fig 1 ie trouts ; Fig 1 iThe analysis of melanophores demonstrated that these cells were present in wild-type and cobalt blue  whereas in yellow- and white-albino rainbow trouts they were absent  revealedp < 0.05)  .p < 0.05). No significant differences were found between wild-type and yellow-albino as well as between cobalt blue and white-albino phenotypes.The comparison of pituitary morphology showed that juveniles and adults of cobalt blue and white-albino presented a smaller pituitary compared to wild-type and yellow-albino rainbow trout . The valgh), prolactin (prl), deiodinase 1 (deio1) and 2 (deio2) genes did not present any difference between cobalt blue and wild-type phenotypes (tshb) and thyroid-releasing hormone (trh) compared to the wild-type (2) in both phenotypes  and a general profiling of neuroendocrine hormones is being currently considered.Although a more detailed analysis is still required, particularly in terms of ontogeny, structure, and hormone secretion of pituitary, an intriguing aspect of cobalt blue trouts lies in their resemblance to the sea run form. This stage is characterized by a blueish color on dorsal region, silvery/whitish color on the belly and undersides, and an elongated body shape, which arises through the smoltification process during transition from freshwater to marine environment . This prrolactin –40. Baserolactin  and controlactin ,43, it i hormone –43. The loci to be expressed. The analysis of chromatophores revealed that white-albino trouts were devoid of melanophores, as the yellow-albino phenotype, and presented a reduced number of xanthophores. In medaka, another population of chromatophores called leucophores responsible for the white color have been described, however these cells are absent in zebrafish and rainbow trout [Compared to yellow-albino trouts, the white-albino is a rarer phenotype with few descriptions in fish species . These tow trout ,46,47. Ttshb and trh genes. Comparative studies among color variations in trout can be useful for endocrinology studies related to pigmentation process, particularly to ontogeny and specification of chromatophores, to growth performance, and also to pituitary development.In conclusion, this study shows that the cobalt blue rainbow trout presents dominant inheritance in relation to wild-type and co-dominance with dominant yellow-albino trouts; in this last case a fourth phenotype called white-albino was generated. The allele responsible for the reduced number of xanthophores is supposedly a mutation in a gene related to the regulation of HPT , possibly upstream S1 TableStandard length  and total body weight  of juveniles and adult fish used for pituitary comparison analysis. Data are represented by the average ± SD. Numbers between brackets represent the sample size per color phenotype.(DOCX)Click here for additional data file.S2 TableGene names, primers sequence, amplicon size in base pairs and GenBank accession numbers of sequences used for primers design.(DOCX)Click here for additional data file.


The structural and electronic properties of a monolayer and bilayer blue phosphorene/graphene-like GaN van der Waals heterostructures are studied using first-principle calculations. The results show that the monolayer-blue phosphorene/graphene-like GaN heterostructure is an indirect bandgap semiconductor with intrinsic type II band alignment. More importantly, the external electric field tunes the bandgap of monolayer-blue phosphorene/graphene-like GaN and bilayer-blue phosphorene/graphene-like GaN, and the relationship between bandgap and external electric field indicates a Stark effect. The semiconductor-to-metal transition is observed in the presence of a strong electric field. Two-dimensional 2D) materials such as graphene , transitD materiaAmong the above-mentioned 2D semiconductor materials, blue-P monolayer has been prepared by epitaxial growth on Au (111) substrates for the first time in 2016 . Z. ZhanEg) with the vertical external electric field (Eext) in the blue-P/g-GaN vdW heterostructures are evaluated and conducted by using the first-principles calculations with vdW-corrected exchange-correlation functional.In this work, the electronic structural properties and the variation tendency of the bandgap energy  , which ieraction  is descrx/y. The evolution of the total energy difference as a function of δx and δy is shown in our previous studies  [Eb = 70 meV] [Eb = 25 meV] [Several structures shown in our previous work have been studied as a benchmark to obtain the most stable structure of the bilayer heterostructures . The opt studies . Figure  studies . Figure  studies . The Eb  60 meV] , blue-P/ 25 meV] .Fig. 1a Eg of 1.26 eV and 1.075 eV calculated by using GGA, respectively. For the HSE06 method, the Eg is 2.2 eV and 1.91 eV, respectively. For both heterostructures, the minimal-energy states in the conduction band are near M point and the maximal-energy states in the valence band are at K point, the two points are not at the same crystal momentum in the Brillouin zone. Thus, the bandgap is an indirect band gap for both semiconductor heterostructures. The Eg of monolayer-blue-P/g-GaN heterostructure decreases 0.63 eV compared with the monolayer-blue-P (1.89 eV), while the Eg of bilayer-blue-P (1.118 eV) shrinks 0.043 eV in contrast to bilayer-blue-P/g-GaN heterostructure. The band bending can be achieved from the difference between the Fermi levels of the blue-P with the g-GaN system and the free-standing blue-P [EF = W − WP, where W is the work function of the composed system (blue-P/g-GaN), and WP is the work function of the pristine blue-P. The ΔEF of − 1.17 eV and − 0.81 eV for the monolayer-blue-P/g-GaN heterojunctions and the bilayer-blue-P/g-GaN heterojunctions are obtained respectively, as shown in Fig. Figure g blue-P : ΔEF = WEext on the electronic structure, the band structures are calculated with different Eext for the blue-P/g-GaN heterostructures. As reported in previous work, the geometrical structure of the heterostructure can be neglected, but the band structure changes greatly under different Eext [Eg as a function of the Eext from − 1.0 eV/Å to 1.0 eV/Å. The direction of Eext from top (g-GaN layer) to bottom (blue-P layer) is taken as the forward direction. It is clearly shown that monolayer-blue-P/g-GaN and bilayer-blue-P/g-GaN heterostructures exhibit a bandgap modulation with the Eext. For monolayer-blue-P/g-GaN, in the case of the forward Eext, the Eg increases linearly with the increasing Eext ≤ 0.4 eV/Å (L-increase range). The monolayer-blue-P/g-GaN obtains its maximum Eg when Eext = 0.5 eV/Å and shows little change when Eext is in the range 0.4 < Eext < 0.6 eV/Å (saturation range), which enhances the band offsets so as to promote the separation of electron-hole pairs. The initial enlargement in Eg is attributed to the counterbalance of Eext to some extent by the built-in electric field (Eint). The Eg comes to a linear decrease range with increasing Eext > 0.6 eV/Å (L-decrease range). Thus, the heterostructure shows a metal behavior when it is subjected to a stronger electric field. This is originated from the dielectric breakdown as well as charge tunneling. In contrast, the Eg declines linearly with increasing Eext (L-decrease range) under a reverse Eext, caused by the conduction band minimum (CBM) band edge shifting toward to the valence band maximum (VBM). However, when Eext = − 0.7 eV/Å, the bandgap begins to decrease sharply, which may be due to the breakdown. When Eext < − 0.8 eV/Å, the blue-P/g-GaN heterojunction experiences a transition from semiconductor to metal . These results reveal that both Eg and semiconductor to metal transition of the blue-P/g-GaN heterostructure is dependent on electrostatic gating, which could be used in high-performance electronic and optoelectronic devices. In addition, the effect of Eext on the Eg between the bilayers of blue-P and g-GaN heterostructure is the same as the single layer but with a smaller electronic field for transition from semiconductor to metal.The heterostructure is often subjected to an external electric field to tune its electronic properties while applied to nanoelectronic devices. In order to study the influence of the ent Eext . Figure Eext of 0.3 eV/Å, 0.5 eV/Å, − 0.3 eV/Å, and 0.7 eV/Å are shown in Fig. Eext, the Eg increases to 1.651 eV and 1.757 eV. This indicates the quasi-Fermi level of the g-GaN monolayer is shifted downward, and the quasi-Fermi level of blue-P monolayer is lifted upward. However, in Fig. Eext, the Eg decrease to 0.888 eV and 0.49 eV. The quasi-Fermi level of g-GaN moves upward, and the quasi-Fermi level of blue-P moves downward. The results show that the bandgap varies linearly with the applied vertical Eext, indicating a giant Stark effect [Eext, the subband states of the valence and conduction valence would undergo a mixing, leading to a field-induced splitting of the electronic levels. The electrostatic potential difference induced by the external field considerably changed the electronic structures near the Fermi level [To explore the effect of electric field on the band structure, the relation between the energy band structures and the external electric field are calculated. The band structures of the monolayer-blue-P/g-GaN heterostructures with k effect . Upon apmi level .Eext value of 0.3 eV/Å, 0.5 eV/Å, − 0.3 eV/Å, and − 0.7 eV/Å, respectively. Upon applying a forward Eext, as exhibited in Fig. Eext induces electrons to accumulate/deplete at the opposite side, as visualized in Fig. EVBM rise, while the quasi-Fermi level of blue-P monolayer and ECBM decrease, resulting in a linear reduction on bandgap. Simultaneously, electrons are transferred from blue-P to g-GaN under a reverse Eext. It is found that the amount of the transferred charge increases with the increase of electric field intensity. Figure Eext modulates the electronic property, the integrated charge density difference of the monolayer-blue-P/g-GaN heterostructure as a function of the perpendicular distance is calculated, displayed in Fig. Eext = 0, the charge density difference of the heterostructure is obtained by ∆ρ = ρheterostructure−ρg-GaN−ρblue-P. The change of the plane-average charge density difference at interfaces indicates that the electrons were transferred from the g-GaN layer to blue-P layer across the interface, whereas the holes remained in the g-GaN side. The surface averaged differential charge with an electric field is calculated for 0.3 eV/Å and − 0.3 eV/Å. The Eext can exert influence on transferring charges in the heterostructure. It can be described as [To make it clear that how ribed as \documentx, y, z) point in the supercell of the monolayer-BP/g-GaN heterostructure with and without Eext, respectively. The direction of charge transfer induced by the negative (blue line) Eext is opposite to that of the positive (red line) Eext. The integrated charge density quantitatively illustrates that the amount of transferred charges increases with the strength of the Eext. The value of the charges transfers for the blue-P/g-GaN heterostructure with 0.3 eV/Å of Eext is larger than that of 0 eV/Å and − 0.3 eV/Å, because the positive external electric field localizes the charges along the direction of the applied field, confining the charges to g-GaN planes.where In order to distinguish the contributions of blue-P and g-GaN in the band structure, the projected state density of the heterostructures is calculated and shown in Fig. Eg of monolayer-blue-P/g-GaN and bilayer-blue-P/g-GaN can be continuously tuned by Eext, and the relation between Eg and Eext indicates a Stark effect. The Eg becomes zero at − 0.8 and 0.9 eV/Å for monolayer-blue-P/g-GaN, and − 0.5 and 0.7 eV/Å for bilayer-blue-P/g-GaN, indicating a transition from semiconductor to metal.In summary, the structural and electronic properties of the monolayer-blue-P/g-GaN and bilayer-blue-P/g-GaN vdW heterostructures are investigated by using first-principle calculations. The results show that the monolayer-blue-P/g- GaN heterostructure is an indirect band gap semiconductor with intrinsic type II band alignment. The band offset and


Mental health is reportedly influenced by the presence of green and blue space in residential areas, but scientific evidence of a relation to psychotic disorders is scant. We put two hypotheses to the test: first, compared to the general population, psychiatric patients live in neighborhoods with less green and blue space; second, the amount of green and blue space is negatively associated with the duration of hospital admission. The study population consisted of 623 patients with psychotic disorders who had been admitted to the psychiatric ward of an academic hospital in Utrecht, The Netherlands from 2008 to 2016. Recovery was measured by length of stay. Structured patient data was linked to socio-economic status and the amount of green and blue space in the residential area. Associations were assessed by means of regression models controlling for confounding factors. Compared to the general population, psychiatric patients had a significantly lower amount of green space in their neighborhood. This result was not confirmed for blue space. Furthermore, no significant associations were found between green and blue space and the duration of hospital stay. In conclusion, previous studies focusing on other mental disorders, like anxiety or depression, found positive mental health effects of green and blue space in the neighborhood. We were not able to confirm significant effects among our study population on duration of admission, however. Future research focusing on psychotic patients could investigate the influence of exposure to green and blue space on other influences and outcomes on mental health. A high burden of disease is caused by mental disorders , particuThis study addresses that gap in the literature by investigating the possible protective effect of living near green or blue space among patients with diagnosed psychotic disorders in the province of Utrecht, The Netherlands. Specifically, it assesses the relation between green space or blue space in the residential environment of patients that have been diagnosed with a psychotic disorder and admitted to an academic hospital. We hypothesized that psychiatric patients live in neighborhoods where the absence of green or blue space is more pronounced than in areas occupied by the general population. Then we tested whether the availability of green or blue space is negatively associated with the duration of admission (as a measure of recovery) for the psychotic disorder.In 2013, the Province of Utrecht had 640,610 addresses and 1,245,294 inhabitants , of whomThe health data originate from the UMC Utrecht clinical databases. To guarantee privacy, data from patients who used the inpatient services of the psychiatric department of the UMC Utrecht were anonymized and stored in the UMC Utrecht research database for analysis. We selected patients with the diagnosis ‘schizophrenia spectrum and other psychotic disorders’ according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). As dependent variables for subsequent regression models, we operationalized the length of stay as a measure of severity/recovery. The length of stay refers to the number of days for which patients had been admitted to the psychiatric ward at the UMC Utrecht, so the duration could only be calculated for inpatients.This study was approved by the medical ethics committee of the UMC Utrecht (reference number WAG/nt/16/033895).The key explanatory variables were the availability of green and the availability of blue space, both given in percentages, near the patient’s home. Green space refers to agricultural areas, natural areas, or artificially installed greenery; blue space refers to fresh water or salt water bodies. Both green and blue space data were extracted from the most recent Dutch land use database for 2012 with a spatial resolution of 25 × 25 m per raster cell . This laBesides environmental exposure, the analyses considered both individual and area-based covariates. Covariates on an individual level were gender and age (in years). Covariates based on area were urbanicity and socio-economic status. Urbanicity adjusts for urban-rural differences in mental health  and is o2 tests. We carried out non-parametric Spearman correlations between the variables to gain insight into the bivariate associations and to identify problems due to multicollinearity. We selected the Spearman correlation coefficient because our data did not exactly follow a normal distribution. To study the extent to which natural environments (green and blue space) are associated with the severity of symptoms of psychotic patients, we performed a multivariate regression analysis by means of fitting ordinary least squares regression models. We regressed the dependent variable ‘length of stay’ on blue space and green space while adjusting for the covariates including gender, age, socio-economic status and urbanicity. We estimated two different models with increasing complexity. Model 1 included green and blue space and was adjusted for gender and age. Model 2 additionally considered urbanicity and area-based socio-economic status. SPSS 22.0  was used for the data preparation and statistical analysis.We used descriptive statistics to summarize the data. To test the hypothesis that psychiatric patients live in neighborhoods with less green and blue space compared to the general population, we performed a series of ChiOur study population consisted of 623 patients, of whom 70% were male . The mea2 tests showed that psychiatric patients live significantly more often in neighbourhoods with no green space than the general population of the province of Utrecht . This result was not confirmed for blue space. The proportion of psychiatric patients with no blue space within their residential area did not significantly differ from the general population .F-test of the partially adjusted regression model 1  and the fully adjusted model 2  showed statistical significance  known to affect mental health. Third, our sample showed limited variance concerning the availability of green and blue space for inhabitants of Utrecht as well as for patients with schizophrenia and psychotic disorders. Further, the skewness in these two environmental variables made them less powerful as predictors in the analyses. The amounts of green and blue space are calculated around a patient’s home address that was registered by the hospital. But as argued elsewhere , people In view of the mixed findings we suggest three ways to pursue more reliable insight into the interaction between green and blue space availability and psychotic disorders. First, the majority of studies have focused on the natural environment, either in the neighborhood or as operationalized through buffers, rather than using nature exposure during the day . Advances in geotechnology and GPS make it possible to refine exposure assessments in future research. Similarly, historical data on natural environmental exposure has rarely been incorporated in research, although the influence of exposure over the life course might be of great interest ,28.This study explored how natural environments may be associated with psychotic disorders. Our results did not support the increasingly reported mental health benefit of green and blue space. Across our regression models, the associations tuned out to be insignificant, though the direction of the effect was in line with our hypothesis. Nonetheless, this study does contribute to the literature by exploring the relationship between environments and psychotic disorders by analyzing clinical data, which so far has rarely been attempted.


Analysis of public databases and resected HNSCC specimens revealed that HPV status was independent of PD-L1 expression and TMB in HNSCC. However, HPV infection significantly increased T-cell infiltration, immune effector cell activation and the diversity of T-cell receptors. Notably, HPV-positivity correlated with increased immune cytolytic activity and a T-cell-inflamed gene expression profile. This work provides evidence that HPV status can be used to predict the effectiveness of PD-1 inhibitors in HNSCC, independently of PD-L1 expression and TMB, and probably results from an inflamed immune microenvironment induced by HPV infection.Chemotherapy and radiotherapy predominantly improve the clinical outcomes of patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC). Whether this superiority goes on when treated with immune checkpoint inhibitors is still unclear. This study sought to determine the predictive value and potential mechanisms of HPV status for the treatment of programmed cell death 1 (PD-1)/ligand 1(PD-L1) inhibitors. We conducted an integrated analysis of the relationships between HPV status and PD-L1, tumor mutation burden (TMB) and inflammation-related immune cells and molecules, based on the analysis of repository databases and resected HNSCC specimens. The pooled analysis of overall survival (OS) and objective response rate (ORR) suggested that HPV-positive patients benefited more from PD-1/PD-L1 inhibitors than HPV-negative patients (OS: hazard ratio (HR) = 0.71,  Because the majority of HNSCC patients are diagnosed at an advanced stage (stage III to IVB), HNSCC is associated with poor prognosis and a high mortality rate. Traditional treatment strategies typically consist of doublet platinum-based chemotherapy, which is associated with significant toxicity and has limited therapeutic effect2. Novel therapeutic strategies that can broaden the current treatment schedule of HNSCC are urgently needed.Head and neck squamous cell carcinoma (HNSCC) includes cancers of the oral cavity, oropharynx, and larynx, and accounts for approximately 4% of cancers worldwide5. However, the response rate to PD-1/PD-L1 blockade among biomarker-unselected groups is still far from satisfactory. Nowadays, PD-L1 levels are usually determined by immunohistochemical (IHC) staining in clinical practice, but prediction of the response to anti-PD-1/PD-L1 is imperfect owing to dynamic and heterogeneous expression6. Because the immune environment of HNSCC is quite complicated, a more comprehensive understanding is needed to guide new ICI applications.The development of immune checkpoint inhibitors (ICIs) has revolutionized immunotherapy. Anti- programmed cell death 1 (PD-1)/ligand 1(PD-L1) therapy is effective in multiple tumor types, and can bring remarkable clinical benefits with limited toxicity. Several clinical trials have proved that anti-PD-1/PD-L1 therapy results in clinically meaningful antitumor activity and an acceptable safety profile when used to treat HNSCC patients8. The primary clinical data have revealed that the response rate to PD-1/PD-L1 is higher in HPV-positive patients than in HPV-negative patients4. HPV status may be another factor that can be used to classify HNSCC and identify patient suitability. However, the underlying mechanism and the potential association between HPV status and the tumor immune environment have not yet been fully characterized.Human papillomavirus (HPV)-associated HNSCC appears to have distinct biological and clinical features; it is associated with better prognosis than HPV-negative HNSCCIn this study, we attempted to elucidate the relationship between HPV status and immune environment elements that account for the response to PD-1/PD-L1 blocking. We conducted an integrated analysis incorporating HPV status, PD-L1 expression, content of CD8 + T-cell infiltration and tumor mutation burden (TMB), based on data from a multicenter database. Finally, we determined that an HPV-positive status contributes to T-cell infiltration and enhances cytolytic activity (CYT), which results in a better response to anti-PD-1/PD-L1 therapy in HNSCC patients.9 cohorts were retrieved from online data repositories. The TCGA cohort comprises 451 HNSCC patients, 155 with confirmed HPV status. HPV positive was defined as a positive p16 IHC or HPV In-Situ Hybridization (ISH) result or an HPV viral titer over 30. We retrieved their RNA and protein expression profiles, copy number alteration (CNA) information and gene mutation data from the cBioPortal website. The GSE40774 cohort comprises 134 HNSCC patients, and we obtained their associated data from the Gene Expression Omnibus (GEO) database, including detailed information about each patient’s HPV status and RNA sequencing. A total of 52 HNSCC patients had confirmed HPV status, and the associated CNA information and gene mutation profiles were extracted from the MSK-IMPACT cohort as a subgroup. HNSCC-tissue microarray (TMA) cohorts containing a total of 130 tissues were obtained from Shanghai Biochip Co., Ltd (Horac080PG01) and Alenabio Biotechnology Co., Ltd . All patients provided specimens for HNSCC-TMA with written informed consent. Human tumor samples from TCGA and GEO database were available of patient consent and tumor quality. Additional publicly available data sets used in this study are listed in Supplementary Table Data for The Cancer Genome Atlas (TCGA), GSE40774 and Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT)4, KEYNOTE-0123, KEYNOTE-0555, and NCT01693562 (HAWK)10) and the ORR data for 589 patients from six trials . The baseline characteristics of the enrolled trials are summarized in Supplementary Table We carried out a pooled analysis of the efficacy of PD-1/PD-L1 inhibitors in HPV-positive and -negative HNSCC patients. We analyzed the OS data for 425 patients from four trials (CheckMate-14113.Data extraction from eligible studies was performed independently by two authors (Xue-Jun Guo and Qin Zeng). Hazard ratios for the OS analysis were calculated using the Tierney methodology if not immediately available from the primary reportInk4a antibodies . The IHC-stained tissue sections were scored separately by two pathologists blinded to the clinical parameters. The PD-L1 expression of tumor cells and immune cells was evaluated using a three-tiered grading system: tumor cell (TC) 3/immune cell (IC) 3: ≥50% for TC or ≥10% for IC; TC2/IC2: 5–49% for TC or 5–9% for IC; TC0-1/IC0-1: <5% for TC or IC. We assessed the percentage of CD8+ lymphocytes among all nucleated cells in the stromal compartments. Scoring cut-off points were set at 10% or 25% for each core, according to the cell density: low density: <10%; moderate density: 10–25%; high density: ≥25%15. Positive p16 expression was defined as strong and diffuse nuclear and cytoplasmic staining in ≥70% tumor cells16. The patients and experiments included in this study were approved by the Institutional Ethical Board (IRB) of Nanfang Hospital. We confirmed that all experiments were performed in accordance with relevant guidelines and regulations.Samples for HNSCC-TMA were collected using 1.5-mm diameter core needles from tumor regions with the most representative histology of each surgical specimen. Serial sections from the HNSCC-TMA were used for analyzing PD-L1, p16 (HPV) and CD8. Tumor sections were assessed immunohistochemically using PD-L1 , CD8  and p16https://tcga-data.nci.nih.gov/tcga/findArchives.htm). The mutation and CNA data for the MSK-IMPACT cohort were retrieved from the cBioPortal for Cancer Genomics . To assess the mutation burden, the number of mutated genes carrying at least one non-synonymous mutation in the coding region was computed for each tumor. Tumor neoantigens of HNSCC patients in the TCGA cohort were directly obtained from the supplementary materials provided in a previous published study17. If the mutation was predicted to produce a “binder” neopeptide with affinity <500 nM and its corresponding gene expression was greater than 10 Transcripts Per Million (TPM), the mutation would be designated putatively antigenic.The somatic mutation and CNA data for HNSCC patients in the TCGA cohort were retrieved from the TCGA database portal (https://www.ncbi.nlm.nih.gov/geo) respectively. Cytolytic activity (CYT) was defined as the log averages (geometric means) of GZMA and PRF1 RNA expression data in terms of TPM as the previous study suggested17. The T cell-inflamed gene expression profile (GEP) was composed of 18 genes, including CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT1, and TIGIT18. For the calculation of GEP score in TCGA and GSE40774, we used Reads per kilobase of exon per million reads mapped (RPKM) log2 intensity data for each gene, and then averaged the expression for genes in the signature geneset to obtain a signature score per patient.The gene expression data for TCGA cohort and GEO cohorts (GSE40774 and GSE62027) were downloaded from TCGA database portal and GEO repository  test and inconsistency index (I2); values were considered significant when the χ2 P-value was <0.1 or when I2 was >50%. In the absence of statistically significant heterogeneity, the fixed-effect model was used for pooled analysis. Otherwise, the random-effect model was selected. Chi-squared tests were used to analyze differences in the expression levels of PD-L1 and CD8 between the HPV-positive and -negative groups. The correlation between HPV viral titers and immune-related parameters was analyzed by Spearman’s rank correlation. All reported P-values were two-tailed, and for all analyses P ≤ 0.05 was considered statistically significant, unless otherwise specified.Statistical analyses were performed using Review Manager Version 5.2 , GraphPad Prism (version 7.01) and SPSS version 22.0 . Statistical heterogeneity in the pooled analysis was evaluated using the chi-squared (χp = 0.02)  . These results indicate that HPV-positive status may be a potential biomarker for the application of anti-PD-1/PD-L1 therapy, and HPV-related HNSCC patients could benefit from PD-1 blockade.It has been proven that an HPV-positive status is associated with better clinical outcomes for HNSCC patients undergoing chemotherapy or radiotherapy. Therefore, we hypothesized that HPV status could also be used as a biomarker for anti-PD-1/PD-L1 therapy. We conducted a pooled analysis to assess the efficacy of a PD-1/PD-L1 inhibitor in HPV-positive and -negative HNSCC patients. A total of 425 patients who received treatment with PD-1/PD-L1 inhibitors and had confirmed HPV status from four clinical trials were included in the analysis. The pooled analysis revealed that HPV-positive patients experienced greater clinical benefits in terms of overall survival (OS) than HPV-negative patients (hazard ratio (HR) = 0.71; 95% confidence interval (95%CI) = 0.53–0.94; 02) Fig. . Further01) Fig. . These r23. We speculated whether the use of HPV status as a predictive biomarker is dependent on PD-L1 expression. To verify this hypothesis, we conducted an analysis based on gene profiles from The Cancer Genome Atlas (TCGA) and GSE40774 cohorts to compare the RNA profiles of PD-L1 in HPV-positive and -negative HNSCC patients. The results revealed that there was no significant difference in PD-L1 expression between the HPV-positive and -negative groups . A correlation study of HPV viral titers and PD-L1 expression in the TCGA cohort also revealed poor correlation . We further analyzed PD-L1 levels among seven HNSCC cell lines based on the RNA sequencing profiles of the GSE62027 cohort  and found no distinct relationship between HPV status and PD-L1 expression in the HNSCC cell lines  analysis of 207 HNSCC patients from the TCGA cohort revealed no significant correlation between HPV status and PD-L1 protein expression  and HPV, were associated with chromosomal instability and DNA damage repairCD8A RNA levels in the HPV-positive and -negative groups based on gene profiles from the TCGA and GSE40774 cohorts. Our analysis confirmed that HPV-positive patients had higher levels of CD8A than HPV-negative patients in both cohorts . Notably, we also detected a positive correlation between HPV viral titers and CD8A RNA levels . We also created a heat map of the relative RNA expression of immunocyte-related core-enriched genes based on the TCGA and GSE40774 cohorts to identify which kind of immune cells was the predominant subset in HPV-positive tumors. We found that HPV-positive status corresponded strongly with the levels of biomarkers of immune effector cells (effector T (T-eff) cells, natural killer (NK) cells, and B cells), but not with those of immune suppressor cells (regulatory T (T-reg) cell and macrophages)  are proven to be a stable and ideal biomarker for response to PD-1 blockade immunotherapy, we evaluated the . Furthermore, we analyzed the TCR diversity and richness between HPV-positive and -negative tumors based on previously published data19. This revealed that HPV-positive HNSCC had significantly increased TCR diversity  and richness  compared with HPV-negative HNSCC. However, there was no difference in B-cell receptor (BCR) diversity (p = 0.087) or richness (p = 0.477) between the two groups  to identify pathways enriched in specific HPV status. The T-cell receptor (TCR) pathway that functions in immunological recognition was significantly up-regulated in HPV-positive tumors 27. GSEA revealed that the IL-2 pathway, which directly activates the immune response, was significantly upregulated in HPV-positive tumors, whereas the IL-6 and TGFB1 pathways, which negatively regulate the immune microenvironment, were significantly downregulated in HPV-positive tumors -associated cytolytic immune signature—had improved OS when treated with a PD-L1 inhibitorors Fig. . Further28. A heat map of the relative RNA expression levels of the T cell-inflamed GEP showed increased expression in HPV-positive tumors from the TCGA and GSE40774 cohorts  and GSE40774  databases. These findings suggest that HPV-positive HNSCC patients tended to have a T cell-inflamed microenvironment which contributes to PD-1 blockade immunotherapy sensitivity.To obtain a more comprehensive understanding of the relationship between HPV status and the immune microenvironment, we analyzed the T cell-inflamed gene expression profiles (GEPs) that contained 18 genes relating to antigen presentation, chemokine expression, cytotoxic activity and adaptive immune resistance, which were shown to predict response to anti-PD-1–directed therapyrts Fig. . FurtherCD8A, CD4, TGFB1 and CTLA4, were the independent prognostic factors  and clinical samples to clarify the potential mechanisms that account for the HPV-related immunotherapy response, which supported our conclusions.Considering the significant toxicity and limited clinical efficacy of traditional platinum-based chemotherapy, novel therapeutic strategies that can broaden the current treatment regime of HNSCC are urgently needed. The development of ICIs has provided a novel treatment option that could also be useful for recurrent patients. Clinical trials have already proven that anti-PD-1/PD-L1 therapy provides antitumor activity and has an acceptable safety profile when used to treat HNSCC patients, although several studies have reported that HNSCC sufferers respond to PD-1/PD-L1 therapy regardless of their HPV status29. Recent studies have demonstrated that the improved prognosis of HPV-positive HNSCC patients is related to the high responsiveness of these tumors to chemotherapy, radiation and target therapy31. These studies also showed better prognosis in HPV-positive HNSCC patients at every line of therapy, which supported our hypothesis that HPV status may serve as both a prognostic and predictive biomarker in HNSCC. In our study, 4 trials were included in the OS analysis, of which all patients were at second line therapy or above. Our results showed prolonged OS in patients with HPV-positive HNSCC. The analysis of ORR also suggested that HPV-positive HNSCC showed higher response rates than HPV-negative HNSCC, supporting the favorable OS. These results indicate that HPV status may function as both a prognostic and predictive biomarker for ICI treatment and promote an improved prognosis of HPV-positive HNSCC.It has been proposed that HPV-positive HNSCC have a better prognosis than those that are HPV-negative33, and higher response rates have been observed in HNSCC patients with high levels of PD-L1 expression11. In this study, we demonstrated that as a predictive biomarker, HPV is independent of PD-L1 expression. A recent clinical trial on the treatment of recurrent HNSCC with nivolumab revealed that a single-positive PD-L1 or p16 result could predict a better response to nivolumab, whereas a double-positive result could not, which partly corroborates our results4. We speculated whether it was possible to determine if HPV status can serve as a specific biomarker to predict the response to ICIs in PD-L1-negative HNSCC patients. In addition, our study demonstrated that HPV infection promoted T-cell infiltration and produced an inflamed microenvironment, which may subsequently induce PD-L1 expression. The dynamic changes of PD-L1 and TIL in HPV-positive tumors potentially overcome the immune ignorance in those with PD-L1-negative tumors. These mechanisms are the most likely explanation for patients with low or no PD-L1 expression that respond well to anti-PD1/PD-L1 therapy.The expression of PD-L1 is a useful predictive biomarker for checkpoint inhibitors according to clinical trials24. It is well known that chromosomal instability is correlated with an increase in gene mutations. Furthermore, foreign viral antigens from specific viruses were also found to enhance immunogenicity34. To investigate HPV-induced responses to anti-PD-1/PD-L1 immunotherapy, we analyzed the relationships between HPV status and TMB, tumor neoantigens and CNA in HNSCC databases, but found no correlations. Possible explanations may be that other agents predominantly affect the TMB in HPV-positive HNSCC, or that only some specific HPV oncoproteins (such as HPV E7) can increase the TMB or alter tumor aneuploidy. These findings suggest that HPV-induced responses to anti-PD-1/PD-L1 therapy may be independent of tumor genomic alternations.Previous studies have reported that the HPV-16 E7 oncoprotein was found to induce centrosomal abnormalities, thereby disrupting mitotic fidelity and increasing the risk of chromosome missegregation and aneuploidy36. Previous studies have identified the relationship between HPV infection and T-cell-inflamed phenotype37. These findings also supported the improved prognosis of patients with HPV-positive HNSCC that probably resulted from activated immune cell subtypes. Our study also verified that HPV-positive status was significantly correlated with lymphocyte infiltration  and cytolytic activity based on an integrated analysis. However, we also discovered that tumors with HPV infection showed up-regulation of the TCR pathway as well as increased TCR diversity and richness, suggesting enhanced immune recognition. Meanwhile, we observed that HPV positive status was correlated with an increased T cell-inflamed GEP, which was demonstrated to predict the response to anti-PD-1 therapy. Therefore, our study extended the understanding of the effect of HPV status in the immune microenvironment and furtherly verified these pre-clinical perspectives through the analysis of anti-PD-1 therapy for HNSCC trial patients.It has been suggested that virus-infected cancer cells communicate with stromal cells through the secretion of cytokines and chemokines, or by releasing tumor exosomes to alter the tumor microenvironmentin vivo- and in vitro-based experimental information to determine the molecular mechanisms underlying HPV-induced immune activation. Our next study will focus on determining the biological mechanisms underlying the relationship between viral infection  and immune surveillance in HNSCC. Secondly, we demonstrated that the HPV-induced immunotherapy response was independent of PD-L1 expression in HNSCC patients. However, we failed to conduct a stratified analysis of the efficacy of HPV status for predicting anti-PD-1/PD-L1 treatment in a subgroup of PD-L1-positive and -negative patients. This limitation arose because the primary data extracted from published studies lacked individual results for HPV and PD-L1 status. Further studies should therefore focus on the value of HPV status for predicting the response to anti-PD-1/PD-L1 therapy in HPV-negative patients or patients with a low TMB.Our study has several limitations. Firstly, although we established the relationships between HPV status and PD-L1 expression, tumor mutation profiles and the prevalence of inflammation-related immune cells and molecules, there is a lack of In conclusion, our study proved that anti-PD-1/PD-L1 treatment is more effective in HPV-associated HNSCC patients. HPV status may be an independent predictive factor in addition to PD-L1 expression and TMB in HNSCC, which activates the immune microenvironment by recruiting infiltrated T-cells and promoting their CYT.supplementary Dataset