C19orf57 (BRME1), a gene previously correlated with positive response to cisplatin in cervical cancer, among the most significantly differentially expressed genes between HPV+ and HPV- PDXs. In vivo experiments demonstrated a possible relationship between increased C19orf57 expression and superior anti-tumor responses of PDXs to cisplatin, which should be investigated further. These findings highlight the value of PDXs as models for HPV+ and HPV- HNSCC, providing a resource for future discovery of predictive biomarkers to guide treatment selection in HNSCC.There are currently no clinical strategies utilizing tumor gene expression to inform therapeutic selection for patients with head and neck squamous cell carcinoma (HNSCC). One of the challenges in developing predictive biomarkers is the limited characterization of preclinical HNSCC models. Patient-derived xenografts (PDXs) are increasingly recognized as translationally relevant preclinical avatars for human tumors; however, the overall transcriptomic concordance of HNSCC PDXs with primary human HNSCC is understudied, especially in human papillomavirus-associated (HPV+) disease. Here, we characterized 64 HNSCC PDXs (16 HPV+ and 48 HPV-) at the transcriptomic level using RNA-sequencing. The range of human-specific reads per PDX varied from 64.6%-96.5%, with a comparison of the most differentially expressed genes before and after removal of mouse transcripts revealing no significant benefit to filtering out mouse mRNA reads in this cohort. We demonstrate that four previously established HNSCC molecular subtypes found in The Cancer Genome Atlas (TCGA) are also clearly recapitulated in HNSCC PDXs. Unsupervised hierarchical clustering yielded a striking natural division of HNSCC PDXs by HPV status, with  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide . In the Mice harboring patient-derived xenograft (PDX) tumors have emerged as more translationally relevant preclinical cancer models compared to mice implanted with cell line xenograft tumors, due to the closer resemblance of PDXs to the primary human tumors from which they are derived . We and To date, HPV+ HNSCC tumors have been underrepresented in studies of HNSCC PDXs, most likely because these cancers are often treated nonsurgically and biopsy material is limited for the development of such models. In addition, multiple reports suggest that the engraftment rate of HPV+ HNSCC primary tumors in immunodeficient mice is substantially lower than that of HPV- tumors –12, withC19orf57, a gene previously correlated with positive response to cisplatin in cervical cancer  (C19orf57 expression [log2(counts/million) = 2.13]. The dramatic difference in cisplatin sensitivity at the extremum of C19orf57 expression suggests a potential relationship which should be further investigated with larger sample sizes.We next sought to investigate any potential relationship between  = 3.95] . MoreoveC19orf57 expression and cisplatin sensitivity, which could be investigated further.Developing preclinical HNSCC models that most closely reflect human HNSCC tumors is a critical step in identifying therapeutic targets and testing potential treatments for patients with head and neck cancer. While PDXs are increasingly recognized as more translationally relevant avatars compared to cell line xenografts, there are still relatively few published HNSCC PDX collections in the literature, and those that do exist have undergone varying degrees of molecular characterization . In partXenome, Disambiguate, or XenofilteR) [Compared to cancer cell lines or fresh surgical specimens, the presence of murine stroma in the PDX tumor microenvironment is a theoretical limitation to the analysis of high-throughput sequencing data of both DNA- and RNA-based measurements. As a result, groups working with PDXs may opt to remove mouse-identified genetic material from that which is human-specific, using one of multiple computational methods published over the last decade  , 28, 29.ofilteR) , 24. HowIn this study, we demonstrate a remarkable transcriptomic similarity of HNSCC PDXs to human tumors by recapitulating four well-characterized HNSCC molecular subtypes from TCGA in our PDX models. These four HNSCC subtypes—basal, mesenchymal, atypical, and classical—were originally identified from a cDNA microarray study of 60 human tumors , and subOur characterization of 16 HPV+ PDXs represents the largest reported HPV+ PDX cohort in a single study to date. Multiple groups have reported on the difficulties of successfully engrafting HPV+ tumors into mice, which is further hindered by a general scarcity of HPV+ tumor tissue to implant due to the widespread use of primary radiation or chemoradiation as curative treatment for this patient population , 11. Giva priori consideration of PDX clinical characteristics to probe for overlapping transcriptomic signatures which may indicate shared biology or treatment susceptibility. This approach naturally separated our PDXs by HPV status, with every HPV+ model clustering in a single subgroup composed exclusively of HPV+ PDXs. Importantly, none of the other demographic or clinical variables assessed were significantly associated with this expression-based clustering . Such a stark transcriptomic division, identified solely based on molecular characteristics, supports the already ample evidence that HPV+ and HPV- HNSCC are distinct disease entities. This finding also reinforces the potential of harnessing PDX transcriptomics to uncover unique biologic targets in both HPV+ and HPV- HNSCC.Using hierarchical clustering analysis, we further leveraged RNA-Seq data to subcategorize our PDX cohort based on patterns of gene expression. We took an unsupervised approach without Analysis of differential gene expression between PDX subtypes is potentially therapeutically relevant, as RNA-Seq allows for identification of actionable alterations which may guide treatment selection. For example, one study of 37 PDXs derived from difficult-to-treat breast cancers leveraged a multi-level “-omics” approach to identify targetable vulnerabilities at the DNA (whole-genome sequencing), RNA (RNA-Seq), and protein (RPPA) levels . We idenC19orf57 as a differentially expressed gene which warrants further investigation in HNSCC patients treated with cisplatin. C19orf57 encodes break repair meiotic recombinase recruitment factor 1 (BRME1). This protein plays a key role in meiotic recombination by facilitating homology-directed DNA repair via recruitment of recombinases such as RAD51 to DNA double-strand breaks in gametes [C19orf57 and cisplatin [C19orf57 expression was associated with improved survival in the setting of cisplatin therapy relative to the survival of patients whose tumors expressed lower levels of C19orf57 [C19orf57 expression in predicting cisplatin response in HNSCC, and interactions with HPV status, has not been explored.Furthermore, we identified  gametes . A studyisplatin . SpecifiC19orf57 . This obC19orf57 was expressed at levels 6-fold higher in HPV+ PDXs compared to HPV- PDXs, a finding consistent with two prior reports that found significantly higher C19orf57 expression in primary patient samples of HPV+ OPSCC compared to HPV- OPSCC [C19orf57 expression also demonstrated the largest response to cisplatin treatment. It is worth mentioning that this PDX had substantially higher expression of C19orf57 than the other five PDXs tested with cisplatin, with ~2.5-fold higher C19orf57 counts/million than the PDX with second-highest C19orf57 expression. In addition, the PDX with the most robust cisplatin response was one of the two HPV+ PDXs tested, and responded substantially better to treatment than the other HPV+ PDX with lower C19orf57 expression. These results suggest that HPV status may potentiate the value of C19orf57 expression for cisplatin response prediction in HNSCC. However, there are several notable limitations to conclusions about C19orf57 drawn from these in vivo PDX experiments. One limitation of our analysis is the relatively low number of PDXs tested with cisplatin in this study, and the observation that HPV+ HNSCC may demonstrate a superior baseline response to DNA-targeted therapy  than HPV- HNSCC, potentially due to a delay in the repair of DNA damage [C19orf57 expression with non-response to this agent. For these reasons, there is insufficient evidence at present to definitively implicate C19orf57 as a predictive biomarker for cisplatin response, though our data demonstrate that analyses of the PDX transcriptome have potential for elucidating clinical response to treatment. Future studies which characterize the degree of dose-dependence between C19orf57 expression and cisplatin response in both HPV+ and HPV- HNSCC are warranted to determine whether this transcript may hold utility in the clinical setting.In our study, V- OPSCC , 34. TheV- OPSCC . IdentifA damage –38. SecoGene expression-based stratification of HNSCC has not yet materialized as a feasible strategy for treatment selection in head and neck cancer patients. Our transcriptomic analyses of a large collection of HPV+ and HPV- HNSCC PDXs provide valuable characterization of these preclinical models at the mRNA level, demonstrating the strength of PDXs in recapitulating human biology. Our results are a valuable resource for future investigation and may be useful for identifying novel transcriptomic biomarkers of therapeutic response and new mechanistic targets in HNSCC.S1 TableList of r values for correlation comparisons of expression of the top 2,000 most variable genes for each PDX in the unfiltered RNA-Seq dataset (mouse and human mRNA reads) versus the filtered RNA-Seq dataset (only human reads).(PDF)Click here for additional data file.S2 TableList of r values for the 3.8% of transcripts (76/2000) with r <0.99 at the gene-level. Correlations are comparing expression level of each of the top 2,000 most variable genes across the PDXs before and after filtering for mouse reads.(PDF)Click here for additional data file.S3 TableGene Ontology (GO) annotated pathway analysis to assess for common biological functions between the 76 genes with gene-level correlation r values <0.99. Skeletal system development was the only GO term with q-value <0.5.(PDF)Click here for additional data file.S4 TableCorrelation of demographic and clinical characteristics of PDXs with cluster membership reveals that HPV status and tumor site are the only variables significantly correlated with hierarchical gene expression-based clustering . Age, sex, and stage were not significantly associated with expression-based clustering in any analyses conducted.(PDF)Click here for additional data file.


Background and Objectives: Romania faces one of the highest cervical cancer burdens in Europe though it is a preventable cancer through population screening by cytology and human papillomavirus (HPV) detection. Also, it has one of the highest incidences of human immunodeficiency virus (HIV) infection. HPV and HIV coinfection are frequently encountered. The aim of study was to establish the prevalence of HPV infection among HIV-positive women in Southeast Region of Romania, to genotype high risk HPV types -and to correlate the results with clinical data and cytological cervical lesions. Materials and Methods: 40 HIV-positive women were screened for HPV types and for cytological cervical lesions. The findings were evaluated in correlation with CD4 cell counts, HIV viral load, age at first sexual intercourse, number of sexual partners, vaginal candidiasis, and Gardnerella using statistical methods. Results: 19/40 (47.5%) women were positive for HPV types, 63.15% infected with single HPV type and 36.85% with multiple HPV types. The most frequent types were type: 31 (42.1%), 56 (31.57%), 53 (15.78%). On cytology, 34 (85%) women were found with NILM of which 38.23% were HPV-positive. Fifteen percent of women had abnormal cytology , and all of them were HPV-positive. Through analyzing the value of CD4 count, women with CD4 count ≤ 200 cells/μL were found to be significantly more likely to be infected with HPV; meanwhile there was no correlation between the detection of HPV types and HIV viral load. Candida or Gardnerella were more often associated with HIV-positive women with HPV, than in women without HPV. Conclusions: Infection with HPV types is common among HIV-positive women in the Southeast Region of Romania and it is associated with age at the beginning of sexual life, number of sexual partners, CD4 value, vaginal candidiasis, and Gardnerella infection. HPV infection is the most common sexually transmitted infection in young women. Its natural evolution is to regression and to spontaneous clearance in case of a competent immune system. According to the study of Ho et al. 1998), HPV infection may regress spontaneously within one year in 70% of cases and in 24 months in 91% of them [998, HPV Over 100 different HPV genotypes have been described, more than 40 of them infecting the anogenital tract. Of these, approximately 1/3 are associated with cervical cancer and anal neoplasm. The anogenital HPV’s are generally divided into two categories regarding their potential risk to develop cancer: low oncogenic types and medium-high oncogenic types. The high-risk HPV (HR-HPV) genotypes are generally associated with high-grade precancerous lesions and invasive cancer; the association between oncogenic HR-HPV types and cervical cancer has been well-established, while low-risk HPVs are frequently found in asymptomatic or benign conditions such as genital warts ,3,4,5.Persistent HR-HPV infection is responsible for over 90% of cervical cancers, but prophylactic vaccination against HPV infections including high-risk genotypes can reduce cervical cancers .In HIV infection, premalignant cervical lesions have a higher prevalence, a greater persistence, and a higher tendency to relapse than in the general population ,8,9.Cervical cancer is the most common cancer among HIV-positive women, being up to five times higher than in HIV-negative women . Since 1This close correlation between HPV and HIV infection is so obvious, leading to absolute international recognition by being awarded with the Nobel Prize in Medicine 2008 at the same time for “the parents” of these two viruses: Harald zur Hausen “for his discovery of human papillomaviruses causing cervical cancer”, and Françoise Barré-Sinoussi and Luc Montagnier “for their discovery of human immunodeficiency virus” . Unlike For years, Romania stands in the first position in the international statistics reports regarding the incidence and mortality of cervical cancer, even from the first year (2012) of implementation of the National Cervical Cancer Screening Program. The increased efforts of cervical cancer screening by cytology determined favorable results, beginning with 2015, when Romania managed to no longer be the leader in international statistics reports .According to the International Agency for Research on Cancer, in 2020, cervical cancer in Romania was the third most prevalent cancer in females, following breast and colorectal cancer in statistics. In Europe, Romania also stands in second place regarding estimated age-standardized incidence rates of cervical cancer for 20–54 aged females ,15.The most effective way to prevent cervical cancer is through the Babes-Papanicolau cervicovaginal cytological examination (Pap cytology test) associated with the detection of HR-HPV strains. Although the etiology of cervical cancer is multifactorial, HPV infection is crucial in the vast majority of cases. Regarding the fact that HPV infection is a sexually transmitted disease, it is known that the risk increases with an increasing number of sexual partners and with the early onset of sexual life. For primary prevention of cervical cancer, there is an available HPV vaccine.It is estimated that three of four HIV-positive women are infected with HPV. The prevalence of HPV infection among women with HIV is directly associated with HIV viral load and CD4 levels ,17.Antiretroviral therapy (ART) lowers HIV viral load, CD4 levels increase and it decreases the incidence of opportunistic infections and certain cancers. Studies on the impact of ART on the natural history of premalignant cervical lesions were inconclusive ,19.As HPV infection becomes more persistent, its possibility for spontaneous clearance decreases and the probability of evolution towards premalignant lesions or cancer grows . HoweverIn our study, we analyzed the prevalence of HPV infection among HIV-positive women in the Southeast Region of Romania, genotyped HR-HPV types, and correlated them with clinical data and cytological cervical lesions.Data were obtained by a descriptive study analyzing demographics of HPV and cervical lesions in HIV-positive women from Constanta, Southeast Region of Romania. Between September 2018 and February 2020 (18 months), 40 HIV-positive women undergoing cervical cancer screening were included in the study after considering the inclusion and exclusion criteria.Samples were collected from the Day Clinic of Constanta’s Clinical Infectious Diseases Hospital. After collection of demographic data, cervical samples were used both for screening cytology of cervical lesions and for determining the HPV strains. Blood samples were collected in order to determine CD4 counts and HIV viral load. Also, medical history was retrospectively retrieved from hospital medical records.Participation of subjects was entirely voluntary and written informed consent was required for inclusion in the study. All personal data were removed from samples to ensure patient confidentiality. A standardized questionnaire was used to gather demographic data. Other parameters identified and assessed in the questionnaire were time of first sexual intercourse, number of sexual partners, and history of sexually transmitted infections.Cervical cytobrush samples were collected from the cervical transformation zone of all women. Samples preservation, transporting, and processing for liquid-based cytology were performed according to the instructions of the reagents manufacturers. For samples preservation transporting Cell Solutions General Preservative  was used. Exo-/endocervical cells suspended in fixative liquid were collected by centrifugation , transferred in monolayer onto a glass slide and stained with Papanicolaou staining method with Harris’ hematoxylin, Orange G and EA polychromatic solutions according manufacturer’s recommendations . Pap smears were examined by at least three trained cyto/pathologists, independently of HR-HPV results, and classified according to the Bethesda System for Reporting Cervical Cytology (2014): Negative for intraepithelial lesion or malignancy (NILM); atypical squamous cells of undetermined significance (ASC-US); low-grade squamous intraepithelial lesions (LSIL); atypical squamous cells but cannot exclude high-grade lesions (ASC-H); or high-grade squamous intraepithelial lesions (HSIL) .Blood samples were collected by venous puncture in vacutainers, processed according to kit producer and analyzed for CD4 and HIV viral determination. CD4 was determined by lymphocytic immunophenotyping using flow citometry with acoustic focusing cytometer , reagents from AQUIOS Tetra-1 Panel . HIV viral load evaluation was performed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) method using 7500 Fast Real TIME Systems  and Xpert HIV-1 Viral Load test reagents  which include ribonucleic acid (RNA) extraction, purification, reverse transcription, and complementary deoxyribonucleic acid (cDNA) real time quantitation.® DNA Mini Kit, Qiagen, Redwood City, CA, USA). Using High Papilloma Strip of Operon  qualitative detection of HR-HPV types were made from DNA samples by reverse blot technique. To determine HPV status, DNA samples were genotyped for HR-HPVs  using PCR Thermal Cycler .Cervical cytobrush samples were vortexed and separated using lysis buffer. DNA was extracted from the lysis buffer using the DNeasy Tissue extraction kit according to the manufacturer’s protocol. . We computed descriptive statistics  for continuous variables (age) and inferential statistics with Chi-square for categorical variables. Variance was analyzed using one variable selected as independent (HPV) versus the other remaining dependent variables collected: CD4 level (≤200 cells/μL vs. >200 cells/μL), HIV viral load (<50 copies/mL vs. >51 copies/mL), age at first sexual intercourse (≤18 years vs. >18 years), number of sexual partners (>2 partners vs. ≤2 partners), vaginal candidiasis (yes vs. no), Gardnerella (yes vs. no)). Women aged between 18 and 60 years old, HIV-positive, sexually active, and had signed informed consent were included in the study. We excluded women with a history of cervical precancerous lesions or other malignancies, total hysterectomy, pregnancy, and absence of written informed consent. After inclusion-exclusion criteria, we obtained a group of 40 representative cases. Odds ratios were calculated to identify groups with significantly increased odds of HPV. Significance level was set at Data of cervical PAP cytology, HPV types, and clinic-epidemiological questionnaire responses are available for 40 HIV-positive women from South-East Romania. The age ranged between 20 and 54 years old and the mean age was 31.77 years old. The statistical analysis was performed on factors presented in At least one type of HPV was detected in 19 cases (47.5%) including 12 cases with one HPV type (63.15%) and 7 cases (36.85%) with multiple HPV types. The most frequent HPV types were: 31 (8 cases—42.10%), 56 (6 cases—31.57%), 53 (3 cases—15.78%), 39, 51 and 52 HPV types that were identified in 2 (10.52%) cases each. A total of 16, 18, 68 and 73 HPV types were detected in 1 (5.26%) case each .From the total of 40 studied cases, 34 women had no cytological abnormalities and were reported as NILM, while 6 presented cytological lesions.In the HPV-positive group (19 out of 40 cases) 13 women were reported as NILM. In this group of HPV-positive women, six had cytological lesions (three of them with ASC-US and three with LSIL) . In the In the group of HPV-negative women (21 cases out of 40) no cytological abnormalities were identified and they were reported as NILM.Of 33 cases with CD4 > 200 cells/μL, 4 cases were reported with abnormal Pap cytology (2 with ASC-us and 2 with LSIL) and the rest of them (29 cases) were reported as NILM.In the CD4 ≤ 200 cells/μL group two cases with abnormal Pap cytology were reported: one with ASC-US and one with LSIL.Forty HIV-positive women were tested for HPV presence (19 were found positive and 21 negative) and level of CD4 (7 were found with CD4 ≤ 200 cells/μL and 33 with CD4 > 200 cells/μL) . A link In the group of HPV-negative cases , only one case had CD4 ≤ 200 cells/μL.In the group of HPV-positive patients  only 6 (31.57% of women out of 19 positive cases) had CD4 ≤ 200 cells/μL.In the group of CD4 > 200 cells/μL (33 out of 40 cases), 20 women were HPV negative. In the same group (CD4 > 200 cells/μL) 11 cases had only one type of HPV, while 2 cases had 2 types of HPV .p < 0.05)  , Table 1p > 0.05).The value of HIV viral load in HIV-positive women was another parameter checked for any correlation with HR-HPV presence. Only one out of 21HPV-negativewomen had a detectable HIV viral load, while in the group of HPV-positive women, 4 out of 19 had a detectable HIV viral load , Table 1p < 0.001). The duration of HIV infection for the group of HPV-positive women was between 13 and 27 years, with a median value of 20 years and an interquartile range = 8 years, and the duration of HIV infection for the HPV-negative group was between 6 and 29 years, with a median value of 19 years and an interquartile range = 10.50 years. By comparing the mean values of the duration of HIV infection between the two groups of HPV-positive and HPV-negative women, we found that there are no significant differences between their values (p > 0.05).Analyzing the duration of antiretroviral therapy for HPV-positive and HPV-negative women, we found that the mean duration of therapy for HPV-positive was 12 years, while the mean duration of antiretroviral therapy for the HPV-negative group was 19 years with a statistically significant difference between the two groups (p < 0.05).Number of sexual partners was an important factor for the detection of HR-HPV types. Women with two or more previous sex partners were predisposed to HPV infection. The risk of having HPV was greater than 4.13 times in the group of women with more than two sexual partners, compared to the group of women with one or two sexual partners. These differences have statistical significance (p < 0.05).The younger the age when sexual life begins, the higher the risk of being detected with HPV types. Fourteen of the 19 women with detectable HPV types began their sexual life before the age of 18. Only 8 of the 21 patients with undetectable HPV types started having sex before the age of 18. The risk that a woman who began her sexual life before the age of 18 would develop an HPV infection was 4.55 times higher compared to women who began asexual life after 18 years old. The differences between the two groups of patients have statistical significance (p < 0.05). The same situation was noticed in the case of Gardnerella infection, which was also associated with a five times higher risk of having HPV than the group of women without Gardnerella (p < 0.05).Vaginal discharge did not influence the detection of HPV in HIV-positive women but the identification of Candida in vaginal discharge presented a 5.4 times higher risk of having an HPV infection than the women without Candida  suggested that as HIV-positive women live longer, many of them develop abnormal epithelial changes as cervical cancer precursors sometime during their evolution of HIV infection [000 suggeIn our study, we found that 15% of HIV-positive women have cytological cervical precancerous lesions but a study conducted by Halichidis et al. in 2014 on nearly 100 HIV-positive women from Constanta Region (South-East Romania) identified that 54% of them had abnormal cytology . The scoMassad et al., (2001) reported that 73% of HIV-positive women had at least one abnormal Pap test compared to 42% in HIV-negative women, during a 5-year follow-up . Also, iThe worldwide prevalence of HPV infection in women without cervical abnormalities is 11–12% with higher rates in sub-Saharan Africa (24%), Eastern Europe (21%), and Latin America (16%) . We founDelmas et al. (2000), on behalf of the European Centre for the Epidemiological Monitoring of AIDS, reported that abnormal epithelial lesions were detected in the initial Pap test in 24.2% (113/467) of HIV-positive women .Studies show that cases of persistent HPV infection in the absence of detectable cytological changes are frequent. We found that 68.42% of HIV-positive women have been detected with HPV infection in the absence of cytological cervical precancerous lesions, explicable by the persistence of HPV infection and leading to the necessity for meticulous monitoring. Ellerbrock’s findings indicate that the persistence of HPV infection in HIV-positive women conferred an increased risk for developing preinvasive cervical lesions and he found that an additional 26%  will develop a cervical lesion after 54 months of follow-up [Regarding HPV genotyping, we found that HPV 31 and 56 are the most frequent HPV types in HIV-infected women in the South East Region of Romania. The meta-analysis conducted by Clifford in 2017  identifiWomen with CD4 count ≤ 200 cells/μL are significantly more likely to be infected with HPV despite long-term ART. Similar results were obtained by Ellerbrock, Palefsky, Johnson, and Vermund ,34,35. AWe detected a higher prevalence of HPV among sexually active women, even those with a single long-term partner, suggesting that sexual activity may be an important factor ininfection and the persistence of HPV infection. Also, the younger the age at which sexual life begins, the higher the risk of developing HPV infections. It seems that isolation of Candida or Gardnerella from vaginal discharge is more often noticed in HIV-positive women with HPV, than in HIV-positive women without HPV. However, it is well-known that HIV-infected women are more likely to have prevalent vaginal candidiasis, HR-HPV, and abnormal Pap cytology test .Cervix infection with HPV and bacteria or fungi in correlation with the existence of HIV infection can lead to chronic inflammation, which favors HPV-related cervical precancerous lesions.This descriptive study showed that the most frequent HPV type was 31 and the only cytological lesions were ASC-US and LSIL . Women with CD4 < 200 cells/μL were more likely to be HPV-infected (nine times higher than the group with CD4 > 200 cells/μL). The risk of HPV infection was4.132 times higher for women with two or more sexual partners and 4.55 higher for those with an early sex life debut (under 18 years old). An association between Candida or Gardnerella infection with HPV was five times more frequent.Other studies, on a larger group of HIV-positive women, have shown a higher percentage of association between HPV, vaginal infections, and cytology changes compared with our study. Studies conducted on general populations of women show a smaller percentage of those associations compared to this study.Representing a small local study with a limited number of cases, further extensive studies are needed to achieve an appropriate conclusion.The high prevalence of cervical precancerous lesions in HIV-positive women warrants the need for regular Pap cytology screening and co-testing for HPV detection in order to diagnose cervical cancer early when treatment could also be effective. This systematic screening for cytological premalignant cervical lesions must be continued for all women, especially for those with immunosuppression, combining HPV detection with Pap cytology as well as trying to include co-testing for HPV types in the National Screening Program.


Cervical cancer is a type of cancer caused by human papillomavirus (HPV).To determine the relationship between awareness of cervical cancer and HPV infection and attitudes towards HPV vaccine among women aged 15-49 years.Cross-sectional study conducted at Karabük Training and Research Hospital, Turkey.500 women who visited the gynecology outpatient clinic of a public hospital between July 15 and December 31, 2019, were selected through random sampling. Data were collected using a sociodemographic questionnaire comprising nine questions (created by the researchers), the HPV and Cervical Cancer Awareness Questionnaire and the Carolina HPV Immunization Attitudes and Beliefs Scale.The relationship between the awareness questionnaire and the beliefs scale was explained through simple effect modeling of a structural equation. The women’s knowledge score regarding cervical cancer and HPV infection was 4.69 ± 4.02 out of 15. Women were afraid of being diagnosed with cervical cancer and HPV infection, but they did not have sufficient information. They had poor information about the HPV vaccine, did not know how to obtain the vaccine and did not have enough information about its benefits and harmful effects. Women who were afraid of getting cervical cancer, and who thought that they were at risk, had more information about the HPV vaccine.Women need information about cervical cancer, HPV infection and the HPV vaccine. Midwives, nurses and physicians who provide healthcare services in gynecological follow-ups should provide information to women about the HPV vaccine and cervical cancer. With 569,847 new cases and 311,365 deaths worldwide in 2018, cervical cancer is expected to be the fourth most common type of cancer and the fourth leading cause of cancer death among women. Out of these deaths, 90% occur in underdeveloped or developing countries. In 2018, 2,356 new cases and 1,280 deaths related to cervical cancer were seen in Turkey.2 It has been estimated that the lifetime probability of acquiring HPV exceeds 80% among women and 90% among men.3 Different groups of HPVs exist, with different epithelial tropisms  and life-cycle strategies. Many HPVs are classified as low risk (lrHPV) because they are very rarely associated with neoplasia or cancer in the general population. These lrHPVs typically cause indeterminate/undetectable infections or benign papillomas that can last for months or years but are eventually cleared by the host’s immune system. High-risk HPV (hrHPV) types are the cause of many major human cancers, including almost all cases of cervical cancer, a large proportion of other anogenital cancers and an increasing number of head and neck tumors.4 HPV infections can be temporary or permanent. Most cervical HPV infections (around 90%) are cleared by cell-mediated immunity within one to two years of exposure. In lrHPV infections, clearance occurs within a shorter period than in hrHPV infections. Among all HPV infections, 5%-10% cause persistent disease.5Human papillomavirus (HPV) is the most common sexually transmitted infection.5Cervical cancer has a long preinvasive period due to lesions associated with persistent hrHPV infection. Early diagnosis of these preinvasive lesions using screening methods , effective treatment of these lesions and administration of HPV vaccines can prevent this disease. Most cases of cervical cancer occur in women who have never been screened or were screened poorly.6 As cervical cancer has a long preinvasive period, it can be diagnosed and treated early by means of the screening programs that have been developed. For this reason, evaluating society’s attitudes and beliefs about cervical cancer and HPV vaccine and increasing the level of knowledge are important in terms of preventive medical practices.HPV vaccination has the potential to greatly reduce the morbidity and mortality associated with genital HPV infections and is recommended by the American Society of Obstetrics and Gynecology (ACOG) for all women and men aged 9-26 years.10Greater awareness among sexually active women aged 15-49 years regarding cervical cancer will decrease the rate of occurrence of this disease and increase the levels of knowledge about HPV vaccines and the vaccination rate.The aim of this study was to determine the relationships between knowledge of cervical cancer, awareness of HPV infection and attitudes towards HPV vaccines among women aged 15-49 years.11 However, the sample used in this study comprised 500 women, as calculated through G-power analysis with a 95% confidence interval and 5% margin of error, assuming 75.2% prevalence. Data were collected at the gynecology and diseases outpatient clinic of a public hospital between July 15 and December 31, 2019.This cross-sectional study was planned with the aim of determining the levels of knowledge about HPV and cervical cancer, levels of knowledge regarding preventive measures, health-related beliefs and awareness about HPV vaccines among women aged 15-49 years. The study population comprised women  within this age group who were living in the province of Karabük, Turkey.To collect data, a questionnaire on sociodemographic characteristics comprising nine questions was prepared in line with the literature by the researchers. In addition, the HPV and Cervical Cancer Awareness Questionnaire and the Carolina HPV Immunization Attitudes and Beliefs Scale (CHIAS) were used. Permission for this study was obtained from the Bülent Ecevit University Human Research Ethics Committee  and from the institute at which this research was conducted. After obtaining permission from the women who agreed to participate in the study, data were collected through face-to-face interviews. The study was conducted in accordance with the Declaration of Helsinki.14 Computer-assisted data analysis was used for the basic evaluation (correlations and frequencies) on the study data. We found that relationships between pairs of scales were explained through first-order factor analysis. A computer-assisted analysis program was used for factor analysis. Pearson’s correlation analysis was used to determine the relationships between scales and subdimensions.15 The data obtained were evaluated with a 95% confidence interval and a significance level of P < 0.05.The statistical analysis for this study was done with the aid of the SPSS 20 computer software . Given that the skewness and kurtosis values of the data remained within the +2.0/-2.0 range limit, the data were considered to follow normal distribution.12 In the validity-reliability study for use of this awareness scale in Turkish, conducted by Özdemir and Kısa, Cronbach’s alpha coefficient was 0.71.16 In our study, the Cronbach’s alpha coefficient of the awareness scale for cervical cancer and HPV infection was 0.81. The Cronbach’s alpha coefficient of the CHIAS was determined by McRee et al.17 Accordingly, the alpha values for “harm”, “obstacles”, “effects” and “uncertainty” were 0.69, 0.69, 0.61 and 0.66, respectively.17 Cronbach’s alpha was 0.62 in the validity-reliability study for use of the Carolina HPV Immunization Attitudes and Beliefs Scale in Turkish, conducted by Sunar and Süt.18 In our study, the Cronbach’s alpha of the CHIAS was 0.80; the alpha values for the subdimensions “harm”, “obstacles”, “effects” and “uncertainty” were 0.73, 0.75, 0.74 and 0.70, respectively.The Cronbach’s alpha coefficient of the awareness scale for cervical cancer and HPV infection was determined as 0.91 by Ingledue.The sociodemographic characteristics of the women participating in the study (n = 500) are shown in The mean knowledge score from the first 15 questions on the awareness scale for cervical cancer and HPV infection was 4.69 ± 4.02. More than half of the women provided incorrect answers to the information questions about cervical cancer and HPV infection in the first section. At the same time, the mean knowledge scores were also low.16Regarding the perceived threat due to cervical cancer, perceived sensitivity and severity scores, which are subdimensions of health beliefs, were evaluated. The subdimension score for perceived sensitivity was determined to be a maximum of 45.The mean perceived sensitivity score in our study was 25.32 ± 6.38. Among the participants, 50% were worried about getting cervical cancer and HPV. Conversely, 50% of the women stated that they did not have any information about prevention of cervical cancer and the measures to be taken against HPV infection.Similarly, the mean perceived severity score was 17.74 ± 4.03. The perceived severity scores of the participants ranged from 6 to 30 points. Among the women, 41.2% saw HPV infection as a life-threatening disease, whereas 38.2% saw cervical cancer as a curable disease.19 The subdimension scores of the CHIAS were as follows: harm = 13.8 ± 3.37; obstacles = 8.53 ± 1.97; effects = 4.61 ± 1.30; and uncertainty = 4.42 ± 21.22.For each item derived from the previous versions of the CHIAS, the expressions were changed to reflect the perspective of a young adult rather than a parental perspective. In this process, the sentence format used by Dempsey et al. was taken as an example.In our study, the relationships between the subdimensions of the HPV and cervical cancer awareness questionnaire and those of the CHIAS were explained through simple effect modeling from a structural equation model (SEM). The adaptation values were as follows: minimum discrepancy (CMIN) = 34.911; degrees of freedom (df) = 13; minimum discrepancy/degrees of freedom ratio (CMIN/df) = 2.685; root mean square error of approximation (RMSEA) = 0.058; comparative fix index (CFI) = 0.938; and goodness-of-fit index (GFI) = 0.981. Because CMIN/df was not within the required limits, correction indices were examined. The “effects” subdimension of CHIAS provided a correction in accordance with the modification index, with the item of levels of knowledge of cervical cancer and HPV: the F1 and F2 dimensions.The analysis was repeated by removing the “effects” subdimension item from the model. Then, the adaptation values were as follows: CMIN = 8.617; df = 8; CMIN/df = 1.077; RMSEA = 0.012; CFI = 0.998; and GFI = 0.994. All of the adaptation criteria were thus met within the desired limits.The nonstandard path coefficient of F2 was 0.152, and this was statistically significant (P < 0.001). The standard path coefficient for this item was 0.249. The nonstandard path coefficient of the perceived severity subdimension was 1, and this was statistically significant (p < 0.001). The standard path coefficient for this item was 0.851. The nonstandard path coefficient of the perceived sensitivity subdimension was 1.366, and this was statistically significant (P < 0.001). The standard path coefficient for this item was 0.736.The nonstandard path coefficient of the “harm” subdimension was 1, and this was statistically significant (P < 0.001). The standard path coefficient for this item was 0.62. The nonstandard path coefficient of the “obstacles” subdimension was 0.336, and this was statistically significant (P < 0.001). The standard path coefficient for this item was 0.357. The nonstandard path coefficient of the “uncertainty” subdimension was 0.223, and this was statistically significant (P < 0.001). The standard path coefficient for this item was 0.382. The nonstandard path coefficient for the levels of knowledge of cervical cancer and HPV was 0.174, and this was statistically significant (P < 0.001). The standard path coefficient for this item was 0.149 ; Table 2Pearson’s correlation was used because it showed parametric distribution between the awareness scale for cervical cancer and HPV infection and the subdimensions of the CHIAS . There w21 Through use of these screening programs for cervical cancer, mortality and morbidity due to this disease are gradually decreasing.21 The important factors in treating this disease are early diagnosis and women’s awareness. The degree of cancer or the size of the lesions that can be treated are decisive in the treatment process.22 Although cervical cancer is preventable, reports in the literature have demonstrated that, despite knowing about cervical cancer, women are not aware of the factors involved in its development.24Cervical cancer is a type of cancer that is monitored regularly through screening programs around the world, including in Turkey, and it can be treated quickly when detected.23 In a study by Ozan et al., 336 women who visited a gynecology outpatient clinic were assessed regarding their level of knowledge of cervical cancer and HPV, and it was observed that although 86.6% of them knew about cervical cancer, only 33.6% knew about HPV infection.24 Pehlivanoğlu et al. found that 26.8% out of 295 women who visited a family medicine outpatient clinic had never heard of the Pap smear test and 43.4% did not know about HPV infection.20In our study, the women’s knowledge score about cervical cancer and HPV infection was low (4.69 ± 4.02), contrary to the findings in studies in the literature. Although 50% of the women were afraid of getting cervical cancer and HPV infection, they did not have any information about preventing this infection. In a study conducted by Montgomery et al. to determine the level of knowledge of cervical cancer and HPV, the knowledge score was 7.39 among 149 women.25Although the women in our study had heard about HPV vaccines, their attitudes and knowledge regarding HPV vaccination was inadequate. This situation might have originated from the women’s low awareness of cervical cancer and inadequate knowledge of HPV infection. The level of knowledge of HPV vaccines that we found in our study was consistent with data in the literature.13Pelullo et al. conducted a study among 556 nursing students, with the aim of examining their knowledge and attitudes regarding HPV vaccines. They found that although almost all the students had heard about the vaccine, only 36.5% were aware of its risk factors.9 Although 87.7% of those students knew that an HPV vaccine for women exists, only 52.4% were aware of the existence of an HPV vaccine for men.9In a study by Yılmaz et al., in which 624 nursing students were examined in terms of their knowledge, behavior and attitudes in relation to HPV vaccines, their levels of knowledge regarding the vaccine were lower than their levels of knowledge regarding HPV infection.26In our study, the subdimension scores for “obstacles” “harm” and “uncertainty” in relation to getting the HPV vaccine were low. This might have been due to lack of knowledge about the vaccine, lack of vaccine availability, the women’s lack of awareness about the vaccine effects and unavailability of the vaccinees. This result was consistent with data in the literature.13In our study, significant relationships were found between the responses to the questionnaire regarding levels of knowledge of cervical cancer and HPV infection and the subdimensions of the CHIAS. Women who were worried about cervical cancer had higher levels of knowledge about HPV. Meanwhile, a significant relationship was found between women who thought they had a high probability of getting cervical cancer or HPV infection and the total knowledge score and the “harm” subdimension score. There were significant relationships between women’s fear of being infected with HPV virus and the “effects” and “obstacles” subdimension scores of the CHIAS. This might have been due to inability to cover the cost of the vaccine, lack of a vaccination program across the country and lack of information about where women can obtain the vaccine. This result was consistent with data in the literature.27Knowledge about HPV vaccines increased with the perceived sensitivity, and there was a positive relationship between them. Conversely, a negative relationship was observed between the level of knowledge of cervical cancer and HPV infection and the “harm” subdimension. As the level of knowledge increased, the number of women thinking that the vaccine was harmful decreased. These results were consistent with data in the literature.13Pelullo et al. reported that there was a positive relationship between the levels of knowledge of HPV vaccines among the students in their study and these students’ awareness of the risk factors.10Giuseppe et al. explored HPV awareness among 1,348 adolescent girls and young women and reported that those who saw themselves at risk of cervical cancer and HPV infection had higher levels of knowledge about HPV vaccination.27In a cross-sectional study, in which Napolitoni et al. examined women’s knowledge and attitudes regarding HPV infection and vaccines, a positive significant relationship was found between women carrying and/or knowing about HPV risk factors and their levels of knowledge and attitudes in relation to HPV vaccines.In our study, women’s knowledge and attitudes towards cervical cancer and HPV infection were found to be inadequate. This inadequacy had an effect on their levels of knowledge regarding HPV vaccines. The highest score on the scale of knowledge about HPV vaccines was 56, but the mean score of these women was 33.37 ± 5.05. Through this result, it was seen that women did not have enough information about HPV vaccines and that HPV vaccine-related education was needed. It was also found that women were not getting vaccinated because of their lack of knowledge about vaccine access, its effects and its cost. Considering the efforts made towards ensuring widespread use of cervical cancer screening programs, similar strategies and programs need to be developed for HPV vaccine programs, in order to provide greater immunity against HPV infection. Further qualitative and quantitative studies are needed in order to determine HPV vaccine awareness in Turkey.


Furthermore, advanced MRI techniques, such as intravoxel incoherent motion and perfusion MRI as well as application of artificial intelligence and radiomic techniques, have shown promise in treatment response monitoring and prognostication. The following review will consider the contemporary role and knowledge on imaging in HPV+OPC.The clinical behaviour and outcomes of patients with oropharyngeal cancer (OPC) may be dichotomised according to their association with human papilloma virus (HPV) infection. Patients with HPV-associated disease (HPV+OPC) have a distinct demographic profile, clinical phenotype and demonstrate considerably better responses to chemoradiotherapy. This has led to a reappraisal of staging and treatment strategies for HPV+OPC, which are underpinned by radiological data. Structural modalities, such as CT and MRI can provide accurate staging information. These can be combined with ultrasound-guided tissue sampling and functional techniques (such as diffusion-weighted MRI and  Imaging is key to the diagnosis, staging and monitoring of treatment response in OPC and there is increasing recognition of the influence HPV status has on imaging findings.Human papilloma virus-associated oropharyngeal squamous cell carcinoma (HPV+OPC) is now recognised as distinct from non-HPV-related squamous cell carcinoma (SCC) of the head and neck (HPV-HNSCC). This is due to its unique epidemiology, histology, clinical behaviour, and outcomes. In particular, HPV+OPC is associated with a significantly improved chemoradiotherapy (CRT) response. These demonstrate a tropism for oropharyngeal tonsillar crypts, which have a unique structure that is thought to facilitate viral entry into an otherwise immune privileged site. Once the virus has gained access to oropharyngeal tissue, it downregulates tumour suppressive pathways and upregulates p16 transcription .cription .8,9 Furthermore, p16 IHC correlates well with HPV in situ hybridisation (ISH) techniques, is more cost-effective than detection of HPV oncogene E6 and E7 transcripts and is more specific than HPV DNA polymerase chain reaction (PCR) testing for transcriptionally active HPV infection. As a result, p16 IHC has been included in the eighth edition of the American Joint Committee on Cancer (AJCC) guidelines as the principle marker of HPV status. However, in recognition of the fact that p16 positivity is not always HPV driven, many well-resourced centres use a combination of techniques (e.g. p16 IHC with ISH or PCR) to increase specificity and prevent false positives.HPV+and HPV-OPC differ in their microscopic appearances, which can be used to guide diagnosis . In addi There is also geographic variation, with a higher (and rising) prevalence in high-income Western countries; e.g. in the United States and Western Europe, HPV+OPC is thought to account for around 70% of OPC, compared to around 22.4% internationally.In contrast to other forms of HNSCC, which are strongly associated with tobacco and alcohol excess, HPV+OPC typically affects younger patients, with or without a smoking history and may relate to certain sexual behaviours. In contrast, nodal disease in HPV+OPC may be extensive and cystic. As a result, patients may present with cervical nodal metastases of unknown primary origin, potentially mimicking a second branchial cleft cyst.Primary neoplasms in HPV+OPC usually arise from the lymphoepithelial tissues of the oropharynx , but may be very small and therefore clinically occult.The incidence of HPV+non-oropharyngeal HNSCC is variably reported, but an international histopathological study found 4.4% of oral cancer and 3.5% laryngeal cancers tested positive for HPV markers.e.g. a recent meta-analysis found HPV+laryngeal and hypopharyngeal SCC were associated with improved survival metrics , but HPV+ oral cavity SCC was associated with a worse disease-free survival (hazard ratio of 1.81). In nasopharyngeal SCC, approximately 20.6% are HPV+ and there is an association with non-endemic, Epstein-Barr virus (EBV) negative, lower grade (WHO Grade 1) disease, which is postulated to reflect a distinct subtype. Elsewhere within the upper aerodigestive tract, infection by high-risk HPV33 is associated with the development of HPV-related multiphenotypic sinonasal carcinoma, which typically has a relatively indolent course.The prognostic significance of HPV+non OPC HNSCC appears to be site dependent;  The changes in the eighth edition predominantly pertain to nodal (N) staging in HPV+ OPC (reflecting the improved prognosis); e.g. multiple ipsilateral metastatic lymph nodes are re-classified as N1 rather than N2b disease. This has an impact on clinical staging groups that can be used to guide treatment  to stages I or II (using the eighth edition) .16,28 reatment . might be reduced without reducing cure rates. Following surgical techniques, such as transoral robotic surgery (TORS) or transoral laser microsurgery (TLM), it may be possible to decrease the adjuvant RT dose or omit concurrent chemotherapy in those without extracapsular spread or positive margins and this is currently being investigated. Other trials looking at RT dose reduction and treatment modification based on response to induction chemotherapy are currently underway. However, deintensification remains controversial, particularly following the publication of two trials that found reduced survival rates in the groups of HPV+OPC patients treated with a less toxic alternative to cisplatin chemotherapy (cetuximab). In particular, the trials found 2 and 5 year overall survival rates were 97·5 and 84.6% respectively in the cisplatin groups, compared with 89·4 and 77.9% respectively in the cetuximab groups. Application of deintensification arms to the subgroup of HPV+OPC with the lowest clinical risk (limited neck disease and non-smokers) is likely to be important in future trials.Currently, standard treatment for OPC is usually independent of HPV status . HoweverGiven the histological and phenotypic differences between HPV+ and HPV- OPC, and its impact on staging and management, there has been considerable interest in using imaging as a non-invasive means of predicting HPV status prior to pathological diagnosis. Similarly, Chan et al found HPV+ primary tumours (compared to HPV- tumours) were significantly more likely to be smaller  at presentation, exophytic (73% compared to 63%), with well-defined borders (58% compared to 47%), and were significantly less likely to demonstrate ulceration (10% compared to 20%) or necrosis (9% compared to 19%).Whilst imaging cannot replace histopathology in differentiating HPV+ and HPV- OPC, structural differences in the appearances of the primary tumour have been identified. Cantrell et al reviewed the pre-treatment CT scans of patients with OPC, and found HPV+ primaries were significantly less likely to demonstrate local muscle invasion (6% compared to 26%) and displayed a non-significant tendency towards greater enhancement, exophytic growth and well-defined borders .37 Sim On structural imaging, it can be challenging to differentiate cystic change from necrosis; therefore, Goldenberg et al suggested a series of CT criteria in an effort to aid differentiation , but a higher specificity (73.3%) for HPV+ status in OPC. The same group also found specificity could be improved (100% compared to 73.3%) by using an additional criterion of low density (<25 Hounsfield units) change within small (<1.5 cm) lymph nodes. Both Chan et al and Cantrell et al. (using the Goldenberg criteria on CT and MRI respectively) found cystic lymphadenopathy to be significantly more common in HPV+ OPC (36–45%) than HPV- OPC (9–32%). However, there was no relationship between nodal distribution and HPV status.Cystic lymphadenopathy is a recognised feature of HPV+ OPC  and has been proposed as an imaging biomarker.ntiation .41 A s As a result, such cases are typically investigated as a carcinoma of unknown primary, including with 18F-fludeoxyglucose positron emission tomography-CT, which has been found to have a high negative-predictive value for malignancy of 96% . Positive staining can be helpful in differentiating cystic HPV+ OPC metastases from branchial cleft cysts. Whilst such testing has been shown to correlate well with histological findings, issues remain including a lack of consensus regarding cut-off values for p16 positivity and there is a risk of false negative results (up to 21%), particularly if there is a paucity of material. There are also issues surrounding obtaining sufficient material for a cell block (up to 48% adequacy in one study). Ultrasound-guided core biopsy represents an important adjunct to FNA with a higher sensitivity and sample adequacy. Its use may be additive in the setting of non-diagnostic ultrasound-FNA or where HPV testing is required; a meta-analysis found, compared to FNA, core biopsy was able to achieve greater specificity (99% compared to 96%), accuracy (96% compared to 93%), and NPV (95% compared to 90%) in detecting head and neck malignancy. Nevertheless, this should be balanced with the appreciation that core biopsy is a more traumatic technique with a slightly higher risk profile than FNA, although this may be mitigated with smaller gauge core biopsy needles.Approximately, 44% of patients with OPC present with a neck mass and ultrasound-guided fine needle aspiration (ultrasound-FNA) is frequently the first-line diagnostic procedure.18F-FDG PET-CT and diffusion-weighted imaging (DWI) are the most frequently employed. 18F FDG PET-CT yields metabolic information based on glycolytic activity, whilst quantitative DWI with apparent diffusion coefficient (ADC) values provides information on lesion microstructure, such as cellularity.Various imaging techniques can provide functional and quantitative information in patients with HNSCC, but 18F FDG PET-CT, there is conflicting evidence with respect to its ability to differentiate HPV+ from HPV- OPC primaries, with Tahari et al finding significantly higher glycolytic parameters max SUVpeak and SUVmean) in HPV- OPC, but Clark et al finding no differences in the SUVmax between the HPV+ and HPV- groups. In contrast, studies have more consistently demonstrated greater glycolytic parameters in HPV+ OPC nodal metastases. For example, Clark et al found that a nodal SUVmax of >10.8 could reliably predict p16+ disease.For  A threshold value of mean ADC for HPV+ OPC has been proposed  and yielding high sensitivities and specificities (83.33 and 78.57% respectively). However, the relationship has not been observed in all studies and higher and more variable ADC values have been found in HPV+ OPC, which may relate to intratumoral micronecrosis. Furthermore, investigation into the relationship between lower ADC and nodal disease in HPV+ OPC has yielded conflicting results.. It is important to recognise that the methods of region of interest (ROI) contouring and the incorporation of cystic components is likely to influence the observed ADC values.With respect to quantitative DWI, several studies have found lower pre-treatment ADC values in HPV+ OPC primary tumours, which may relate to tumour-infiltrating lymphocyte content.Whilst not employed in routine clinical practice, advanced imaging and data analysis techniques have been employed in the evaluation of OPC. As a result, maps reflecting separate diffusion and perfusion components can be created. Lower ADC and tissue diffusion coefficient (Dt) values have been observed in primary tumours and metastatic lymph nodes of patients with HPV+ OPC, with Dt values achieving good levels of sensitivity (85.7–94.9%) and specificity (61.9–64.7%) when combined with risk factors, such as smoking and alcohol intake. However, perfusion-related parameters, including the perfusion fraction (f) and perfusion-related diffusion coefficient (D*), have not been found to be discriminatory. Due to the paucity of data and time consuming nature of the IVIM sequence, its use remains primarily in the research domain.Intravoxel incoherent motion (IVIM) MRI is an extension of DWI  which accounts for sources of incoherent motion of water molecules within voxels (notably arising from moving blood within capillaries). The approximately linear relationship between gadolinium concentration and T1 signal can be used to derive perfusion and permeability metrics that are thought to reflect the tumoral microenvironment. However, its role in predicting HPV status is unclear. In particular, one study found the DCE MRI metrics volume transfer constant (Ktrans) and flux rate constant (kep) (reflecting capillary permeability) were significantly lower in HPV- and epidermal growth factor receptor (EFGR)+ oral cavity SCC and OPC and a Ktrans cut-off value of 0.18 min−1 could be used to predict HPV+ status with a sensitivity of 80% and specificity of 85.7%. However, a more recent study found no difference in DCE parameters between HPV+ and HPV- OPC. Given the inconsistencies, further data are required before it can be relied upon to aid prediction of HPV status.Dynamic contrast-enhanced (DCE) MRI involves rapid imaging of a bolus of contrast passing through a volume of tissue using 3D spoiled gradient echo sequences. Furthermore, textural histogram parameters have been found to differ significantly, depending on HPV status on pre-treatment CT studies, with Lee et al finding the entropy parameter could be used to predict priary tumour HPV status with an accuracy of 80% in the training and 75% in the validation data sets. Additionally, evaluation of ADC-derived histograms for HPV- oral cavity SCC and OPC have been found to display a more normal distribution  as compared to HPV+ oral cavity SCC and OPC, which is characterised by greater kurtosis and skewness . Despite promising data, the use of radiomics remains at an early stage with persistent challenges pertaining to reproducibility and generalisability, which limit its use to the research domain.Radiomics involves the extraction of quantitative data from imaging studies that cannot be appreciated by the human eye and can be facilitated by artificial intelligence (AI) techniques. Radiomics-based phenotyping (using 6 of 170 radiomic features) derived from segmented contrast-enhanced MRI studies has shown promise in differentiating HPV+ from HPV- OPC on MRI (with an area under the receiver operator characteristic curve of 0.982 on a training data set and 0.744 on the test data set).Whilst HPV+ OPC has an excellent prognosis, there remains significant interest in identifying imaging biomarkers that could help identify tumours which are less likely to respond to CRT. This would help guide more proactive surveillance and to prompt more intensive or modified treatment plans such as radiotherapy dose escalation or surgery.e.g. positive surgical margins at the primary site) are an indication for adjuvant CRT. In such cases, the intended deintensification benefits of surgery are lost and patients undergo triple (rather than double) modality therapy. Therefore, predicting which cases are likely to have surgically unfavourable features pre-operatively using imaging is of clear benefit. MRI has been shown to reliably predict positive surgical margins (with a sensitivity of 87.5% and specificity of 92.3%) based on pharyngeal constrictor muscle obscuration or invasion on MRI. MRI may also be used to identify invasion of other key muscular structures, such as the extrinsic tongue muscles, pterygoid muscles and eustachian tube or high risk anatomical variants, such as a retropharyngeal internal carotid artery that might contraindicate TORS.TORS and TLM can be employed as part of a treatment deintensification strategy in HPV+ OPC; however, discovery of prognostically unfavourable histological features  nodal metastases have been found to correlate with a higher risk of treatment failure and poorer disease-free survival (DFS) in HPV+ OPC (hazard ratio of 2.21). In addition, the nodal distribution may be important since the presence of retropharyngeal lymphadenopathy (coupled with a more advanced stage) has been associated with a reduced time to distant metastatic failure in 266 patients with HPV+ OPC (combined C-index of 0.84).Cystic metastases have been found to be associated with an improved prognosis in studies of HNSCC, but this finding can be accounted for by the confounding inclusion of patients with HPV+ OPC. As a result, an additional (N3b) stage was added for HPV- HNSCC (including HPV- OPC) in the eighth edition of the AJCC staging manual. Since early data were unclear, ENE was not included as a separate nodal stage in the eighth edition of the AJCC manual for HPV+ OPC. However, more recent evidence suggests a correlation between ENE and poor outcomes in HPV+ OPC treated surgically, with An et al finding ENE was associated with worse 3 year OS , and Bauer et al finding ENE-positivity was associated with a hazard of death of 1.90; 95% CI: 1.35–2.67) compared to ENE-negative cases. The clinical definition of ENE in the latest AJCC staging manual is based upon physical examination rather than radiological findings, but structural imaging has been used to predict ENE based on a number of criteria  as compared to HPV- oral cavity or all HNSCC (87%) and postulated that the lower reliability might be due to the inclusion of central necrosis in the diagnostic criteria. As a result, it remains prudent to exercise caution when predicting ENE on imaging in cases of HPV+ OPC.ENE is known to be an indicator of poor prognosis in HPV- HNSCC (with higher rates of recurrence and distant metastasis).criteria . A recen18F FDG PET-CT has been found to outperform CT in predicting time to locoregional recurrence based on SUV metrics [metabolic tumour volume (MTV), total lesion glycolysis (TLG) and SUV2.5in HPV+ OPC. Furthermore, several studies have found poorer outcomes to be associated with elevated glycolytic parameters, including higher tumoral and/or nodal SUVmax, MTV and TLG levels. However, some conflicting data exist, including one study that found elevated TLG (>135.3 g) to be associated with favourable survival statistics (particularly for HPV OPC). It is possible that AI techniques could aid risk stratification following pre-treatment 18F FDG PET-CT in future. It is proposed that this relates to histological findings of higher stromal content, lower cellularity and micronecrosis, all of which are known to result in greater treatment resistance. Such studies also demonstrated an association between lower pre-treatment ADC values and favourable post-treatment survival outcomes in OPC and other HNSCC; however, until recently, little data were provided on HPV status. It has since been demonstrated that the association between low pre-treatment ADC values and improved outcomes is largely lost when the confounding factor of HPV+ OPC  is corrected for. For example, Cao et al found HPV- OPC primary tumours had significantly greater pre-treatment ADCmean (1.48 µm2/ms) compared to HPV+ OPC primary tumours (ADCmean of 1.34 µm2/ms), but this was not replicated in metastatic lymph nodes.Evaluation of pre-treatment ADC values in patients with HNSCC has revealed an association between higher ADC values and poorer post-CRT outcomes. Bogowicz et al found an association between a 3-feature radiomic signature indicative of a more heterogenous CT density distribution and poorer local control . However, as with prediction of HPV status, there remains limited reliable evidence for radiomics in HNSCC prognostication, with a dearth of validation and reproducibility studies.Radiomic techniques have been applied to prognostication with promising results. For example, Song et al developed radiomic risk score classifiers for patients with HPV+ and HPV- OPC based on pre-segmented CT studies and found that radiomic signatures generated from the data could be used to predict HPV status (accuracy of 76%) and DFS (hazard ratio of 1.97).It is worth noting that a degree of caution is required when interpreting quantitative data and thresholds derived from medical imaging, since differing methodologies, patient cohorts and analysis techniques can influence values obtained.e.g. dose painting) or deintensification of treatment. Alternatively, it may be performed post-treatment, in order to detect early residual or recurrent disease, since timely intervention with salvage surgery is required before locoregional disease becomes irresectable. In the latter setting, there is an intrinsic trade-off between imaging early enough to detect remediable treatment failure and imaging late enough for treatment-related tumour shrinkage to manifest, so that failure is not declared prematurely.Imaging following the initiation of treatment for HPV+ OPC has been performed in clinical studies to enable adaptive radiotherapy techniques such as rapid intensification  and D (using IVIM) values as well as more rapid tumoral regression. Conversely, lower intratreatment ADC and higher pre- and intratreatment D values, as well as larger reductions in f, have been associated with regional treatment failure. For example, Marzi et al found that patients with regional failure had higher mid-radiotherapy D values (1.28 × 10−3mm2/s compared to 1.09 × 10−3mm2/s) as well as reductions in f and D*×f from the baseline. In patients with HPV+ OPC, Ding et al found complete response to treatment correlated with lower pre-treatment ADC and D values and relatively greater changes in mid-treatment ADC, D and f , achieving an AUC of 0.87, sensitivity of 0.63, specificity of 0.85 and accuracy of 0.78. DCE MRI has been applied to a mixed (p16+ and p16-) cohort in the context of a multiparametric approach, finding that persistently low blood volume derived from DCE data is associated with a higher risk of distant failure.Since a reduction in tumour dimensions may be delayed, evaluation of intratreatment imaging response has focused on quantitative techniques DWI, IVIM and DCE.18F FDG PET-CT, studies carried out mid treatment (e.g. 3 weeks following commencement) have also been found to be of utility in identifying patients at risk of treatment failure in HNSCC. For HPV+ OPC, reductions in nodal metabolic tumour volume on mid-treatment scans have been identify a subgroup of patients at low risk of locoregional-failure but higher risk of distant-metastatic-failure.With respect to early post-treatment imaging of OPC, both qualitative and quantitative metrics can be evaluated, but the choice of modality and timing of imaging are important considerations.18F FDG PET-CT in the post-CRT setting and offers advantages over CT due to superior contrast resolution. In particular, well-described features visible on conventional anatomical MRI sequences can aid differentiation of recurrence from treatment-related change  and local failure. Relative to CT, MRI is particularly helpful in this situation, given its superior ability to differentiate between tumour and peritumoral oedema Both CT and MRI may be used to evaluate post-treatment changes in nodal volume, with greater percentage reductions associated with treatment success.At the primary site, measured reductions in tumour size on MRI are important in confirming response.The optimum timing of post-CRT MRI has recently been examined by Connor et al. In this study, nodal response to therapy  in HPV- OPC was most significant at 6 weeks following completion of CRT and had changed little by 12 weeks. Conversely, in HPV+ OPC, there was significant continued reduction in nodal size between the 6 and 12 week scans. In view of this, earlier post-CRT MRI may be appropriate for HPV- OPC, but 12 week scanning remains appropriate for HPV+ cases with nodal disease.18F FDG PET-CT is widely used for the identification of treatment failure following CRT for Stage III and IV OPC and other HNSCC, since structural imaging is often difficult to interpret in the setting of treatment related anatomical distortion. It is particularly helpful for ruling out disease owing to excellent negative-predictive values .. It is also capable of detecting distant metastatic disease  remain problematic and can lead to unnecessary invasive procedures. This is compounded in the setting of HPV+ OPC where there is evidence to suggest that the positive predictive value of a 12 week post-CRT 18F FDG PET-CT is particularly low (30% in one study). Furthermore, nodal lesions in HPV+ OPC have been shown to demonstrate a different pattern of involution, with a more rapid initial decrease in size followed by a longer time to a complete resolution (particularly for cystic lymphadenopathy). This may be addressed by serial or later 18F FDG PET-CT scanning. There is now a trend for 18F-FDG PET-CT to be performed at a later time interval following HPV OPC treatment, and a study by Liu et al, the number of false positives were reduced by carrying out a repeat 18F FDG PET-CT (at 16 weeks) in cases of incomplete nodal response at 12 weeks.Post-CRT 18F FDG PET-CT metrics have been used to predict survival and recurrence in HNSCC. In general, higher levels of SUVmax following treatment and a reduced change relative to the pre-operative SUVmax are indicators of poor prognosis. MTV has also been utilised in HNSCC, with Murphy et al finding a larger post-radiation volume being associated with a poorer prognosis. Some authors have identified cut-off values in patients with HPV+ OPC, but there is variation in the literature. For example, Vainshtein et al used post-treatment SUVmax thresholds of 6.5 for the primary tumour and 2.8 for the neck to define complete response, with a high NPV (91–98%), albeit with a low sensitivity and PPV (0–33%). Chan et al used a lower SUVmax threshold of 2, which was associated with a high NPV of 91.7% but lower sensitivity (75% and PPV of 37.5%). There is evidence that 18F FDG PET-CT has a lower PPV in HPV+ HNSCC (20%) compared to HPV- disease (62.5%). This may be due to the unique response of HPV+ OPC to treatment, with an increased cytotoxic T-cell-based response  and greater radiosensitivity .Post-treatment  Post-CRT increases in ADC metrics following CRT have been associated with disease-free survival in HNSCC; in particular, a rise in post-treatment ADC as well as an increased change in ADC relative to baseline correlated with a favourable prognosis. For example, Connor et al found HNSCC patients with higher post-treatment ADCmean values 6 and 12 weeks following RT were associated with increased 2 year disease free survival rates. At 6 weeks, an optimum threshold of 1405 10–6 mm2/s was derived  and at 12 weeks an optimum threshold of 1840 10–6 mm2/s was derived .DWI and ADC maps can be used as a surrogate marker of microstructural treatment-related tumoral changes. In particular, therapy-induced tumour necrosis is thought to lead to an increase in diffusivity and therefore ADC values.However, interpretation alongside anatomical MRI sequences is imperative since treatment-related scarring may also produce low ADC values.18F FDG PET-CT or there may be visible residua on CT or MRI with reduced 18F-FDG uptake  within blood specimens have shown early promise in prognosis and post-treatment surveillance. Such assays may provide complementary data and increase the specificity of imaging findings.Although still at an early stage, serological assays capable of detecting HPV-related antibodies (HPV+ OPC has emerged as a distinct clinical entity and an understanding of HPV+ OPC. Whilst identification of HPV status through tissue sampling remains the gold standard, pre-treatment imaging features may enable early identification to refine diagnostic and therapeutic approaches, whilst recognition of the expected post-treatment evolution will help guide investigation for residual disease. Moreover, appreciating the impact of HPV+ OPC on imaging biomarkers will allow their appropriate application for prognostication and treatment monitoring.


U.S. military veterans have been heavily impacted by the opioid overdose crisis, with drug overdose mortality rates increasing by 53% from 2010–2019.The risks for overdose that veterans face need to be understood as resulting from an ongoing interaction among biological/physiological, psychological, and social/structural factors.Addressing drug overdose in the veteran population requires accessible and non-judgemental, low threshold, wraparound, and holistic solutions that recognise the complex aetiology of overdose risk for veterans.U.S. military veterans have been heavily impacted by the opioid overdose crisis, with drug overdose mortality rates increasing by 53% from 2010–2019. Risk for overdose among veterans is complex and influenced by ongoing interaction among physiological/biological, psychological, and socio-structural factors. A thorough understanding of opioid-related overdose among veterans, one that goes beyond simple pharmacological determinism, must examine the interplay of pain, pain treatment, and stress, as well as psychological and social experiences—before, during, and after military service. Comprehensive efforts to tackle the overdose crisis among veterans require interventions that address each of these dimensions. Promising interventions include widespread naloxone distribution and increased provision of low-threshold wrap-around services, including medications for opioid use disorder (MOUD) and holistic/complementary approaches. Interventions that are delivered by peers – individuals who share key experiential or sociodemographic characteristics with the population being served – may be ideally suited to address many of the barriers to opioid-related risk mitigation common among veterans. Community care models could be beneficial for the large proportion of veterans who are not connected to the Veterans Health Administration and for veterans who, for various reasons including mental health problems and the avoidance of stigma, are socially isolated or reluctant to use traditional substance use services. Interventions need to be tailored in such a way that they reach those more socially isolated veterans who may not have access to naloxone or the social support to help them in overdose situations. It is important to incorporate the perspectives and voices of veterans with lived experience of substance use into the design and implementation of new overdose prevention resources and strategies to meet the needs of this population. DurinSpecifically, this current crisis needs to be understood in the context of the relatively recent history of deindustrialisation, suburbanisation, and widening social and structural inequality that shaped the post-WWII landscape in the U.S. ,13. EconIn the early 1990s, when U.S. soldiers were returning from the first Gulf War conflict, Operation Desert Storm, an influential and lasting convergence of pharmacological, medical, social, economic and technological factors helped catalyse what would become an unprecedented rise in opioid-involved overdose ,17. Over2.1.Exit Wounds: A Survival Guide to Pain Management for Returning Veterans and their Families [deserved because of claimed improvements in functionality and quality of life. These arguments took advantage of veterans’ concerns about being denied healthcare and socioeconomic support post-service, and leveraged the narrative that those who served the nation were entitled to the best care available [Abundant research, journalistic investigations, and court cases have now documented the role played by Big Pharma and some medical professionals in creating the opioid overdose crisis that emerged in the 1990s. In particular, the industry capitalised on the American Pain Society’s 1995 designation of pain as the “Fifth Vital Sign” as a rationale for the aggressive marketing and widespread prescription of opioids . In 1998Families , which fvailable .2.2.In addition to high rates of service-related injury treated with opioid analgesics in the recent veteran population, medical PO use among active Department of Defense (DOD) personnel also increased – by more than 500% between 2002 (2%) and 2008 (11%). Illicit drug use, including misuse of prescription drugs, rose considerably as well, from 3% in 2002 to 12% in 2008 such that, by 2008, more than 1 in 10 service members was legally or illegally taking opioids . During 2.3.About fifteen years into the new millennium, a spate of new measures intended to curb liberal PO prescribing and PO diversion was implemented in the face of mounting evidence of opioid-related harms . Efforts2.4.The overdose rate among veterans steadily climbed beginning in the late 1990s due to many of the factors discussed above, a trend that has continued largely unabated according to recent published reports ,7. Amongn = 65, 741) found that the majority appeared to have at least one substance use disorder in addition to OUD and many had multiple substance use disorders  [At the same time, while opioid-involved overdoses continued to climb, data demonstrated that the majority of drug-related fatalities  involved multiple substances, especially benzodiazepines, alcohol, and more recently, stimulants . A retrozepines) . In recezepines) ,45. AnotFor many veterans, military service and recreational culture involves moderate to heavy alcohol use, and the use of alcohol greatly elevates the risk of overdose when combined with opioids ,47. Rate3.biology of injury and neurological dysfunction in chronic pain), but also requires consideration of the role of mental health concerns  and interpersonal relationships and life events  [Veterans’ opioid use initiation is frequently grounded in service-related injury, which for some, leads to long-term opioid use for chronic pain management . Therefo points)  in syner3.1.Veterans exhibit high rates of mental health disorders  ,61–64, a3.2.Military sexual trauma is a case study in how psychosocial trauma can be a precondition for OUD and, by extension, risk for overdose. There is a high prevalence of military sexual trauma among both men and women veterans , and a r3.3.Social isolation is a significant concern among the veteran population . Mental 3.4.Research suggests that opioid-related overdose has become the most common cause of mortality among many homeless populations , and hom3.5.Suicide is a highly pressing issue among veterans, especially those who served in Afghanistan and Iraq, and an unknown proportion of fatal and non-fatal overdose events in veterans may be the result of suicide attempts. Suicide risk has been shown to be higher among OEF/OIF/OND veterans than among the general population , and the3.6.Another significant barrier and potential risk factor for overdose among veterans is a fundamental lack of access to healthcare services and low rates of utilisation of the VA in particular. As noted in the introduction, while fewer than 50% of veterans used at least one VA benefit (such as the GI Bill or GI mortgage), only 6.1 million of the total 20.3 million veteran population (30%) have used VA healthcare. This is concerning on several fronts. First, much of the scientific knowledge about health risks among veterans who use opioids comes from VA samples, which represent less than half of all military veterans  at most.4.In this final section we describe promising interventions that have been implemented to address overdose among veterans. Following the BPS framework, we review innovative and timely efforts to tackle the overdose crisis among veterans by addressing physiological, psychological, and socio-structural dimensions of risk. Veterans represent a population with a unique potential for being at the vanguard of public health innovation and novel intervention implementation. Despite its early involvement in liberal PO prescribing and iatrogenic dependence, the VA has in more recent years provided naloxone access across its facilities and has implemented innovative peer support programs for veterans with SUD. Nevertheless, in light of the ongoing overdose health crisis and the high numbers of veterans who continue to experience opioid-related morbidity and mortality, additional initiatives are needed. For example, while medication for opioid use disorder (MOUD) is the gold standard of evidence-based OUD treatment, only 38% of persons with OUD across VA healthcare facilities receive MOUD, and barriers such as stigma and discrimination hinder uptake . One rec4.1.Interventions that are delivered by peers who share key experiential or sociodemographic characteristics with the population being served may be ideally suited to address overdose and many of the barriers to opioid-related risk mitigation discussed above. The presence of a peer  can serve to diminish the sense of shame or stigmatisation that veterans may feel when interacting with persons without personal experience of substance use ,103. A n4.2.For those veterans who are not connected to the VA, there are very few culturally sensitive and low-threshold interventions available, and with a few exceptions, harm reduction services have not been systematically tailored for and targeted to the veteran population. Community-based care models, often employing peers, and have demonstrated their capacity to effectively engage some of these underserved populations by extending culturally sensitive outreach beyond clinical settings ,112. The4.3.Perhaps the VA’s biggest accomplishment in the effort to mitigate veterans’ overdose risk is the creation of a system-wide naloxone access and distribution program. In the 2000s, as the opioid crisis was rapidly escalating, public health advocates and community activists (many working at harm reduction agencies) established early methods for distributing naloxone directly to people who use and/or inject drugs, focussing on getting naloxone directly to people who use drugs in community settings . Low-thrWhile the VA must be commended for making naloxone widely available in its facilities, next steps should include adopting a comprehensive BPS-informed approach to overdose prevention that attends to the psychological, social, structural and practical barriers faced by the most vulnerable groups of veterans. The VA has embraced multi-modal, non-pharmacological approached to pain care which should help prevent iatrogenic dependency in the course of pain management . In addiAdditionally, it is critically important that opioid safety messaging and naloxone reach those veterans who use opioids and/or other drugs irregularly – e.g. on weekends or holidays –who may have a false sense of safety about their risk for overdose. Given current illicit drug market conditions, even casual users of illicit drugs are at very real risk of overdose, as reports of fentanyl contamination of cocaine and counterfeit pills, including POs and benzodiazepines, are increasingly frequent throughout the U.S. In light of this risk, opioid safety messaging must also emphasise the importance of using fentanyl test strips  or other available drug checking technologies to test all illicit drugs for the presence of fentanyl before consuming them. Whenever possible, messaging should also emphasise the importance of using drugs in the presence of a trusted friend who could administer naloxone and call 911 in an overdose event.via mail delivery from the service Next Distro, an organisation that distributes harm reduction supplies by mail to rural locations across the country [To help optimise feasibility, acceptability and effectiveness, these community care and peer outreach models of overdose prevention should be adapted for the local implementation context and available resources. Where possible, it may be advantageous for overdose prevention initiatives to partner with existing community-based agencies and/or veteran service organisations, co-locating the delivery of harm reduction materials such as naloxone and drug testing technologies (e.g. fentanyl test strips). These promising interventions are not necessarily specific to veterans, though organisations that service veterans are obvious places from which to distribute naloxone and other harm reduction supplies to at-risk groups of veterans. For veterans in rural areas, it is now possible to obtain naloxone and other safer drug use supplies  country ; efforts country .4.4.Perhaps the best suggestions for how to prevent overdose among veterans are provided by veterans themselves who have lived experience of opioid and other drug use. Veterans we have interviewed in our research have emphasised the need for additional low-threshold, peer-delivered services to address many of the ongoing substance-related challenges they and other veterans face ,16. EnsuVeterans with whom we have worked have also spoken directly to overdose and affirmed their central goal of keeping their fellow veterans alive and safe from disease and accidental and preventable causes of death. Helping to equip veterans with the resources to actualise their life goals may be the best treatment of all. Concretely, veterans suggested expanding short- and long-term MOUD treatments and making harm reduction resources and supplies, such as naloxone and fentanyl test strips, more readily available . A disinVeterans also expressed concern that their potential advancement within the military would be limited if they accepted treatment such as MOUD . For exa5.Despite the efforts detailed above to curb opioid and other substance-related mortality among veterans, the opioid overdose public health crisis persists, with the highest number of overdose fatalities ever reported during 2020–2021 among the general population. Many veterans face difficult challenges combining problems of pain management, substance use, service-related mental and physical health problems, and the day-to-day challenges associated with housing, employment and relationships, all of which can heighten risk for drug overdose. As we have highlighted in this review, veterans’ drug-related problems, and in particular, overdose risk, must be understood in light of their distinct and evolving life contexts and situations – as the product of ongoing interactions across physiological, psychological social, and structural domains. Pain management needs must be understood alongside the larger complex of issues veterans face over the civilian/military/veteran career, acknowledging the physiological, psychological, social and structural factors at play. Addressing and putting resources into veterans’ basic needs, including stable housing, meaningful employment, and holistic, low- threshold, integrated health and healing services are central.Ultimately, this review suggests the importance of adopting a holistic view in conceptualising overdose risk and in mitigating drug-related harms. Our review of research literature and epidemiological findings demonstrates that veterans’ vulnerability to opioid-related overdose is multifaceted and involves far more than the opioid dependence resulting from long-term management of pain with opioids. Interventions need to address BPS dimensions of risk with multimodal and multidisciplinary approaches to health and well-being.


Cervical cancer burden is still high in low- and middle-income countries, including Botswana. Persistent human papillomavirus (HPV) infection is the leading cause of cervical cancer. Accurate knowledge of HPV diversity associated to cervical cancer in sub-Saharan Africa may provide accurate understanding of the natural history of HPV infection in these contexts. The goal of this review was to consolidate existing evidence on cervical HPV infection and to conduct a pooled analysis of data from all eligible Botswana studies. After a successful review of twelve studies on cervical HPV genotypes that met the inclusion criteria, HPV-16 genotype was the most frequently discovered in women with pre-cancerous and cancer lesions, followed by HPV-18. HPV-16 in HIV-positive women with precancerous lesions to cancer is between 45% and 47.7%, and between 4.5% and 26.1% for HPV-18. With reference to other HPV genotypes, the proportion of HPV-35 and HPV-58 (13-16%) seems relatively consistent among the studies, however HPV-58 appears to be more common in HIV-positive subjects compared to HIV-negative women. Indeed, HPV-45 seems to be frequently detected in women with cervical cancer compared to women with precancerous lesions. Regarding the low-risk HPV genotypes, an appropriate breakdown has been provided. In conclusion, the current prophylactic vaccines against HPV-16 and HPV-18, which have demonstrated good immunogenicity in HIV-infected populations, may still prevent infection and ultimately cancer. Human papillomavirus (HPV) is the main etiological factor for cervical cancer onset and progression among women . HoweverThe impact of HIV on cervical cancer is demonstrated by the significantly higher incidence of cervical cancer in HIV-positive than in HIV-negative women despite antiretroviral therapy (ART) use . This suIn Botswana, a landlocked country in Southern Africa, cervical cancer is the leading cause of cancer death in women , with moUnderstanding the prevalence of the HPV genotypes in Botswana is essential for formulating HPV vaccination policy, quantifying possible impact of vaccination, and allocating resources for screening triage to prevent cervical cancer. Therefore, the purpose of this article is to examine available data on the prevalence of HPV genotypes (with or without cancer) in this peculiar context.This scoping review aims to provide an overview of published articles on the prevalence of cervical HPV genotype distribution in Botswana and its relationship with cancer and HIV, when possible and/or available. Two search engines, PubMed and Google Scholar, were utilized in the procurement of widely available literature. All publications were included that matched the selection criteria “HPV” and “Botswana” published until August 2022. The titles of the references from the identified publications were also scanned for keywords matching our selection criteria and included if they met one of them. The search was repeated to identify the consistency of search terms and results. Two authors (LT and GMP) independently reviewed the titles, abstracts, and full articles of the retrieved studies.Studies conducted in Botswana that reported the presence and prevalence of HPV genotypes were included. The inclusion was restricted to the papers published in English language until August 2022. Studies that reported HPV genotypes from anal, penile and oral sites were excluded to focus on cervical cancers HPV genotypes.Twelve studies from Botswana met the criteria for inclusion, with a total of 2,284 young subjects (18 to 22 years of age) who were free of cervical lesions , 20, 25;HPV molecular studies are an important focus of research in Botswana as well as the results of the collaboration between local academics/researchers and those of the University of Pennsylvania . The curRegarding HPV genotypes other than HPV-16 and/or HPV-18, the percentage of HPV-58 appears to be higher in HIV-positive people than HIV-negative subjects in Botswana , 26, 31,Concerning technical aspects of the HPV genotyping among the studies reviewed, a genotyping bias for HPV-18 could be assumed. In fact, when Abbott Real-Time High-Risk HPV platform is used, the rate of HPV-18 detection decreases significantly , 2. A prIn summary, evidences from this review show that there is a significant presence of HPV among Botswana women, including the HPV genotypes targeted by the quadrivalent vaccine. Thus, it may be anticipated that the national HPV vaccination program in Botswana, which achieves vaccine coverage as high as 90.0%, will significantly reduce HPV burden and HPV-associated malignancies in the next few years. However, the 9-valent HPV vaccine that offers more protection against HPV than the quadrivalent HPV vaccine is highly recommended. Furthermore HPV surveillance studies should be done to monitor the evolution and prevalence of HPV infection and disease.Important limitations of this work are that data across different studies are not always fully comparable because of differences in technical approaches for sample collection and genotyping, including the fact that the number and HPV types detected vary according to the method chosen. Additionally, HIV status is not always indicated, the severity of lesions is different and not always comparable, and ages are different across the studies. In addition, one of the papers  did not In conclusion, this paper reviewed the available literature on cervical HPV genotypes in Botswana. The results highlighted that young women have a slightly lower prevalence of HPV-16, which rises with age. Independently from HIV status, cervical cancer patients have concurrently higher levels of HPV-16 and HPV-18, in Botswana. HIV-positive individuals seem to have greater HPV-58 rate than HIV-negative individuals , 26, 31.LT and GP designed the project and prepared the tables. LT wrote the manuscript with ideas and contributions from GP. PR, RK, MK, ER, SG, and DR-M critically revised the manuscript. All authors approved the final version of the manuscript.