Routine cervical cancer screening is important for women living with HIV (WLH) due to the greater incidence and persistence of high-risk HPV (HR-HPV) infection. HR-HPV self-sampling has been proposed to overcome barriers to in-office cervical cancer screening in underserved populations. However, little is known about baseline knowledge of HR-HPV and the acceptability of HR-HPV self-sampling among WLH. This paper describes WLH’s experiences and needs regarding cervical cancer screening, specifically HR-HPV self-sampling, and seeks to reconcile their experiences with the views of their providers. In total, 10 providers and 39 WLH participated in semi-structured interviews and group discussions, respectively. Knowledge of cervical cancer and HR-HPV was generally limited among WLH; when present, it was often due to personal experience of or proximity to someone affected by cervical cancer. Most WLH were not familiar with HR-HPV self-sampling but, despite some of the providers’ skepticism, expressed their willingness to participate in a mail-based HR-HPV self-sampling intervention and highlighted convenience, ease of use, and affordability as facilitators to the uptake of HR-HPV self-sampling. The experiences identified can be used to guide patient-centered communication aimed at improving cervical cancer knowledge and to inform interventions, such as HR-HPV self-sampling, designed to increase cervical cancer screening among under-screened WLH. Women living with HIV (WLH) bear a disproportionate risk of high-grade cervical intraepithelial neoplasia CIN2+) and invasive cervical cancer due to greater incidence and longer persistence of high-risk human papillomavirus (HR-HPV) infection in this population ,2,3. Per+ and invCervical cancer is preventable through early detection with regular Papanicolaou (Pap) and/or HR-HPV screening. Current guidelines recommend initiating cervical cancer screening at HIV diagnosis but not before age 21. Cervical cytology is performed annually for three years, then every three years if normal in women age 21–29. In women age 30 or greater, follow-up cytology and HR-HPV co-testing is performed every 3 years if the initial co-testing result is negative . While tTo improve cervical cancer screening rates, self-collection of cervicovaginal samples for HR-HPV testing has been proposed as an alternative to traditional office-based screening . PrimaryMy Self-Sampling, HPV Awareness, Results, and Empowerment” (MySHARE) intervention, which will examine the acceptability of HR-HPV self-testing among WLH. These preliminary data describe the knowledge, experiences, and needs of WLH regarding cervical cancer screening and seek to reconcile patient experiences with the views of their providers. Provider bias has been shown to have a significant impact on patients’ health care decision making. It is important to understand the screening needs among WLH to inform tailored interventions to maximize engagement, patient satisfaction  was absolutely cured or something.” . Others shared a desire to expand their knowledge of cervical cancer and HR-HPV: “I don’t know too much information about cervical cancer, but I really would like to know more about it.” . While many of the participants were able to identify that cervical cancer is cancer related to their reproductive organs , only a handful could correctly distinguish what a cervix was and where it was located. There was also little to no knowledge of HR-HPV transmission or the role of HR-HPV in the development of cervical cancer. A few mentioned discussing cervical cancer and HR-HPV with their health care provider .Among women who claimed some awareness and knowledge of HR-HPV, many credited the educational brochures and pamphlets made available in the waiting room of their doctors’ offices and advertisements and commercials about the HPV vaccine (Gardasil): “I haven’t seen that much about HPV. Sporadically I would see commercials but I think that, in my remembrance, they were geared more towards teenagers.” . Most were aware that HR-HPV is a sexually-transmitted infection to which WLH are more susceptible .The women unanimously expressed the need to expand their current understanding of cervical cancer and HR-HPV. They identified peer group discussion, media, community outreach events, and providers as potential avenues to increase this dialogue among the community of WLH:“We have our groups and stuff. We take notes down and they give us flyers, different information […] to keep us [knowledgeable]. I always keep my flyers and read over them.” “I usually go to health fairs and I get information that way.” “I’ve never even seen anything, [not even] like that pamphlet in my doctor’s office, you know about HPV. Like I said, this is the first time I’ve ever heard of it… [There is a need for] educating physicians on educating their patients, even if they are not there [at their appointment] for that… outside of what we’re there for, to speak to them as far as HPV.” Despite their low-level understanding of cervical cancer and HR-HPV, most WLH placed a high priority on cervical cancer screening: “I was going to say [that], to me, it’s a priority because I am HIV positive. […] It is still easier to get some infection even if I’m taking my medicine as usual so it’s a priority [for me] to get a Pap smear test every time required.” .The providers’ perceptions of the women’s awareness and knowledge of cervical cancer and HR-HPV accurately reflected that shared by WLH in the FGDs. The providers recognized that knowledge about cervical cancer among WLH could be improved and that it is vital for WLH to also understand the association between cervical cancer and HR-HPV (Community Advisory Member). Providers correctly believed that unless women had a history of cervical cancer or HR-HPV, their awareness and knowledge on these topics remained generally limited. However, providers underestimated the priority level of cervical cancer screening and prevention among WLH, assuming they would have many competing priorities .When asked about current cervical cancer screening recommendations for WLH  , the women provided mixed responses. While some accurately indicated that screening should be completed annually  indefinitely, others believed that it should be completed twice a year and/or only up to the age of 65 years. Despite current guidelines/expert opinion to initiate screening no earlier than age 21, many women thought screening should be initiated on coitarche , with a handful indicated that screening should be completed as early as the onset of menarche .Interestingly, while providers universally agreed that cervical cancer screening for WLH is within the scope of primary care providers, they displayed varying knowledge of the screening standards specific to this patient population. While a few providers accurately recognized that (1) WLH should have a Pap test at the time of initial HIV diagnosis, then every 12 months  and (2) that screening  should continue through a WLH’s lifetime  , others were not aware of specific screening recommendations for WLH:“So I don’t think the guidelines are any different for women with HIV. Generally, it used to be every three years [that] this has to be done and now it’s every five years, unless, of course, they have something abnormal. […] Oh okay, is that the current recommendation for those with HIV? I didn’t even know that.” (Nurse Practitioner)Most participants indicated that they had regular cervical cancer screening . However, a handful of women explicitly stated that they only completed cervical cancer screening tests when they had symptoms or if it was directly recommended by their provider . Some of the women similarly voiced that if cervical cancer screening was not brought up by their physicians, they would not request to be screened .WLH felt that providing easily accessible general education on cervical cancer and HR-HPV prevention would increase screening intention and behavior . They felt that education targeting all ages of WLH and emphasizing their increased risk of cervical cancer and HR-HPV would be welcome and effective.Providers also recognized the importance of physician recommendation for screening and agreed that WLH are generally open to screenings and compliant with regular cervical cancer screening . They further shared their perception that older WLH were less interested in regular screenings compared to younger WLH (Physician). Like WLH, providers emphasized the importance of education for young WLH :“Right now, our team is having a hard time engaging the young adults living with HIV in care for that [cervical cancer screening]. […] Once we get them in the office or [on a] phone call, we can talk to them more about the risks for cervical cancer and why it’s important for them to do the screening.” (Nurse Practitioner)While WLH had little awareness or knowledge of HR-HPV self-sampling, some inferred that self-sampling would be similar to cervical cytology . Others supposed it would be similar to a urine test . Participants who had previously completed an at-home self-test for sexually-transmitted diseases or infections (STDs/STIs) were more likely to accurately describe the HR-HPV self-sampling process:“I would think that they would maybe send a Q-tip or something and put it inside of a tube […] and you send it back. […] I think I had something done like that before, […] I think I had like an anal test done.” Most providers had a similar lack of awareness or knowledge of HR-HPV self-sampling and equated it to other STD/STI self-tests:“In my mind, it would be something pretty simple and not very invasive; not time-consuming. I mean, almost like swabbing in your cheek or in your mouth for other things, but this time, you’re swabbing the vaginal area.” (Community Advisory Member)WLH felt HR-HPV self-sampling was feasible and acceptable due to its convenience , privacy, simplicity, and ease of use . Many were motivated by the health benefits of HR-HPV testing  and some elaborated that knowing how to correctly do the test and being empowered to take care of their bodies would serve as additional facilitators . Affordability and provider recommendation were also mentioned as important factors . Although the majority of our FGD participants expressed willingness to try HR-HPV self-sampling (80.5%), a few shared their preference for in-person screening with their provider .Most providers agreed that HR-HPV self-sampling was not frequently offered to WLH . Some providers recognized the use of HR-HPV self-sampling for patients with gender dysphoria or a history of sexual abuse:“I know that some providers offer the test, [but] I don’t know exactly how they’re doing it. […] [Some providers are] giving sampling kits to patients with gender dysphoria who don’t want to do the Pap; or it’s too like traumatic to do it so they’ll give [their patients] something that they can take home to do or that they can do in the clinic on their own.” (Physician)Providers had mixed opinions on whether HR-HPV self-sampling should be offered in the U.S. and whether acceptability and uptake among the WLH would be high. Most providers were concerned about incorrect use of the kit and the capacity for follow up and triage to further testing or treatment in the case of an abnormal test . Overall, providers believed that HR-HPV self-sampling would be feasible for WLH. The providers highlighted the “convenience” and “privacy” that self-sampling would offer its users (Nurse Practitioner).In our study, WLH were overwhelmingly willing to try remote self-sampling, emphasizing “one less trip to the doctor’s” . Many of them felt self-sampling was especially crucial in situations such as the COVID-19 pandemic where patients do not feel in-person visits are safe . The women strongly favored a mail-based intervention , highlighting its convenience and privacy:“I think mailing it out to them works best because [after] receiv[ing] it, [they can] just do it in the privacy of their own home, then mail it back in. Just [include] a self-addressed envelope; [they can] put it in there and no one [will] know what is in that envelope [when they] mail it back.”. “Because [they] have kids and [are not] able to get out, mailing them [an HPV self-collection kit] would be one way for them to get it and [to] get [screening] done.” Many providers were concerned that remote self-sampling and the self-sampling process would be an added burden for both providers and patients . They feared mailed kits would not be returned promptly or at all. These concerns, however, were contradictory to what the majority of our FGD participants shared.This qualitative study describes the experiences and knowledge of cervical cancer and HR-HPV among WLH in the greater Washington-Baltimore area and examines the feasibility and acceptability of HR-HPV self-sampling for this cohort. To our knowledge, this is the first qualitative study to examine these questions among a diverse, urban population of WLH and their providers. Since providers of health care and social services directly influence their patients’/clients’ decision making, health-seeking behaviors, and access, their perspectives will undoubtedly influence the acceptability and feasibility of HR-HPV self-sampling.The present study found that, overall, WLH have negative  attitudes towards cervical cancer and acknowledge the severity of the disease. Both providers and WLH expressed that cervical cancer and HR-HPV knowledge among WLH is limited. Patients and providers agreed that this knowledge should be improved through patient education to help women prioritize cervical cancer screening despite competing needs. Our study revealed provider recommendation as a major driver of cervical cancer screening. While our cohort had little or no prior knowledge or experience with HR-HPV self-sampling, they were very accepting of such testing. Providers were also unfamiliar with HR-HPV self-sampling and recognized its appeal to patients, despite the administrative and clinical challenges for follow-up of abnormal results.Similar to prior literature, our study revealed knowledge gaps regarding HR-HPV and cervical cancer among WLH ,15,16. FOur study highlights the importance of provider recommendation in cervical cancer screening, with mixed responses to provider-led decision making. Although FGD participants ultimately felt responsible for their own health, they deferred to their providers’ recommendations and felt they would be less likely to bring up cervical cancer screening unprompted. Similar findings were reported by Beach et al. : 86% of Our study is the first to explore the acceptability of HR-HPV self-sampling from both patient and provider perspectives. While some of the providers believed that HR-HPV self-sampling would be burdensome, our FGD participants were extremely receptive to the concept of remote self-sampling and believed they would complete this testing if the self-collection process were simple with clear instructions. Most women, in our study, were familiar with other self-testing options  which may further explain their receptiveness to HR-HPV self-sampling. Findings from our study also distinctly emphasized the need for designing interventions that offer remote HR-HPV self-sampling, especially to women with limited access to cervical cancer screening, so that they can complete self-collection at a time and place that is convenient and appropriate to them.There are several limitations of the current study. First, the findings described herein reflect primarily the views and experiences of WLH residing in the urban WBMA and may not be generalizable to that of WLH located in more rural areas. Additionally, since most women in our FGDs were over the age of 50, our findings may not be generalizable to a younger population. Lastly, we conducted our FGDs without video, in order include those without camera-enabled devices or internet connection. Therefore, visual cues were not available to the discussion moderator. Despite these limitations, this study adds important information to the literature related to cervical cancer prevention efforts among vulnerable populations and provides valuable insights that can help shape policies and programs aimed at addressing the health needs of WLH, as defined by the women and the providers that serve their community.As outlined in the Healthy People 2030 objectives, increasing the number of women who are screened based on the most recent guidelines  will redWLH are a particularly vulnerable population that faces several barriers to cervical cancer screening ,54,55. H


Ultrasonic guided wave monitoring is regularly used for monitoring the structural health of industrial pipes, but small defects are difficult to identify owing to the influence of the environment and pipe structure on the guided wave signal. In this paper, a high-sensitivity monitoring algorithm based on adaptive principal component analysis (APCA) for defects of pipes is proposed, which calculates the sensitivity index of the signals and optimizes the process of selecting principal components in principal component analysis (PCA). Furthermore, we established a comprehensive damage index (K) by extracting the subspace features of signals to display the existence of defects intuitively. The damage monitoring algorithm was tested by the dataset collected from several pipe types, and the experimental results show that the APCA method can monitor the hole defect of 0.075% cross section loss ratio (SLR) on the straight pipe, 0.15% SLR on the spiral pipe, and 0.18% SLR on the bent pipe, which is superior to conventional methods such as optimal baseline subtraction (OBS) and average Euclidean distance (AED). The results of the damage index curve obtained by the algorithm clearly showed the change trend of defects; moreover, the contribution rate of the K index roughly showed the location of the defects. With the increase in the service life of oil and gas pipes, various kinds of defects or damage will occur gradually. The ultrasonic guided wave testing technique ,2,3,4, wIn the continuous monitoring of a pipe, there is still a probability that the instrument will produce large noise while working normally;Slight differences in the monitoring signals exist at different times of the day, and due to the influence of temperature on the materials, the guided wave propagation is affected too .During testing for pipe defects, monitoring by ultrasonic guided waves has a higher signal-to-noise ratio (SNR) and sensitivity compared with detection ,9. The tThe traditional solution to these problems is to increase the excitation power and transducer coupling efficiency at the instrument level and to establish the temperature compensation at the signal preprocessing level . HoweverDuring monitoring signal processing research, the pipe signal recorded by an instrument, in which the temperature influence has been compensated by optimal time-domain stretch method , is usedFeature space decomposition reduction technology has been found to have great potential in enhancing the representativeness of data . PrincipIn order to improve the sensitivity of detecting small defects, the adaptive principal component analysis algorithm (APCA) is proposed in this paper. Based on the traditional PCA, an adaptive link is added to the selection process of the principal component to enhance the sensitivity of the monitoring algorithm. In For the guided wave signal collected by the instrument, due to the noise caused by the instrument or the transducer, the guided wave signal is usually preprocessed before the application of the monitoring algorithm to identify the defect. It mainly includes Butterworth data filtering and Hilbert envelope extraction, the application of both of which has been proved to improve the signal-to-noise ratio and to reduce the influence of the end surface and weld ,27. The the bias b. An exaT is the thickness of pipe. In addition to that, the original signals and test signals are also temperature compensated to avoid the interference of temperature; this compensation method [In the first stage, we collected original signals on the undamaged pipe and carried out the above preprocess. In the second stage, the test signals were collected on pipe with different cross section loss ratio (SLR) of hole defects; the SLR refers to the ratio of defect area to the cross section of pipe, and it is used to measure the degree of defect and can be expressed as the following Equation (3):n method  can refem and that each signal contains n sampling points and is regarded as a vector Suppose that the number of collected original signals is n-order covariance matrix C is constructed for the First, an U obtained by the operation is an n × n characteristic matrix, and the matrix S is a diagonal matrix composed of eigenvalues U as the vector, we will get n eigenvectors k-dimensional matrix k vectors of matrix n-k vectors of matrix U, and it also supports the residual subspace (RS), which is used to measure the residual between the test signals and the principal component model. Then, the damage evaluation index should be established, which is based on the above two subspaces.The matrix According to the previous discussion, the PCA mapped the data into PCS and RS, and then two statistics were introduced—Hotelling or (SPE) ,30—to mok eigenvalues C. a is the confidence level , and The SPE was used to measure the deviation size of the test signal in the RS. It is defined as the following Equations (10) and (11):In this formula, Under normal conditions, the projection of the original signals in RS should be very small. When the damage occurs in the pipe, the observation sample will deviate from the PCS and increase its projection in RS, the information size and deviation size will change significantly, and the k principal components are selected, the CPV is obtained by Equation (14):When PCA is used to construct the principal component matrix, the number of principal components must be determined, which will directly affect the performance of defect monitoring and diagnosis. If the number of principal components is too small, the PCS contains too little information, which makes the  defects ,32. The  defects . When thGenerally, CPV ∈  is used to balance principal component information and dimension reduction points . HoweverTo begin with, for the original signals without defects and test signals with defects, in previous The process of this method is demonstrated with our experimental signals. The example used was a straight pipe with a defect at 1 m. The signals of this pipe include 100 sets of original signals without defects and 140 sets of test signals with different defects :A comprehensive original sensitive index considering PCS and RS is defined by Equation (20):We can achieve a better sensitivity by referring to OSI when selecting the principal components. The processing flow of this adaptive method can be seen intuitively in The selection results of principal components in this dataset are shown in TDI means a better recognition effect of the APCA method. Therefore, we studied the relationship between the number of principal components, TDI, OSI, and CPV, as shown in Obviously, a larger The feature extraction effect of signals was also compared with different numbers of principal components in As shown in As seen in At the same time, the damage indexes It can be seen from Qa, and thus will lead to an insensitive damage index. As can be seen from the previous During the sampling process of the instrument, a very small part of the signals may be disturbed by electromagnetic noise, which are defined as outlier samples. The useless residual information of outlier samples will cause the rise of In this step, the Pauta Criterion  is used When the After this processing, the SPE become lower, and the damage index As shown in SPE and In step 4, after the latest test signal is collected, consider it as a vector In order to avoid the influence of a single index on the results, the comprehensive damage index K is defined by Equation (25):In this section, the guided wave signals of a straight pipe, bent pipe, and spiral pipe are presented, along with comparisons of the APCA, OBS, and the AED algorithm. The excitation parameters of the guided wave in several experiments are also reported.f = 64 kHz, diam = 0.72 m); under this condition, a superior echo waveform was recorded in this experiment. For the bent pipe experiment, because the SAFE method is only related to the cross-section shape and because Vinogradov’s Field testing on a carbon steel pipe indicated that higher frequencies (128 kHz) provided better performance in penetrating past U-bends [In the first experiment, a straight pipe  was used, and the semi-analytical finite element method (SAFE) was used to solve the dispersion curve ,37; the  U-bends , the samIn the experiment, the transducer was fixed permanently in the process of collecting original signals and test signals, and the defect was judged by comparing test signals with the original signals collected on the same pipe. The transducer worked in the pulse-echo mode, which means it acted as both a guided wave transmitter and a guided wave receiver.As shown in the straight pipe experiment conditions in The original signals of the first hole were collected in the pipe without any defects. After the first hole was expanded to an SLR of 0.6%, a second hole defect 1.5 m away from the transducer was produced in the same pipe. The original signals of the second hole were collected on the pipe with a hole. The guided wave was reflected at the first hole, which resulted in an energy reduction when the guided wave transmitted to the second hole.The guided wave monitoring equipment  was used to acquire guided wave signals of the pipes. The excitation signal was a 128 kHz sinusoidal signal modulated by a Hanning window with a certain bandwidth, as shown in In order to show the experimental results clearly, we calculated the recognition accuracy of the OBS, AED, and APCA methods on the same original signals and test signals. The accuracy is obtained by Equation (26):The other two methods are the following:OBS: First, we calculated the mean value of the original signals, then we subtracted it from all the signals; the max value of the subtraction result was then taken to represent a sample. The threshold was the max value of the subtraction result of original signals. The damage index can be obtained by Equation (28):AED: First, we calculated the mean value of the original signal, then we took the Euclidean distance between this and all signals. The damage index and threshold can be obtained by Equation (29):After the above processing, we can also obtain a damage index curve with a threshold of 1, as is shown in As can be seen from the above From It can be observed from Most of the experimental conditions have been mentioned above; however, a few conditions were different for this experiment. The experimental conditions for the spiral pipe (carbon steel) can be clearly seen in Due to the introduction of the weld, the guided wave echo amplitude of a defect is lower, and the signal-to-noise ratio will also be affected.Compared with the straight pipe, the pure guided wave mode on the spiral pipe is more difficult to excite in the spiral pipe.The collection time and temperature of signals were basically the same as those in Similar to the straight pipe experiment, the final experimental results are shown in As can be seen from the above Again, most of the experimental conditions of the bent pipe have been mentioned above; however, there were just a few different conditions used. As shown in Multiple reflections occur between two welds when the guided wave propagates; moreover, the propagation of guided waves in the bend region is often accompanied by the mode conversion and dispersion.The guided wave will focus on the outer surface of the elbow region, making it more difficult to monitor the inner surface .The acquisition time and temperature of signals were basically the same as those in The pipe and defect information is shown in The final experimental results are shown in As can be seen from the above N means the sampling points, and In order to identify the location of defects, the defect information was determined by calculating the contribution rate of each sample point to by Zhang , the timCompared with the result of the spiral pipe, the SNR of the straight pipe was obviously higher, and This paper discusses an APCA method for the monitoring of pipe defects. We established an effective damage index by selecting the number of principal components adaptively and decomposing the characteristics of the original signals. Moreover, the contribution diagram given by the algorithm enriched the defect location information. The results of the above experiments demonstrate that the recognition sensitivity of the APCA method is higher than that of the OBS and AED methods. At the same time, the APCA method also did not misjudge the undamaged test signals in three experiments. In future research, our group will conduct on-site long-term experiments when conditions permit, so that the APCA-based method can be used more effectively for monitoring the health of pipes in service.


Here, accumulating evidence has shown that targeting enzymes including poly (ADP-ribose) polymerase, ataxia telangiectasia and Rad-3 related, DNA-dependent protein kinase catalytic subunit, and checkpoint kinase 1 can radiosensitise HNSCC cells which should be taken forward in further preclinical studies, with the goal of optimizing the future effective RT treatment of HNSCC.Incidences of head and neck squamous cell carcinoma (HNSCC) have been on the rise in the last few decades, with a significant risk factor being human papillomavirus (HPV) type-16/18 infection, particularly in the development of oropharyngeal cancers. Radiotherapy (RT) is an important treatment modality for HNSCC, where it promotes extensive cellular DNA damage leading to the therapeutic effect. It has been well-established that HPV-positive HNSCC display better response rates and improved survival following RT compared to HPV-negative HNSCC. The differential radiosensitivity has been largely associated with altered cellular DNA damage response mechanisms in HPV-positive HNSCC, and particularly with the signaling and repair of DNA double strand breaks. However, other factors, particularly hypoxia present within the solid cancer, have a major impact on relative radioresistance. Consequently, recent approaches aimed at enhancing the radiosensitivity of HNSCC have largely centered on targeting key proteins involved in DNA repair, DNA damage checkpoint activation, and hypoxia signaling. These studies have utilised  Tobacco and alcohol use are the common risk factors associated with HNSCC. Furthermore, infection by high-risk (type-16/18) human papillomaviruses (HPVs) causes development of HNSCC, particularly in cancers of the oropharynx (~40%-60% incidence). Importantly, patients with HPV-positive HNSCC are known to have a better prognosis and improved survival rates associated with an improved response to radiotherapy (RT) and chemotherapy as compared with HPV-negative HNSCC. This reflects the different molecular mechanisms and biological characteristics underlying the oncogenic processes.Head and neck cancer describes several different malignant tumors that develop in or around the throat, larynx, nose, sinuses, and mouth. They usually develop in the squamous cells of the mucosal surfaces and therefore are often referred to as head and neck squamous cell carcinoma (HNSCC). In the UK, HNSCC is the 8th most common cancer with 12,200 new cases every year, the majority occurring in men (69%). Since the early 1990s, HNSCC incidence rates have increased by ~30% in the UK. One year survival rates among HNSCC subtypes are reportedly highest in salivary gland cancer (~60%) and lowest in hypopharyngeal cancer (~20%). While X-rays are used as a conventional RT treatment for HNSCC, major advancements have been achieved with the introduction of proton beam therapy and the development of 3D conformal stereotactic RT, which allow a better and safer treatment for the patients. The introduction of adaptive RT as a special form of image-guided RT has also been significant. Nevertheless, all RT techniques target and damage the DNA within the cancer cells leading to the therapeutic effect. Whilst RT promotes largely the formation of DNA base damage and DNA single-strand breaks (SSBs), more important is the induction of potentially toxic DNA lesions including DNA double strand breaks (DSBs) and complex DNA damage (CDD), containing multiple DNA damage types including DNA base damage, abasic sites, and SSBs and DSBs within close proximity . These DNA lesions trigger the activation of the cellular DNA damage response (DDR), involving several cellular proteins and pathways that attempt to repair and restore the integrity of the DNA. Depending on which cell cycle stage the cell is in, DSBs can be resolved though the activation of two different repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR)-13. The ultimate aim of RT is to create sufficient and/or persistent DNA damage in the cancer cells that exceeds the cellular capacity for repair, which ultimately triggers the cell death response or blocks their potential to replicate. This goal can be reached more effectively by combining RT with chemotherapeutics, specifically those that inhibit the DNA repair or cell cycle machinery.Treatment of HNSCC invariably include surgery, RT, and chemotherapy, either as a single treatment or in combination. The choice of one treatment over another is strongly influenced by the stage of disease at diagnosisIn this review, we describe the effects of RT on DNA damage and the cellular DDR pathways responsible for DNA repair. We also provide an up-to-date summary of the biological mechanisms critical in controlling radiosensitivity of HNSCC, as well as current radiosensitisation strategies being explored for improving the outcome of HNSCC patients post-RT.,15. Consequently, high LET radiation will display an enhanced biological effect. This is in part mediated by increases in the formation of DNA DSBs and CDD that represent a greater challenge to the cellular DNA repair machinery, whereas the majority of DNA lesions generated by low-LET radiation are DNA base lesions and SSBs that are repaired relatively more efficiently. Particle beam therapy  displays another distinct advantage in that the radiation dose can be delivered directly to the cancer cells with limited exposure of the surrounding healthy tissue. This is due to the deposition of maximum energy in depth at a well-defined region, the Bragg peak, which in turn will lead to less RT-related side effects when compared to conventional RT using low-LET photons. However, particle beam therapy exhibits increases in LET at and around the Bragg peak, which can generate a different DNA damage spectra that contributes to an increased biological response. As a consequence, there is still a significant amount of biological uncertainty when utilising this therapeutic approach,18. Nevertheless, the main goal of RT is to either cause sufficient cellular DNA damage or enhance the persistence of the damage to greatly exceed the cells capacity for repair, thus promoting cell death.RT is a non-invasive treatment method that has certain advantages over chemotherapy or surgery, especially when the cancer is not resectable, and it is the elective treatment for the majority of HNSCC. In order to kill cancer cells, RT damages the DNA either directly or indirectly through ionisation of water molecules. Low linear energy transfer (LET) radiation, including X-rays and γ-rays, deposits a relatively small amount of energy over a short distance, whereas high LET radiation, including charged particles, will deposit a large amount of energy within the same short distance,20. The DNA base damage is recognized and excised by damage-specific DNA glycosylases, which along with AP endonuclease-1 (APE1), incise the phosphodiester backbone to create an SSB flanked by 3’-OH and 5’-deoxyribosephosphate (dRP) ends. Poly (ADP-ribose) polymerase-1 (PARP-1) binds to the SSB due to its high affinity for the lesion and protects the DNA ends. DNA polymerase β (Pol β) then excises the 5’-dRP ends and simultaneously inserts a new nucleotide. This is followed by X-ray cross-complementing protein 1 in complex with DNA ligase IIIα (XRCC1-Lig IIIα) which ligate the SSB ends.The cellular DDR is an elegant signaling network able to detect and repair DNA damage, with different DNA repair pathways activated depending on the type of lesion 0/G1 phase where NHEJ pathway is employed, or in S/G2 phases where a sister chromatid is available to enable HR. NHEJ can be further divided into two sub-pathways. Canonical-NHEJ (c-NHEJ) utilises the Ku70/80 heterodimer that bind the DSB ends, followed by the DNA-dependent protein kinase catalytic subunit (DNA-Pkcs) and X-ray cross-complementing protein 4 in complex with DNA ligase IV (XRCC4/LigIV) that ligate the DSB ends. In contrast, alternative-NHEJ (a-NHEJ) is initiated by the C-terminal binding protein-interacting protein (CtIP) and MRE11/RAD50/NBS1 (MRN) complex that resect the DNA ends, followed by PARP-1 binding, gap filling by a DNA polymerase, and ultimately ligation by XRCC1-Lig IIIα . HR is an error-free DSB repair mechanism, and similar to a-NHEJ, is initiated by the CtIP-MRN complex that promotes DNA end resection and requires breast cancer protein 1 (BRCA1). The 3’-single stranded DNA generated is then bound and stabilized by replication protein A (RPA), replaced with the recombination protein RAD51 in a BRCA2-dependent manner, which then undergoes homology search and strand invasion into the sister chromatid. DNA synthesis in the presence of RAD52/RAD54 results in branch migration and formation of Holliday junctions that are resolved by DNA resolvases that ultimately restore genome integrity. In the case of CDD, which can include a mixture of DNA lesions within a short distance on the DNA molecule, it is likely that the repair of these sites requires more than one DNA repair pathway to resolve the damage. Interestingly, we have recently demonstrated that high-LET proton irradiation generates a high frequency of CDD sites consisting of abasic sites and SSBs, which have a high dependency on PARP-1 for repair.In the case of DSBs, the damage is firstly recognized by the ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad-3 related (ATR) protein kinases that stimulate phosphorylation of the histone H2AX variant (forming γH2AX). Cells then activate either NHEJ or HR depending on whether the cell is in G. HPV-positive HNSCC are known to display high radiosensitivity compared to HPV-negative HNSCC as well as higher patient survival rates due to that. Starting from these clinical observations, several investigations have been performed to unravel the molecular and biological mechanisms responsible for controlling the radiosensitivity of HNSCC, and particularly the role played by HPV in enhancing intrinsic radiosensitivity. To date, it has been largely demonstrated that patient-derived HPV-positive HNSCC cell lines are more sensitive than HPV-negative HNSCC cell lines to irradiation in vitro, which is consistent with the clinical observations. This suggests that these HNSCC cell lines are good models for investigating the nature of the differential radiosensitivity. As for the mechanism involved, several reports have shown that there is a defect in expression of key DSB repair effector proteins and/or defects in the efficiency of the DSB repair pathways post-irradiation. Firstly, five HPV-positive HNSCC cell lines were demonstrated to harbour more persistent levels of 53BP1 and γH2AX foci and display a marked G2/M arrest in response to radiation compared to five HPV-negative HNSCC cell lines. This finding was replicated in another study comparing two HPV-positive with two HPV-negative HNSCC cell lines, where the HPV-positive HNSCC cell lines demonstrated delayed resolution of γH2AX and 53BP1 foci and delayed repair of radiation-induced DSBs post-irradiation revealed through the neutral comet assay. A third study attributes the deficiency in DSB repair in two HPV-positive versus one HPV-negative HNSCC cell line to decreased BRCA2, DNA-Pkcs, and 53BP1 protein expression in HPV-positive HNSCC cells, which ultimately led to NHEJ and HR signaling deficiency as demonstrated through the lack of DNA-Pkcs and BRCA2/RAD51 foci, respectively post-irradiation. It is interesting to note that there is evidence that HPV-positive HNSCC cells also show upregulation of several proteins involved in BER and SSB repair, including PARP-1, PNKP, Pol β, and XRCC1. Whilst this does not appear to relate to the observed increased radiosensitivity, it suggests a compensatory mechanism of increased BER activity through loss of DSB capacity. These data are supported by analysis of the TCGA database, which demonstrated increased gene expression of at least XRCC1 and PARP-1 in HPV-positive HNSCC. Interestingly, it was further shown that shRNA-mediated downregulation of multiple BER genes enhanced the radiosensitivity of one HPV-positive HNSCC cell line and not of a corresponding HPV-negative HNSCC cell line.HPV is a circular, double stranded DNA virus and a member of the papillomaviridae family. Of the over 200 types of HPV identified, HPV-16 and HPV-18 are the two that mainly contribute to oncogenesis and are the most commonly found types in HNSCC. The oncoprotein E7 also leads to the accumulation of p16, which is used as a biomarker of HPV-positive HNSCC. p16 overexpression in HPV-positive HNSCC cells causes initiation of cell cycle progression, but independently has been suggested to have a direct impact on DDR, specifically through HR by reducing RAD51 foci formation post-irradiation. Interestingly, p16 has been recently demonstrated to cause downregulation in the protein levels of the E3 ubiquitin ligase TRIP12 leading to deficiencies in DSB repair. Targeting TRIP12 using siRNA in HPV-negative HNSCC cells led to an impairment of HR by inhibiting the recruitment of BRCA1 to DSBs post-irradiation. Cumulatively, these data clearly show the impact of HPV in reducing the efficiency of the cellular DDR in HNSCC cells.In addition to defective DSB repair pathways, differentially regulated cell cycle control in HPV-positive HNSCC cells is well established, primarily due to the impact of the E6 and E7 HPV genes. The E6 and E7 proteins can bind the tumour suppressor protein p53 and retinoblastoma (Rb) protein, respectively, facilitating their ubiquitylation-dependent proteasomal degradation resulting in the cells losing their ability to perform cell cycle arrest or to undergo apoptosis,33. This biological effect is caused by the physical interference of the lack of oxygen with the radiation effects on creating DNA damage, but also through the alteration of cellular pathways and processes. Oxygen is a particularly powerful radiosensitiser. However, it has been shown that HNSCC can reach levels of moderate and severe hypoxia which will ultimately lead to radioresistance and poor prognosis,35. In terms of cellular pathways, the major player mediating the hypoxic response are the hypoxia-inducible factors (HIFs). The HIF-α subunits contain proline residues which are hydroxylated by prolyl hydroxylase domain (PHD) enzymes allowing the von Hippel-Lindau tumour suppressor protein to label it for ubiquitylation and rapid proteasomal degradation under normoxic conditions-38. During hypoxia, the PHD enzymes are inhibited, leading to HIF-α stabilization which in turn leads to transcriptional activation of many hypoxia-related genes,40. Interestingly, it has been observed that five HPV-positive HNSCC cell lines show higher levels of HIF1-α  compared to two HPV-negative HNSCC cells under conditions of normoxia, possibly due to the HPV oncoprotein inhibiting PHD2-dependent hydroxylation of HIF1-α. Under hypoxic (1% oxygen) conditions, relative protein levels of HIF1-α proportionately increased in both HPV-positive and HPV-negative HNSCC cell lines, with the HPV-positive cells still retaining higher levels of the protein.The existence of hypoxia (low oxygen) in human tumors, an important source of radioresistance, was first established in the 1950s-46. Under hypoxia-induced replication stress, ATR is specifically required for phosphorylation of downstream proteins, including checkpoint kinase 1 (CHK1), p53, and γH2AX,47. It has been demonstrated using three HPV-positive and three HPV-negative HNSCC cells, that a short term incubation (1 h) in severe hypoxia (0% oxygen) prior to irradiation, led to increased radioresistance of both sets of cell lines with observed oxygen enhancement ratios of 2.3-2.9. This was associated with an upregulation in the expression levels of hypoxia-responsive genes, the magnitude of which varied between the cell lines used. More recently, in three HPV-negative HNSCC cell lines it was shown that hypoxia (0.1% oxygen) during irradiation increases cell survival and reduces the levels of residual γH2AX foci post-irradiation. Interestingly though, when cells were exposed to a prolonged period of 4-5 days in hypoxia (1% oxygen) prior to irradiation, ATM-deficient HNSCC cells showed increased radiosensitivity associated with a delayed phosphorylation of DNA-Pkcs and delayed NHEJ pathway activation, whilst the other two HPV-negative HNSCC cell lines displayed increased radioresistance. This demonstrated a cell line-specific response following long term hypoxia treatment prior to irradiation. HPV-negative HNSCC usually contain a high frequency of p53 mutations, therefore the concomitant roles played by p53 mutants and HIF-1 in a hypoxia context could partially explain the higher radioresistance capacity observed in these tumors. However, more detailed cellular investigations are required.Whilst the general impact of hypoxia in mediating cellular radioresistance is well acknowledged, the molecular mechanisms affected specifically in HPV-positive in comparison to HPV-negative HNSCC cell lines, and the relationship to radiosensitivity have not been studied in detail. Hypoxia, particularly severe hypoxia (< 0.1% oxygen), promotes genetic instability through activation of ATM/ATR leading to cell cycle arrest, and downregulation of NHEJ and HR DSB repair pathways at a transcriptional and epigenetic level thus conferring a more resistant cellular phenotypein vitro, including the epidermal growth factor receptor (EGFR), PARPs, and several proteins involved in DSB repair and cell cycle regulation -based drugs and small molecule tyrosine kinase inhibitors (TKIs). mAb-based drugs act by binding the extracellular domain of EGFR and inhibiting its dimerization, which ultimately leads to its degradation after cell internalization. The TKI compounds target the intracellular tyrosine kinase domain of EGFR and compete with ATP binding to eliminate EGFR downstream signaling. The mAb cetuximab is an approved chemotherapeutic for use in combination with RT in patients with unresectable, locoregionally advanced HNSCC, as it has been proven that its administration increases survival compared to RT alone. However, a recent study has shown that platinum-based chemoradiotherapy is superior to cetuximab treatments. In fact, it has been observed that cetuximab caused tumor growth delay in hypopharyngeal xenografts possibly due to reduced ERK1/2 phosphorylation. However, laryngeal xenografts did not show any additive effect of cetuximab to RT and instead displayed increased proliferation, suggesting cetuximab may be effective only in a subpopulation of HNSCC. This is further reflected by the recent failure of two phase III studies examining cetuximab in combination with RT in HPV-positive HNSCC,78, which has raised doubts about EGFR targeting in HNSCC. Many other mAb treatments of HNSCC have reached phase III of development and initially showed promising results.EGFR is a member of the ErbB/HER family of tyrosine kinase receptors. It has been linked to cancer progression in HNSCC, and overexpression of EGFR is associated with poor prognosis and its level has been suggested to be a good predictor for patient outcome in HNSCC. A recent study showed that erlotinib was unable to significantly radiosensitise fourteen HPV-negative HNSCC cell lines, although increased radiosensitisation was observed in some cells under specific re-plating conditions that overcame the G2 cell cycle arrest caused by EGFR targeting. Additionally, the recent use of the second generation TKI afatinid in phase III trials in HNSCC gave conflicting results,84. The observed differential responses of TKIs in radiosensitising HNSCC has therefore led to suggestions that their administration is in addition to current therapeutic treatments. Nevertheless, results regarding both mAb and TKIs must be considered cautiously, as recent works have shown that EGFR overexpression is not common in patients with HNSCC and that in general EGFR expression and activity are not well correlated, with many cell lines used in studies not representative of the clinical situation,86.Several TKIs have been investigated in the last 20 years, with gefitinib and erlotinib the first to be tested, however more recent data have questioned their use in HNSCC. For example, erlotinib failed to improve the radiosensitivity of five HPV-negative HNSCC xenograft models through the observed lack of impact on tumour growth. Currently, oxygen-mimetic radiosensitisers, such as the nitroimidazoles, are the most promising compounds for overcoming hypoxia. Whilst etanidazole failed to show benefit in advanced HNSCC patients in combination with RT in a phase III clinical trial, nimorazole has been demonstrated clinically to be an effective radiosensitiser for HPV-negative but not for HPV-positive HNSCC-91. Following these investigations and further results of studies from the Danish Head and Neck Cancer Group, nimorazole has become standard of care to HNSCC patients in combination with RT in Denmark. A phase III trial examining nimorazole efficacy with RT in locally advanced HNSCC is currently ongoing in the UK. Recently, a group of novel nitroimidazole alkylsulfonamides have been tested using a prodrug approach in order to increase solubility and improve drug delivery, with some of them at least showing promising results in their ability to radiosensitise anoxic colorectal cancer cells in vitro.Hypoxia is well known to contribute to poor prognosis and treatment resistance in HNSCC, therefore hypoxic modulation has been investigated particularly in a clinical setting in order to restore sensitivity to treatment. However, oxygenation treatments are not routinely used clinically because there is no ideal technique to quantify tumor hypoxia and no effective treatment to modify hypoxic conditions. Many hypoxia targeting methodologies have been proposed and tested, including hyperbaric oxygen, hyperthermia, and agents targeting tumor blood flow. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a flavone acetic acid analogue that showed promising results as a monotherapy in reducing growth of HPV-negative HNSCC xenografts in mice. DMXAA may also interact with tumour necrosis factor, leading ultimately to tumor oxygenation and increased radiosensitivity. However, the impact of DMXAA and other VDAs in specifically radiosensitising HNSCC has not been reported. In relation to targeting hypoxia-induced proteins, the protein kinase AKT has been shown to be activated under severe hypoxia (0.1% oxygen for 6-72 h) in both HPV-positive and HPV-negative HNSCC cell lines, and that the AKT inhibitor MK2206 alone significantly reduced HPV-positive and HPV-negative HNSCC cell line growth (sensitiser enhancement ratios of ~1.5). Interestingly, MK2206 led to increased radiosensitivity of only the HPV-positive HNSCC cell lines under hypoxic conditions, however the study was limited to analysing the response to a single dose of radiation, and the molecular basis for this effect is currently unclear. A major problem with the clinical translation of these and other strategies is the heterogeneity of oxygen tensions within the hypoxic region of the tumour, where the level of radioresistance also varies. Nevertheless, further studies examining the molecular and biological impact of different levels of hypoxia on the comparative radiosensitivity of HPV-positive and HPV-negative HNSCC cell lines and tumours, and ultimately strategies to overcome radioresistance, are required.Several other agents have been investigated for overcoming tumor hypoxia, including those targeting tumor blood flow and drugs targeting hypoxia-induced proteins. Vascular disrupting agents (VDAs) alter tumor blood flow by increasing vascular permeability through tubulin binding on endothelial cells, whilst possibly activating apoptosis,98. Here, PARP-1 uses NAD+ to catalyse the synthesis of ADP-ribose chains to itself and also to target residues of specific proteins, which acts as a platform for DNA repair protein recruitment. PARP1 is, however, also involved in the repair of DSBs via both HR and NHEJ. There has been significant interest in PARP-1, and particularly PARP inhibitors, because of its synthetic lethal partnership with BRCA1/2 mutations in breast cancers,101. Therefore, PARP inhibitors have been increasingly investigated as a treatment option in HNSCC with some promising results. Relatively earlier studies focused on veliparib which showed in three HPV-negative HNSCC cell lines that the inhibitor (at >5 µmol/L) exhibited high cytotoxicity in the presence of ionising radiation, and was exacerbated with the EGRF inhibitor cetuximab. Similarly, veliparib (at 10 µmol/L) decreased the survival of one HPV-negative HNSCC cell line post-irradiation, but had a ~1.5-fold enhanced effect on two HPV-positive HNSCC cell lines due to their reported DSB repair defect. This is supported by enhanced radiosensitivity of three HPV-positive HNSCC cell lines using high doses of veliparib (10 and 20 µmol/L), although one out of three HPV-negative HNSCC cell lines also displayed marked radiosensitisation. Interestingly though, veliparib only had a relatively mild effect on suppressing growth of HPV-positive HNSCC xenografts.PARP-1 is one of the 17 members of the PARP family, which acts as an important sensor for gaps and SSBs in the DNA and plays a fundamental role in BER, although individual variability in cellular responses was observed. A recent study has shown that olaparib (at 1 and 5 µmol/L) is effective in combination with radiation in decreasing the survival of HPV-negative HNSCC cells, but only those that are SMAD4 deficient. Despite this, only a modest effect of olaparib in combination with radiation, versus radiation alone, was observed on growth of SMAD4-deficient HNSCC xenografts. Nevertheless, we have reported that two HPV-negative HNSCC cell lines were significantly radiosensitised with olaparib (0.1 µmol/L) with dose enhancement ratios (DER) of 1.79 and 3.34. This was in comparison to two HPV-positive HNSCC cell lines, whereby only one of these showed increased radiosensitisation in the presence of olaparib (DER = 1.51). A separate study using five HPV-positive HNSCC cell lines displayed increased radiosensitivity after olaparib (1 µmol/L) treatment, although markedly only in four cell lines following a relatively high radiation dose (6 Gy), suggesting that the radiation dose plays a fundamental role together with the DNA repair capacity of the cells. Using the PARP inhibitor niraparib (1 µmol/L), this was recently shown to equally increase the sensitivity of two HPV-positive and two HPV-negative HNSCC cell lines to conventional radiation (DER = 1.06-1.21) and proton irradiation (DER = 1.12-1.31). Both sets of cell lines displayed higher numbers of residual 53BP1 foci present when niraparib was used in combination with either form of radiation. In contrast, another recent study has demonstrated that niraparib (1 µmol/L) had a greater impact of the radiosensitisation of two HPV-negative (DER = 1.94-2.13) compared to two HPV-positive (DER = 1.36-1.43) HNSCC cell lines. Cumulatively, although positive results are observed when HNSCC cells are treated with PARP inhibitors prior to irradiation, further studies are required to investigate in depth the potential selectivity towards HPV-positive and HPV-negative HNSCC, and therefore provide evidence for PARP as a potentially promising therapeutic target in the clinical setting.Several studies have utilised the PARP inhibitor olaparib as a radiosensitizer, although these have shown mixed results particularly regarding degree of radiosensitisation of HPV-negative HNSCC cell lines, which are deemed DSB repair proficient. Indeed, it has been suggested that HPV-negative HNSCC cell lines are more responsive to olaparib (1 µmol/L) radiosensitisation when the cells are specifically HR-deficientin vitro and in vivo, and so strategies have mostly focused on enhancing radiosensitivity of relatively radioresistant HPV-negative HNSCC. Given that DSBs are sensed by the protein kinases ATM, ATR, and DNA-Pkcs, these enzymes have been investigated as targets for inhibitors to increase HNSCC radiosensitisation. A number of studies have focused on ATR and found this to be a successful approach for increasing radiosensitivity in HPV-negative HNSCC cell lines. In particular, the ATR inhibitor VE821 was shown to enhance the radiosensitivity of a single HPV-negative HNSCC cell line in both normoxia and hypoxia (2% and < 0.02% oxygen). Similarly using the same inhibitor, we have also recently observed radiosensitisation of two oropharyngeal HPV-negative HNSCC cell lines grown as colonies but also as 3D spheroids following both photons and (low-LET) protons. However, our study also demonstrated that a hypopharyngeal HPV-negative HNSCC spheroid model was not radiosensitized in the presence of VE821, suggesting cell and tumour-specific radiosensitisation. Using the ATR inhibitor AZD6738, two studies have demonstrated that HPV-negative HNSCC cell lines  can be radiosensitised following ATR inhibition, including the impact on preventing 3D spheroid growth of one of the cell lines,59. The combination of both ATR and DNA-Pkcs inhibition (KU-0060648) further exacerbated the cell killing effects post-irradiation. In relation to ATM as a target for radiosensitisation in HNSCC cells, the evidence is sparser. A small molecule inhibitor screen identified the compound GSK635416A in enhancing the radiosensitivity of three HPV-negative HNSCC cell lines, which was then demonstrated to act through inhibition of ATM. This study also showed evidence for additive effects of ATM inhibition in combination with PARP inhibitor (olaparib) in enhancing radiosensitivity. Data recently generated in our lab has shown a significant impact of the ATM inhibitor KU-55933 in reducing survival of four HPV-negative HNSCC cell lines  and reducing growth of these 3D spheroid models in response to both photons and protons. Targeting ATM was also shown to enhance the radiosensitivity of one HPV-positive HNSCC cell line, but not a second which was the most radiosensitive cell line used.Targeting the proteins involved in the repair of DSBs, that are potentially toxic lesions induced by RT, is considered a promising strategy in HNSCC treatment. As detailed previously, HPV-positive HNSCC in general have been found to be DSB repair deficient which contributes to their increased radiosensitivity  and IC87361 have both been shown to cause reduced survival of HPV-negative HNSCC cell lines  post-irradiation, the latter study also suggesting that this approach was more effective than the combination of radiation with targeting PARP (via olaparib). Evidence using the DNA-Pkcs inhibitor NU7441 has further demonstrated the effectiveness of targeting DNA-Pkcs in radiosensitising three HPV-negative in addition to three HPV-positive HNSCC cell lines, suggesting that this is a more general strategy for treatment of HNSCC. This study was further progressed in vivo, where a significant delay in growth of a HPV-positive xenograft model was observed. The slow growth of a HPV-negative xenograft model did not allow for an accurate assessment of the effectiveness of the combination of the DNA-Pkcs inhibitor with radiation, although this was shown in two patient-derived xenograft models where tumour growth was significantly suppressed. Finally, our recent evidence has demonstrated that the DNA-Pkcs inhibitor KU-57788 significantly enhanced the radiosensitisation of four HPV-negative HNSCC cell lines, including suppression of the growth of 3D spheroid models, following both photons and protons. We also observed increased radiosensitisation of HPV-positive HNSCC cells and spheroids, and in general identified that targeting DNA-Pkcs was the most effective strategy for radiosensitisation of HNSCC cells in comparison to inhibitors against ATM and ATR. Cumulatively, these data suggest that targeting the key proteins involved in the repair of DSBs, particularly ATR and DNA-Pkcs, may be an effective treatment in combination with RT for HPV-negative HNSCC. Nevertheless, further evidence investigating this strategy in a broader range of HNSCC cell lines in vitro, as well as utilising in vivo models, are necessary.Several studies have identified that DNA-Pkcs inhibition using different compounds has the ability to increase HNSCC radiosensitivity. The DNA-Pkcs inhibitors KU0060648in vitro and tumour growth delay of a HPV-negative HNSCC model in vivo. This is supported by a study using the CHK1 inhibitor CCT244747 in two HPV-negative HNSCC cell lines where increased radiosensitivity was observed, along with delayed growth of an HPV-negative HNSCC xenograft model. However, this study also identified that the triple combination of paclitaxel, CHK1 inhibitor, and radiation was able to significantly reduce xenograft tumour growth. Subsequent studies using the CHK1 inhibitor PF0477736 demonstrated increased radiosensitisation in two out of three HPV-negative HNSCC cell lines associated with loss of distal chromosome 11q, and only one out of two HPV-negative HNSCC cell lines. The latter study in fact showed that four out of five HPV-positive HNSCC cell lines tested were radiosensitised with CHK1 inhibition, which was supported by a follow-up study performed by the same research group in two HPV-positive HNSCC cell lines. Additional evidence acquired in three HPV-positive HNSCC cell lines showed the impact of the CHK1 inhibitors LY2603618 and MK8776 in increasing cellular radiosensitivity, but also that the additive effect of the WEE1 inhibitor AZD1775 was relatively mild. Interestingly, the WEE1 inhibitor alone in combination with radiation only appeared effective in radiosensitising one of the three HPV-positive HNSCC cell lines.Checkpoint activation allows halting of cell cycle progression and is therefore important in responding to radiation-induced DNA damage to allow cells to accomplish DNA repair. Notably, due to disruption of p53/pRb and cell cycle regulation caused by HPV infection, the proteins involved in cell cycle checkpoint activation, particularly CHK1 and WEE1 in the G2/M checkpoint, are of therapeutic interest in causing enhanced RT treatment of HPV-positive HNSCC. However, this approach has also been investigated in pre-clinical studies in HPV-negative models of the disease. Initial evidence using the CHK1 inhibitor SAR020106 showed enhanced radiosensitivity of two HPV-negative HNSCC cell lines . This is supported by a more recent study demonstrating the added benefit of CHK1 (MK8776) and PARP (niraparib) inhibition in enhancing radiosensitivity of one HPV-positive HNSCC cell line, but which was not effective in one HPV-negative HNSCC cell line. In contrast, it was additionally observed that the HPV-negative HNSCC cell line was more susceptible to the combination of PARP and WEE1 (MK1775) inhibition, which was suggested to be due to more potent activation of the G2/M checkpoint caused by WEE1 inhibition that is downstream of CHK1. Noteworthy, a phase I dose-finding multicenter trial has begun this year with the aim of determining a safe dose of the WEE1 inhibitor AZD1775, both for preoperative patients treated with cisplatin and for post-operative patients treated with cisplatin-based chemoradiation.Studies have also examined combinatorial strategies using inhibitors targeting both cell cycle checkpoints, and DNA repair (via PARP). Here, the additive effect of PARP inhibition (using olaparib) in combination with CHK1 inhibitor PF00477736 in reducing radiation-induced survival of two HPV-positive HNSCC cell lines has been shown. This treatment was shown to cause a reduction in HR efficiency post-irradiation, ultimately leading to increased chromosomal damage and cell death. Palbociclib is currently under testing in the UPSTREAM trial in patients with recurrent or metastatic HNSCC who show cyclin D1 amplification and p16 negativity. Ribociclib is a small inhibitor of CDK4/6, which has showed promising safety and efficacy results in a phase I study in patients with local or metastatic recurrence of HNSCC together with cetuximab. However, the impact of ribociclib in combination with radiation on HNSCC cell models has yet to be investigated. Given the promising evidence that targeting the cell cycle can enhance the radiosensitivity of HNSCC cell lines, particularly relatively radioresistant HPV-negative HNSCC, more comprehensive studies are warranted to accumulate this important preclinical data.Another promising therapeutic target for radiosensitising HNSCC is CDK4/6, utilising the inhibitors palbociclib and ribociclib. Palbociclib has been demonstrated to selectively increase the radiosensitisation of HPV-negative, but not HPV-positive HNSCC cell lines both in normoxic and in hypoxic (1%) conditionsin vitro using the large numbers of established HNSCC cell lines that exist, but that variability in the degree of radiosensitisation is observed which is dependent on the specific cell line used. These studies also differ in the precise inhibitors utilised that harbour different selectivity and potency. Therefore, the challenge now is to accumulate preclinical evidence using the most selective and up to date inhibitors in a larger cohort of HNSCC models, to convincingly demonstrate that such approaches can enhance the radiosensitivity of HNSCC. These models should include HPV-positive and HPV-negative cell lines from the oropharynx, but also those originating from different tumour origins, including the larynx, hypopharynx, and oral cavity which are largely HPV-negative, to understand whether or not the strategies investigated can be utilised more generally in treating HNSCC. It is also apparent that more advanced HNSCC models should be employed in this research, including 3D spheroids and well characterized patient-derived organoids, that mimic more precisely the structure and environment of the original HNSCC from which they were obtained. Whilst 3D spheroids can be developed from the current HNSCC cell lines that exist ,106. Additionally, radiosensitisation strategies should be investigated in in vivo models, particularly xenografts of HNSCC, to provide clear evidence that targeted inhibitors are effective at suppressing or preventing HNSCC tumour growth. Another interesting aspect to examine is the impact of particle beam therapy  both alone and in combination with targeted inhibitors. As we detailed earlier, these data are currently sparse, mainly due to limited access to the appropriate resources, although there is some evidence to suggest that protons in particular may have a different impact on the DNA damage spectra induced compared to conventional photon irradiation, and thus may respond differently to combinations with DNA repair and DNA damage checkpoint inhibitors. Similarly, the impact of increasing LET at and around the Bragg peak and the effect of higher-LET radiation, including carbon ions, has yet to be thoroughly investigated. Nevertheless, once this extensive preclinical data has been acquired for clearly identifying effective strategies in combination with RT (photons and particle ions) for HNSCC, this will pave the way forward for future Phase I/II clinical trials.Promising data has been reported demonstrating that the radiosensitivity of HNSCC, particularlyrelatively radioresistant HPV-negative HNSCC, can be increased by inhibitors targeting proteins involved in DNA damage repair  and in DNA damage checkpoint activation (CHK1). These preclinical studies have largely been performed


To investigate the factors associated with the prognosis of spinal cord injury without radiographic abnormality (SCIWORA) accompanied by cervical ossification of the posterior longitudinal ligament (C-OPLL).  We retrospectively investigated 287 patients with SCIWORA associated with C-OPLL, who were admitted within 30 days after trauma to our facility between August 2014 and August 2018. All patients were divided into the good or poor prognosis group. Patient demographics were analyzed. Besides, occupying ratio on CT and spinal cord high signal changes in MRI T2WI were measured and recorded. Multivariate linear regression was applied to analyze the correlation of prognosis with spinal cord high signal changes in MRI T2WI, cause of injury, and occupying ratio.  Occupying ratio of ossification mass was 43.5 ± 10.7% in the poor prognosis group and 27.3 ± 7.7% in the good prognosis group. The occurrence rate of high signal changes in MRI T2WI was 84.2% in the poor prognosis group and 41.3% in the good prognosis group. Poor prognosis was correlated with high occupying ratio and spinal cord high signal changes in MRI T2WI. In the patient with SCIWORA associated with C-OPLL, ROC curve of occupying ratio showed 30% as a predictor for the poor prognosis. Among the 92 patients with occupying ratio ≤ 30%, poor prognosis was observed in 5 cases (5.4%), whereas in the 72 cases with occupying ratio > 30%, poor prognosis was seen in 33 cases (45.8%). Postoperative AIS grade at final follow-up in occupying ratio > 30% group was significantly worse.  Patients suffering from SCIWORA with C-OPLL have poor prognosis when they have higher occupying ratio of ossification mass and spinal cord high signal changes in MRI T2WI. The cut-off value of occupying ratio for predicting the poor prognosis was 30% in patients with SCIWORA associated with C-OPLL. Spinal cord injury without radiographic abnormality (SCIWORA) is a syndrome that involves spinal cord injury (SCI) without evidence of spine fracture or dislocation on plain radiographs or computed tomography (CT), characterized by low energy damage and mostly incomplete quadriplegia –3. BesidThe prevalence of C-OPLL among SCI patients was higher than the general prevalence rate of C-OPLL. C-OPLL may increase the risk of SCI associated with minor injury. There are many reports on SCI associated with C-OPLL; however, the precise mechanism remains unknown. Kwon et al.  reportedWe retrospectively analyzed patients with SCIWORA associated with C-OPLL. According to the neurological outcomes assessed by the American Spinal Injury Association (ASIA) impairment scale (AIS) grade at admission and discharge, we defined absence of improvement of AIS grade as poor prognosis and improved neural function based on AIS grade as good prognosis . All theWe retrospectively investigated 287 patients with SCIWORA associated with C-OPLL, who were admitted within 30 days after trauma to our facility between August 2014 and August 2018. The inclusion criteria were history of trauma, presence of ossification of posterior longitudinal ligament in cervical spine on cervical CT images, and absence of major fracture or dislocation of the cervical spine on X-rays or CT. Patients with a previous history of cervical SCI, surgery, tumor, or tuberculosis were excluded. Finally, 164 patients (mean age: 56.3 years) who had undergone surgical decompression were included in this study. All patients presented symptoms of cervical spinal cord injury (SCI) and MRI manifestations consistent with symptoms secondary to cervical spinal cord compression. For each patient, SCIWORA was confirmed by clinical syndromes, physical examinations, MRI demonstration of cord compression, and CT-confirmed absence of major fracture or dislocation of the cervical spine. Patients with impaired neurological function and persistent cervical spinal cord compression were indicated for surgery [All patients were divided into the poor prognosis group  and the good prognosis group  . The twoThe surgical procedure of anterior cervical ossified posterior longitudinal ligament en bloc resection (ACOE) has been previously reported by us . The surAll patients received antibiotics to prevent infection, 30 minutes before the operation and 24 hours after the operation. The drainage tubes were removed based on the drainage volume. Excessive neck rotation, extension, and flexion were avoided. The patients were encouraged to cough and complete functional exercise. After surgery, all patients were immobilized with a Philadelphia collar for 12 weeks.Postoperative outcomes at final follow-up were assessed and recorded according to the AIS grade . Follow-t-test, while differences in categorical variables were compared using the chi-square test. Then, a multiple logistic regression model yielding odds ratios (ORs) and 95% confidence intervals (CI) was used to identify predictors of poor prognosis  on discharge. Receiver operating characteristic (ROC) curves were drawn, and the area under the ROC curve along with its corresponding 95% CI provided a measure of overall validity. The cut-off value of the occupancy ratio for predicting the postoperative poor prognosis was determined using the ROC analysis and Youden's index [p value < 0.05 was considered to be statistically significant. All tests were two-tailed.Statistical analyses were performed using SPSS17.0 statistical software. The data are expressed as the mean ± standard deviation. Differences in continuous variables were compared using the Student's unpaired 's index . A p valThere were no significant differences in age, gender, comorbidities, preoperative neurologic status, morphology of OPLL, and intraoperative blood loss between the two groups .χ2 test. The average occupancy ratio of patients in the poor prognosis group was 43.5 ± 10.7%, which was significantly higher than that in the good prognosis group (27.3 ± 7.7%).There was no difference in the distribution of cause of injury between the two groups. There was no difference in the distribution of surgical treatment between the two groups. There was significant difference in the distribution of spinal cord high signal changes in MRI T2WI between the two groups according to Multivariate analysis was conducted for spinal cord high signal changes in MRI T2WI, cause of injury, and occupancy ratio. According to the stepwise regression method, the results showed that occupancy ratio and spinal cord high signal changes in MRI T2WI were associated with poor prognosis. The final regression model was as follows:The poor prognosis after SCIWORA associated with C-OPLL was significantly correlated with high occupancy ratio and spinal cord high signal changes in MRI T2WI .p < 0.001, AUC = 0.894, 95% CI 0.839 − 0.950, sensitivity = 97%, specificity = 61%)  .p < 0.001)  in the occupancy ratio > 30% group and 92 patients  in the occupancy ratio ≤ 30% group. There were no significant differences in age, gender, comorbidities, and cause of injury between the two groups . The pro< 0.001) .In 164 patients with SCIWORA associated with C-OPLL, the incidence of poor prognosis is higher in the group with occupancy ratio more than 30% . Among tFor patients with SCIWORA accompanied by C-OPLL, the correlation between occupancy ratio and poor prognosis remains unclear. Kwon et al.  reportedIn this study, the poor prognosis group had higher occupancy ratio and higher occurrence of spinal cord high intensity on MRI T2WI than the good prognosis group. The result of multivariate analysis showed that higher occupancy ratio and higher occurrence rate of spinal cord intensity correlated with poor prognosis. For patients with SCIWORA accompanied by C-OPLL, OPLL was the static factor, and the possible mechanisms were as follows: (1) direct injury from the ossification mass. At the time of traumatic injury, the spinal cord under static compression of ossification mass could be abruptly pinched by ossification mass, resulting in secondary damage of spinal cord, which induced neurological deficits and deterioration . (2) TheHowever, Okada et al.  did not The cut-off value of occupancy ratio for predicting the poor prognosis was 30% in the patient with SCIWORA associated with C-OPLL. In the current study, we found 30% to be the cut-off ratio for predicting the poor prognosis in the patients with SCIWORA associated with C-OPLL. For C-OPLL patients, especially those with occupancy ratio > 30%, their CSF zone was narrowed severely, which may lead to lack of effective buffering and protection.The occurrence rate of high signal changes in spinal cord in MRI T2WI was 84.2% in the poor prognosis group and 41.3% in the good prognosis group, with significant difference. High signal changes in the spinal cord in MRI T2WI originate from dynamic damage of traumatic force and static damage of continuous spinal cord compression. Dynamic damage may be induced by of traumatic force, which abruptly pinches the spinal cord by inner structures of the spinal canal, and causes sudden neurological injury. Static damage may originate from mechanical damage of direct compression from ossification of the posterior longitudinal ligament . Kwon etConsequently, high occupancy ratio and high signal changes in T2W MRI are significant risk factors for developing severe paralysis in patients with SCIWORA associated with C-OPLL. The cut-off value of occupancy ratio for predicting the poor prognosis was 30% in patients with SCIWORA associated with C-OPLL. The present study had several limitations: (1) the number of subjects was relatively small. (2) Multiple factors are associated with the prognosis of C-OPLL. This study only focused on the occupancy ratio and high signal changes in T2W MRI, which may have ignored other possible significant risk factors. Since this study was retrospective and based on a small sample size, large-scale and prospective studies are necessary and warranted.This study evaluated the occupancy ratio and high signal changes in T2W MRI in patients with SCIWORA associated with C-OPLL.The poor prognosis group exhibited a higher occupancy ratio and a higher incidence of high signal changes in T2W MRI than the good prognosis group.The cut-off value of occupancy ratio for predicting the poor prognosis was 30% in patients with SCIWORA associated with C-OPLL.


A face-to-face questionnaire interview was administered, and an anal-canal swab was collected for HPV genotyping.+ T cell count than those with decreased or unchanged  from baseline. In multivariable models, having the current CD4+ T cell count of 350–499 cells/µL , and of ≥ 500 cells/µL  were associated with lower prevalence of any type HPV infection compared with those with < 200 cells/µL. Having taken NVP + 3TC + AZT was inversely associated with any high-risk (HR)-HPV  and any low-risk (LR)-HPV infection , compared with those taking EFV + 3TC + TDF.A total of 766 HIV-positive men were recruited. The HPV prevalence was lower among those with increased CD4+ T cell count at follow-up was significantly associated with lower prevalence of anal HPV infection. Inverse associations between NVP + 3TC + AZT and HR-HPV or LR-HPV infecton were observed.Increased CD4 While some studies showed that increased CD4+ T cell count is associated with lower risk of HPV infection among HIV-positives [Human papillomavirus (HPV) infection is the most prevalent sexually transmitted infection (STI) in the world , 2. Persositives –17, otheositives , 6, 18. ositives . In addiositives , while nositives , 22. Furositives .+ T cell improvement and anal HPV infection, and hypothesized that cART treatments were correlated with HPV infection in the HIV-positive male population.Development of strategies for the prevention and treatment of HPV-related cancers in the anogenital tract requires a better understanding of the risk factors and natural history of persistent HPV infection, especially given that HPV vaccine has not been widely available in China. A cross-sectional study was conducted in male participants from an ongoing HIV cohort to investigate HPV prevalence from anal canal swab samples. We hypothesized an inverse association between CD4+ T cell count when the survey was conducted; (3) willing to have an anal swab sample collected. There were 1,304 HIV infected men being followed-up from the study sites, and 776 (58.7%) were willing to participate in this survey. Ten of them had neither the baseline nor the current CD4+ T cell test results and thus were excluded. A total of 766 participants were included in this analysis. The study was approved by the Institutional Review Board of School of Public Healh, Fudan University, and all the participants provided written informed consent.Participants in this study were those HIV infected men registered in and routinely followed-up by Taizhou City Center for Disease Control and Prevention (CDC) and four district-level CDCs in Taizhou, Zhejiang Province. A survey for anal HPV infection was conducted between 2016 to 2019. The eligible criteria included: (1) aged 18 years or older; (2) had baseline or current test result for CD4+ T cell counts were obtained from the Chinese HIV/AIDS Comprehensive Information Management System. For this survey, participants were face-to-face interviewed by trained local public health staff during the visit, and a questionnaire was used to obtain epidemiological data. Overall, the following information was obtained: age; level of education; marital status with women; sexual orientation; self-reported history of STIs ; number of lifetime sex partners; condom use in the last six months; smoking status and alcohol consumption. Ever smoked was defined as having ever smoked more than 100 cigarettes lifetime [Most of the participants’ socio-demographic characteristics, HIV-related data including cART treatments and multiple CD4lifetime . FrequenOf all 766 participants, 741 participants were under cART based on the recommendation from the manual of national free antiretroviral treatment program for HIV/AIDS in China . Partici+ T cell count was measured at the time of HIV diagnosis, and current CD4+ T cell count was measured at the follow-up which was most close to the time of HPV test, representing the baseline and recent immune status of the participants, respectively. The median time interval was 2.3 years  between these two CD4 measurement. The change of CD4+ T cell counts was also calculated by current CD4 minus baseline CD4. If the result was positive, we defined those participants as having “increased CD4+ T cell counts”; and if the result was negative or zero, we defined those participants as having “decreased or unchanged CD4+ T cell counts”. Baseline and the current CD4+ T cell count were recorded from the system, which had been measured by local CDCs using flow cytometry  according to the manufacturer’s protocol.Baseline CD4For each participant, an anal canal swab was collected by the trained staff. A nylon flocked swab was inserted into the anal canal for 2–3 cm in depth, rotated with gentle pressure against the canal wall while being slowly moved out. The whole process of swab sample collection was required to be no less than 2 min to gain enough cells. Then the swab was kept in a tube with 3 ml liquid medium using PBS as the main component to preserve the cells, and stored at − 20 °C waiting for HPV DNA extraction. HPV genotyping was performed using the HPV GenoArray Test Kit . The test kit detects 21 HPV genotypes using a L1 consensus primer-based polymerase chain reaction assay by the flow-through hybridisation technique with a TC-96/G/H6 HPV DNA Amplification Analyzer and an HMM-2 fast nucleic acid molecule hybridisation instrument. Among the 21 HPV genotypes, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 are carcinogenic types, HPV68 is a probably carcinogenic type, and HPV53 and 66 are possibly carcinogenic types, defined by the International Agency for Research on Cancer (IARC) . Here we+ T cell count or cART. Multivariable logistic regression models were adjusted for age , level of education , marital status , smoking amount (pack year), alcohol consumption , sexual orientation , self-reported history of STIs (no or yes), life-time multiple sex partners (no or yes), condom use in the last six months(no or yes). A two-sided p-value < 0.05 was considered as statistically significant. All statistical analyses were performed using SAS 9.4 statistical software .Chi-Square test and Fisher exact test were used to compare the distribution of categorical variables where appropriate. Unconditional univariable and multivariable logistic regression models were used to estimate the odds ratios (OR) and 95% confidence intervals (CI) in determining the association between the prevalence of HPV infection and CD4+ T cell count was 264 cells/µL , and the median current CD4+ T cell count was 446 cells/µL . About half of the participants were having EFV + 3TC + TDF for cART treatment, 25.1% were EFV + 3TC + AZT, 16.7% were NVP + 3TC + AZT, and all others combined accounted for 8.8%  were included in this analysis. 741 participants were under cART (96.7%). Distribution of the socio-demographic and HIV-related characteristics, as well as the prevalence of HPV infection are shown in Table + T cell count than those with decreased or unchanged  comparing current to baseline tests. The prevalence of HPV was lower among those with longer duration on cART (p = 0.017). Similar differences were observed for HR-HPV and LR-HPV prevalence for most of the above-mentioned characteristics , and of ≥ 500 cells/µL  were inversely associated with prevalence of any type HPV infection compared with those of < 200 cells/µL. Similar significant associations were observed for any HR-HPV infection, but not LR-HPV. Having taken NVP + 3TC + AZT compared with those taking EFV + 3TC + TDF was inversely associated with any HR-HPV infection , and inversely associated with any LR-HPV infection , but not any HPV infection. The association between duration of cART and any type of HPV infection had not been observed. The longer duration on cART was associated with lower HPV prevalence in univariable model, while the association was null in multivariable model  and any HR-HPV (40.3%), after expanding the sample size from the previous cross-sectional study . HIV-pos+ T cell count was inversely associated with anal HPV infection in this HIV male population. Lower HPV and HR-HPV prevalence were not only seen among the participants from the higher current CD4+ T cell count groups, but also among those with an increased CD4+ T cell count from baseline. Yet the effect of CD4+ T cell counts on HPV infection is inconsistent from literature. Some studies have reported that increased CD4+ T cell count were associated with lower risk of HPV infection [We found that higher CD4nfection , 31, 32,nfection , 6, 18. Most studies analyzing the impact of cART on HPV infection reported beneficial effect , 33–35. This study has several limitations. First, sexual behavior characteristic was based on self-reported by participants and may not completely represent actual exposure. Second, the recruitment of participants was based on a convenient sample instead of a random sample. It was possible that the participants differed in behavior or knowledge related to HPV infection from those who did not participate. Third, the testing kit for HPV used in this study was capable of detecting 21 genotypes of HPV, which did not cover all genotypes. Thus the prevalence of HPV might have been under-estimated. However, the kit included the most important genotypes of HR-HPV including 16 and 18, and the most prevalent LR-HPV 6 and 11, we think the measurement of outcome is sufficient in this study. Fourth, this study only included HIV-positive males from four CDCs of Taizhou. Hence, it is necessary to be cautious to generalize the results to all HIV-positive males in China.In conclusion, we observed lower prevalence of anal HPV infection among those HIV-infected men with increased CD4, and those taking NVP + 3TC + AZT. Given the high prevalence of HPV infection among HIV-positive males, a large-scale prospective study on natural history of HPV infection should be conducted in the future to confirm the associations. Besides the impact of CD4 and specific antiretroviral treatments, other host and environmental factors on the persistent HPV infection are of interest for further investigation.