The specificities, however, were lower for dual staining as compared to cytology. Conclusions: Dual staining is safer for decision-making regarding HPV-positive women’s need for follow-up with colposcopy and biopsy, as compared to cytology.The implementation of high-risk human papillomavirus testing (hrHPV testing) as a screening method in substitute for cytology has evoked the need for more sensitive and less objective tests for the triage of HPV-positive women. In a cohort of 1763 HPV-positive women, the potential of immunocytochemical p16 and Ki-67 dual staining as compared to cytology, alone or in combination with HPV partial genotyping, was tested for triage of women attending a cervical cancer screening program. Performance was measured using sensitivity, specificity, and positive and negative predictive values. Comparisons were assessed using logistic regression models and the McNemar test. Dual staining was evaluated in a prospectively collected study cohort of 1763 HPV-screened women. For triage of CIN2+ and CIN3+, NPV and sensitivity, 91.8% and 94.2% versus 87.9% and 89.7%, respectively, were significantly higher using dual staining together with HPV 16/18 positive, as compared to cytology ( Persistent infection with high-risk human papillomavirus (hrHPV) is, with rare exceptions, a prerequisite for developing cervical intraepithelial neoplasia (CIN) and invasive carcinoma . During In developed countries, organized cervical cancer screening programs (CCSP) have existed for decades, and despite increasing exposure to hrHPV in the population, the incidence of cervical cancer is kept at a low level compared to countries without such programs . For quiThe first cohorts of vaccinated women protected against the most carcinogenic HPV subtypes, 16 and 18, have entered cervical cancer screening programs, and the number will increase in the years to come. HPV 16 and 18 account for 70% of all cervical cancers worldwide. However, the vaccinated population will still be vulnerable to infection with 12 other high-risk (hr) HPV subtypes . The oncogenic potential of these hrHPV subtypes is very heterogenic, with a variable prevalence in cervical cancers ranging from 1 to 6% [Immunocytochemical p16/Ki-67 dual staining  for detection of co-expression of p16 and Ki-67 in the same cell has been suggested as a suitable method to increase the specificity and sensitivity for triage of HPV-positive women ,20,21,22n = 850), or cone biopsy (n = 131), diagnosed as normal (34%), CIN1 (25%), CIN2 (7%), CIN3 (31%), Adenocarcinoma in situ (AIS) (1%) or cervical carcinoma (CC) (2%). In case of any uncertainty regarding histologic grading, immunohistochemistry for Ki-67 and p16 supported the decision-making for 70% of the biopsies.The mean age of the study cohort was 46 years . As shown in After the median 391 days , 434 women had an HPV-negative cytology result, defined as HPV negative, regardless of cytology diagnosis. After a median of 368 days , 63 women who had a follow-up with non-conclusive positive cytology, defined as NILM, ASCUS or LSIL, and HPV positive and were excluded from the study. Details regarding study samples are shown in p = 0.29). In The dual stain analysis was performed median of 120  days after sampling. However, regression models showed no statistically significant time dependency for the outcome of the test  than for the corresponding triage with positive cytology and/or positive HPV 16/18; NPVs 88.1% and 91.0% for CIN2+ and CIN3+, respectively, The PPVs for CIN2+ and CIN3+ given a positive triage using dual staining alone or in combination with HPV16/18 were generally lower within this sample than the corresponding PPVs for cytology, but none of these differences were statistically significant. The NPVs were, on the other hand, statistically significantly higher for triage strategies involving dual staining than for cytology. The highest NPV observed was 91.8% (95% CI 89.4 to 93.8%) for CIN2+ and 94.2% (92.0 to 95.8%) for CIN3+. Both were obtained with triage, using dual staining and/or HPV 16/18 positive and were significantly higher (p = 0.001) and the sensitivity (p = 0.017) and specificity (p = 0.003) with regard to detection of CIN3+. For cytology, the differences in sensitivity and specificity between the HPV groups were not statistically significant, For dual staining, logistic regression revealed statistically significant differences between the group of HPV16/18 positive and the group of 12 other hrHPV positive in the sensitivity to detect CIN2+ , the results remained similar to the presented main findings  are biopsied, 402 (of 724) biopsies would be negative, and 82 CIN2+ would be missed. With dual staining replacing cytology, 459 (of 814) biopsies would be negative (+14% vs. cytology), and 49 CIN2+ would be missed (−40% vs. cytology).Demands for a triage test to distinguish between persistent infections with the ability to develop high-grade pre-cancer and harmless transient infections are urgent in order to decrease the costs for over-screening and the burden on colposcopy clinics, as a result of introducing the less specific HPV primary screening for women ≥ 30 years ,16,25.The purpose of the current prospective multicenter study was to evaluate the utility of dual staining as compared to cytology in the triage of HPV-positive cervical samples within an organized CCSP. For predicting cervical pre-cancer, CIN2+, and CIN3+, screening with dual staining, alone or in combination screening with partial genotyping, demonstrated higher sensitivities and higher NPVs, but lower specificities as compared to cytology screening, while the PPV was quite similar for the two, .Comparing cumulative results and HPV detection by Cobas 4800, the results in the current study are similar to the results found by Wright et al. , showingBased on the calculated sensitivities and specificities for triage requiring both positive dual staining and HPV 16/18 would entail nearly 70% more negative biopsies than a similar triage using positive cytology. Additionally, 231 women with CIN2+ would be excluded from taking a biopsy, 17% less than with positive cytology and HPV 16/18.Using a strategy with either positive dual staining or positive for HPV 16/18 as triage would mean 40% less missed CIN2+ and a 14% increase in negative biopsies as compared to using the same strategy with positive cytology. The group with positive dual staining and not positive for HPV 16/18 comprise the 12 other hrHPV positives, the largest group in the current cohort and a growing concern as women vaccinated for the most oncogenic HPV 16/18 enter screening programs. The use of a more sensitive method in screening programs would probably also lead to higher numbers of detected CIN2 lesions with a high regression rate. Many pathologists perceive CIN2 as an equivocal diagnosis, and a meta-analysis has concluded that conservative management with active surveillance rather than immediate ablation of CIN2 was a safe alternative, especially for young women . Dual stThe present study has its strengths and weaknesses. The use of follow-up data from the laboratory information system at the two pathology departments gave an unbiased description of the decision-making the pathologists and cytotechnologists are faced with in daily practice. More than two years of follow-up before the final diagnosis for 20% of the study population indeed, had an impact on the sensitivities and specificities of both methods to detect CIN2+ and CIN3+. On the other hand, comparisons of the two methods in the whole population and in the group diagnosed within 2 years did not show conspicuous differences. The long follow-up was due to an updated version of the algorithm for follow-up testing of HPV-positive women introduced in the screening program during the study period. The new version recommends delayed follow-up in 24 months for women with NILM cytology and positive for 12 other hrHPV. The long duration of follow-up for this group of women might have increased the chance of dropping out of testing and contributed to longer follow-up before a final diagnosis. The explanation for the majority of 9% invalid p16/Ki-67 dual staining results was low cellularity and expected, as the smear for dual staining was reprocessed after the smear for cytology screening. Additionally, samples contaminated with blood and treated with Glacial acetic acid (GAA) have been observed to entail brown artifacts, which may affect the validation of the staining results . This isIn conclusion, based on our findings, positive dual staining and/or positive HPV 16/18 versus negative dual staining and/or negative HPV 16/18 is a safe alternative for the triage of HPV-positive women from an HPV-screened population. Replacing the current cytology screening with dual staining and/or positive HPV 16/18 would result in a wanted outcome with the detection of an increased number of women with CIN2+, but at the expense of an increased number of proven negative biopsies. As compared to the present triage with cytology in combination with partial genotyping, this strategy demonstrated significantly higher sensitivities and higher NPVs. Consequently, dual staining would provide safer decision-making for whether HPV-positive women need follow-up with colposcopy and biopsy or if watchful waiting with cytology-based follow-up is more beneficial.From October 2017 until November 2018, residual material from liquid-based samples fixed in Preservcyt  were prospectively collected from 1763 HPV-positive women who had been triaged with cytology. All women were assigned to primary HPV screening in a pilot study, including two counties in the western part of Norway . ExcludeThe study was approved by the Regional Ethical Committee (REK Sør-øst), Norway, 2017/748. Since the study participants followed the algorithm of an organized CCSP and no intervention took place based on the study result; informed consent was waived.Primary HPV screening was done on the Cobas 4800 system , a qualitative HPV DNA test for the detection of 14 high-risk HPV (hrHPV) in cervical specimens. Cobas 4800 specifically identifies high-risk HPV 16 and 18 and detects the 12 other hrHPV (12 other HPV) as a group. The testing was performed according to the manufacturer’s recommendations, and the protocol provided by Roche was followed.All HPV-positive samples were triaged with liquid-based cytology processed on a ThinPrep 5000 processor , screened by experienced cytotechnicians, according to the Bethesda System  and sign® PLUS Cytology kit . Smears were processed on a ThinPrep 5000 processor, and immunocytochemistry was performed on a fully automated slide-stainer platform  as described in the package insert. Laboratory technicians, who were responsible for the immunocytochemistry procedure, accomplished a 2-day technical training course from Roche. Additionally, cytotechnicians and cytopathologists taking part in the study accomplished a 2-day-course on slide interpretation and a proficiency test under the auspices of Roche.Residual material from LBC samples from 1763 HPV-positive women were used for dual staining, performed at Haukeland University Hospital  by use of the CINtecThe definition of a p16/Ki-67 dual-stained cell is a cervical epithelial cell with simultaneous brown cytoplasmic immunostaining and red immunostaining of the nucleus. Samples were considered dual stained positive if at least one cervical epithelial cell was stained with both a red nuclear stain for Ki-67 and a brown cytoplasmic stain for p16. The screening was performed by cytotechnicians, and findings were confirmed by a cytopathologist, both blinded to the result of the triage cytology. Microscopy was done at a screening magnification of 10× while evaluation of double-stained cells was done at 40×. If no double-stained cells were found, the staining result was considered negative. One hundred and sixty samples (9%) were scored as inconclusive, (i) if the smears had less than four cells per field of vision in a minimum of 10 fields of vision with a 40× objective, (ii) if not at least one cell on the slide contained one red signal (Ki-67) and one brown signal (p16) or (iii) if the sample was contaminated by blood. For 37 (2%) cases, the discrepancy was observed between the cytotechnician and cytopathologist regarding validation of the dual staining result, and a consensus was achieved in a plenary session using a multiheaded microscope.p values < 0.05 were considered statistically significant.Descriptive statistics are presented as counts and percentages (%) for categorical data and as medians and interquartile ranges (IQR) for continuous data. The association between time from index cytology sampling to the p16/Ki-67 dual staining analysis and the odds of a positive test was assessed in logistic regression models, with and without allowing for non-linear association using restricted cubic splines. Sensitivities (SE), specificities (SP), positive (PPV), and negative predictive values (NPV) were estimated and presented with two-sided 95% Wilson confidence intervals (CI). SEs and SPs were compared between different triage strategies within the same sample using the McNemar test. PPVs and NPVs were compared between triage strategies within the same sample using the approach suggested by Leisenring . Further


Access to health care for an aging population with growing needs presents major challenges in northern Sweden’s sparsely populated regions. Few people, the lack of professionals, and long distances make it difficult to provide health care on equitable terms according to the Swedish legislation. Remote treatment (RT) using information and communication technology has been suggested to overcome these difficulties, and person-centered care (PCC) is a desired philosophy to improve the quality of health care. However, there is scarce knowledge about how patients experience RT meetings.This study aimed to describe the experiences of patients with cardiovascular disease revisiting specialist physicians via RT guided by a PCC perspective in northern Sweden’s sparsely populated regions.A qualitative study was conducted based on interviews with 8 patients with cardiovascular disease revisiting their physician through RT, from a digital health room to a health care center or from a health care center to a hospital. The interviews were recorded, transcribed verbatim, and analyzed using inductive content analysis. The results are discussed from a PCC perspective.participation and relationships are important for good and close care via RT.The analysis resulted in 6 categories: good accessibility, safety with good relationships, proximity and distance with technology, habit and quality of the technology facilitating the meeting, cherishing personal integrity, and participation in own care. These categories were interpreted as the theme, participation and relationships are important for good and close care via RT. To improve the quality of an RT meeting, PCC can be applied but needs to be extended to the digital domain—electronic PCC, especially the communication component, as it is the most salient difference from a face-to-face meeting. Important factors that should be considered before, during, and after the RT meeting have been identified.The study shows that  Sparsely populated regions (SPRs) have many things in common across the world such as low population density, long distance to health care and other societal services, being governmentally remotely controlled, and a distinct lifestyle . These rA way to overcome these challenges is to use information and communication technology (ICT), which has been recommended and encouraged by the World Health Organization . An initIt is important to evaluate the implemented methods and techniques because despite its many benefits, the introduction of new technology may lead to new problems, such as patient integrity and safety issues . In a DaThe level of education, previous use of social media and other communication platforms, and being a rural resident are factors that may influence how receptive the informants are to telemedicine ,11. Age treatment that is conducted remotely by means of ICT, including medical advice, examination or treatment, where the patient and therapist are separated in space, but not in time, may be an option. Thus, we considered RT as a subset of the broad concept of telemedicine to limit and clarify the aim of this study  and get away from driving and all that...and because it is so close it is only a couple of minutes to walk there...ID6; female; aged 68 yearsSome informants found it difficult to drive far owing to illness and pain. Reducing travel also meant an economic advantage, as they used their own car to drive to the HCC or hospital:...Say that it cost 150–200 SEK in fuel for a trip and then you get 24 SEK for it in compensation...it is not worth it...ID8; female; aged 53 yearsEven when it was easy to go to the health care unit, the taxi ride to the hospital or HCC could be agreeable with drivers you know. Despite long distances, the journey could be pleasant, and sometimes, it was not difficult to travel, especially when informants also took the opportunity to go shopping or do other errands at the same time:Even so, a trip to [the city] means you can go to the shops before and after and do errands there.ID6; female; aged 68 yearsSome participants indicated that having the meeting with the specialist via RT saved time and that they received help more quickly and avoided worries. RT made the whole day easy and took just 15 minutes compared with the fact that it takes half a day to see a physician in a hospital. Participants felt that time was saved for both themselves and the physicians. Time was also saved for the staff, whose job is to support with connections and keep track of the routines so that the physician’s visit was not delayed. An interviewee said that everyone has the same amount of time and we live in a stressed society; therefore, it was good that care could be provided remotely for people with long distances to health care units. A participant of working age saw benefits in not having to take time off, not missing working hours, and earning income:And if you work and...I do not have to miss so much working time and do not keep on and may not need to compensate for work so much and make changes with colleagues...so there is not much lost work income either...there are financial benefits...ID8; female; aged 53 yearsParticipants in the study felt that the care was equitable and that they received the same assessment as at the HCC or at the hospital for this type of revisit. The RT meeting felt normal and was not different, except that the physician and the patient were not physically in the same room. If they had gone to the HCC or to the hospital to meet the physician face-to-face, the physician would have asked the same questions as asked during the digital meeting:The great thing is that it is equitable...what should I say...it gives just as good results with these technical facilities [video].ID2; male; aged 85 yearssafety with good relationships.In the interviews, it was noted that the patients were familiar with the staff and experienced a comfortable time together in the waiting room, including relatives who gave support in the meeting with the physician, which was interpreted as Participants in the study were familiar with the staff at the health care unit and already knew the physician before the meeting, which created security and a feeling of safety in the meeting. The informants thought that the physician was pleasant and easy to talk to during the meeting. A participant knew his physician only through phone before but felt that the meeting worked very well. It was very important that they had met the physician before, instead of meeting a new unknown physician who did not know anything about them as a person:But had it been a complete stranger then you get a little...then you keep a little distance...if you...you think what the heck is this.ID7; male; aged 78 yearsA good social gathering was experienced when the patients met acquaintances in the waiting room at the HCC unit. It became a pleasant meeting place, similar to going to the neighbor’s house and meeting people you know. The conversations were relaxing, and there were discussions about what had happened since the last time they had met, how it was on the fishing trip, and even some talk about illness. A participant thought that it felt similar to home and he could be himself:...We were standing out there talking and then we entered and then you could have coffee if you wanted and another acquaintance was sitting there and it was no big deal to get there as dressed as when you walk in a village, it feels like home in some particular way, yes...ID1; female; aged 83 yearsClose relatives could be a great support in the RT meeting, which provided a feeling of safety both before and during the meeting. Participants experienced that close relatives were a support when the patient had hearing or memory problems, for example, after a stroke:And so I had [the man] was there to support me if I forgot something or if I lost words...ID6; female; 68 yearsproximity and distance with help of technology.Both proximity and distance were experienced in the RT meeting, a contact that could be perceived both as personal and impersonal. The meeting felt calm and focused on the patient themself, which was interpreted as The image on the screen was large and clear, and the patients were affirmed by the physician on the screen. Participants felt that it was as close as in real life, almost similar to sitting in the same room. Some informants perceived it as if the physician was behind the video screen and that they had eye contact. The patients experienced that the physician could see their reactions and facial expressions:That he or she can look at my face and could see how I think before I answer, I actually think if I’m honest or making up [laughter]...that’s exactly what I think I can do with the grandchildren when I talk to them on Skype.ID1; female; aged 83 yearsThe interviewees experienced that the sound was good when speaking to the physician, without interruption, and that it was easy to communicate. A person described how they went through the medication list in the RT meeting. The patients were also impressed by the possibility that the physician could zoom in on them and listen to their heart and lungs through the digital stethoscope:They listen to my heart and then hear all that, 75 km away, so the physician can sit and listen to my heart, it’s so amazing it’s crazy. Ahh...I think that’s really impressive.ID1; female; aged 83 yearsdistance was experienced by some informants in the RT meeting. It felt different, and some participants found it difficult to be spontaneous and answer the physicians’ questions. For them, it was difficult to see body language and facial expressions:Also, a feeling of If I talk to a person sitting in front of me, I can see their body language, I can joke with the person...I can ask and say things that are almost private but a person who is on a screen is a bit distant because I feel like I can’t really talk.ID 6; woman; aged 68 yearsThe feeling of absence of physical contact was experienced when the physician could not touch the patient and measure the pulse. They thought that physical contact should be the right way to meet the physician, because people become more sensitive if they are close to each other. Some patients were still satisfied and thought that the meeting was normal without physical contact and that the on-site nurses could do the examination. However, a participant wanted a physical meeting:They never asked me what I wanted they just said it would be through video but I would have preferred to meet them there [at the hospital].ID4; female; aged 72 yearsPatients in the study found the personal contact to be perfect even though they met the physician via a computer screen and they were not physically in the room:It was like personal contact even though it was via such a link.ID7; male; aged 78 yearsSome patients felt the opposite, that the physician’s visit was impersonal via RT, and they experienced a feeling of insecurity. Some other participants had problems in getting something out of the meeting; they did not ask their questions because the meeting did not feel personal. A person wanted to meet the physician physically because he had vision and hearing problems, which resulted in the physician feeling like a stranger in the RT meeting. It became uncomfortable; therefore, the person barely remembered the meeting and thought it was something wrong with her but said that the physician was certainly professional:I would rather have a personal meeting, you can reach them in other ways when you have vision problems and sitting close because I would like to comment on things under...and when they were like strangers to me, I couldn’t.ID4; female; aged 72 yearsAnother participant expressed that although the meeting did not feel personal, it worked to meet the physician via the video screen:It’s not this kind of personal, so I feel, but I thought it worked well.ID7; male; aged 78 yearsThe RT meeting was experienced as calm and focused and almost as in a home environment. The patient got easy contact with the physician in a peaceful and quiet way, and nothing was disturbing from the background. The informants thought that the physician was responsive and gave them time to ask questions. This meant that the patients did not feel stressed and felt that the physician was focused on them during the meeting. At an ordinary physical meeting with the physician, people look around at things on bookshelves and other things in the room, but all that disappeared in the RT meeting, which was perceived as positive. A person got the feeling that the meeting was focused as only one could talk at a time:I don’t know if it was because of the technology...You have to be quiet, it felt that way anyway...When one talks, you listen to what he will say...But I don’t think it’s something negative...then you get even more focused than maybe talking at the same time...because then neither of us really listens.ID8; female; aged 53 yearsquality of and familiarity with technology facilitating the meeting.The informants experienced that technical skills facilitating the RT meetings but that technical quality could both support or disturb the meeting, resulting in the interpretation of the category, The interviewees felt that the physicians gave the impression that they were comfortable with the technology. In addition, the patients themselves felt comfortable. It was not strange because they were used to the technology related to using the internet, Skype, or other systems through their work:But you’re used to watching TV so you’re not completely alienated from things like being on Skype with grandchildren on the iPad.ID1; female; aged 83 yearsSome participants also felt unfamiliar and insecure when they had a digital meeting. They knew it was possible to meet the physician via RT but had not had any meetings themselves before. The first time felt special, strange, and stiff because everything was new but, at the same time, exciting. The informants experienced the feeling of not having control, but after a while, they got used to it:And then the physician came, and it felt a little bit strange when you’re not like this [Physically]...but you see a TV screen, but after we had been sitting for a while there was nothing strange about that.ID7; male; aged 78 yearsThere were participants who felt old-fashioned when they would talk via a video screen, but they thought it was a matter of age and habit. Participants hoped for more opportunities to have RT meetings, and they imagined that when they got used to it, it would feel similar to sitting in front of a physically present physician. In addition, a person felt that the physician was uncomfortable with the technology:I think she was uncomfortable in front of...I wonder if she’s done this before. I’m not sure about that...because I found it uncomfortable for her to sit in front of the screen.ID6; female; aged 68 yearsSome interviewees told us in the interview that they got support and help from the staff to start the meeting. The informants also mentioned that when they arrived at the HCC unit, they were directed to a room with a table and a video screen, and the staff started the computer and instructed them about how to use it. In the RT meeting some participants also mentioned how the physician instructed the staff about how to put the stethoscope in place to be able to listen to the sounds from the heart and lungs. An informant said that the physician informed them how the videoconference would be conducted:...She told me where it was and that she was going to ask me a few questions...And I said it’s just to ask questions...I’ll answer as best I can. What I understood, it went as well as possible.ID5; male; aged 75 yearsOverall, the technology worked well, but participants felt that the technique could be disruptive. A participant said that it was a hassle with the sound and it was difficult to hear the lung and heart sounds using the digital stethoscope, but it started to work at the end of the meeting. Another participant could only see half of the physician’s face and thought it felt strange:...At last she got herself on the screen but it just happened that we only saw half of her, I think we saw her from the nose and upward so she sat like in a corner of the picture, and then we talked to her but it felt quite strange to sit and talk to half a person, half a face.ID6; female; aged 68 yearscherishing personal integrity.The patients felt that they did not want to disclose information about themselves at the digital meetings and that it was important to have a private room when they met the physician, which was interpreted as Participants in the study said that several individuals were present during the RT meeting and that they did not want to disclose themselves to people other than the physician. Other people could be present because the health room was also used as a gathering point for the home care service in the area. Participants thought that it was a sensitive situation, and they did not want other people to hear what thoughts, worries, and illnesses they had, even though they knew the staff had a duty of maintaining confidentiality:I may not want so many people listening and hearing what I’ve been thinking about, what illness, what thoughts and what problem I have or concern...ID1; female; aged 83 yearsParticipants pointed out the importance of individual meeting rooms and that people should not pass by all the time. When people passed by, it was difficult to focus on the meeting and maintain confidentiality:Then you sat alone in your own secluded room and it is also quite important.ID8; female; aged 53 yearsparticipation in care.The informants had a desire to be prepared for the meeting with the physician, wanted more information and influence, and saw opportunities for the development of care. These are summarized in the category Participants had written down questions and thoughts they had—such as medications, how they would think ahead, and future follow-ups—and they wanted to be prepared for the RT meeting. It was important to be prepared; otherwise, they had the risk of forgetting half of their thoughts:I probably got answers to all the questions I had; I had written down what to ask for and she answered...ID6; female; aged 68 yearsThere was a request from the participants for more information before the RT meeting. As first-time users, they had heard of an appliance they could talk to and thought that someone would be there to give support and tell them how it worked practically. A participant was concerned that the screen was not switched off after the visit:I felt awfully bad because I thought now it’s on...what if...A lot of these, what if...and standing in [the hospital]...and what if the power is on? All that practical stuff...I walked around and thought about it for a long time.ID6; female; aged 68 yearsThere was a desire for information before the appointed meeting, so that everything could be arranged properly. The participants wondered whether there were any routines at the HCC unit when they got the feeling that no one knew anything about the meeting, for example, who shows the patient the way and who would initiate the meeting via the video screen. A person lacked information and felt that she was not involved in the visit, and she wanted someone to coordinate the visit:But now I understand that it’s an expense for the healthcare system but in this particular case it would only have been the cost of one trip that I would have anyway when I was going to [the hospital].ID4; female; aged 72 yearsJoy and hope for the future were expressed by the participants. Some informants experienced the meeting as fantastic and wished to continue conducting RT meetings—not only for people who lived in SPRs; however, it was people in SPRs who made the most of such visits. It was important that the physician should be known to the participants before the RT meeting and could communicate understandably. Interviewees in this study could see that there were opportunities to develop digital meetings, but it was clear that the need to go to the hospital in more difficult cases was obvious. Developing remote care for mild ailments was welcome. If the physician wanted to do an examination at a distance, they could contact the HCC and order an examination, and after that, the person could see their physician at a distance again. They also saw opportunities for contact via RT with the large hospital to a great extent. A participant thought that it would have been even easy to log in via an app or a smartphone:Everything ends up on the phone, it seems, and it would have been the easiest thing to do through Messenger or whatever way you have for video, it would have been the ultimate, then you do not even have to go anywhere.ID8; female; aged 53 yearsparticipation and relationships are important for good and close care via RT—and was about people experiencing participation and relationships in different ways. When people felt involved and experienced good relationships and reliable technology, a sense of safety and security was created during the RT meeting. When patients felt less involved and the relationship or technology was not satisfactory, a feeling of distance and insecurity provided less good care via RT.A theme was interpreted from the 6 categories—participation and relationships are important for good and close care via RT. Close care can mean more than just geographical proximity. The importance of relationships can be interpreted as the availability to meet those in care that we already know. A digital tool such as a video screen can also convey a sense of proximity.In this study, we investigated the experiences of patients with cardiovascular diseases regarding follow-up meetings with their physician through RT. The inductive analysis showed a common thread throughout the categories that was interpreted as the theme, Introduction section, we further viewed our results considering the 6 categories developed by Sharma et al [establishing a therapeutic relationship, getting to know the person, shared power and responsibility, empowering the person, trust and respect, and communication [Owing to the high demand for PCC, as described in the ma et al  in an ovnication . To simpThis component is based on a partnership with mutual dependency and responsibility between the person and the professionals, and key factors are open communication, a cohesive team, and professionals who possess knowledge and skills to practice PCC .safety with good relationships, and the subcategory, familiarity with the staff. Some were also skeptical about RT with an unknown person, and a participant claimed, “But had it been a complete stranger then you get a little...then you keep a little distance...” Participants preferred to see a physician they were familiar with and trusted in a video consultation, which was confirmed in another study [Our results showed that a therapeutic relationship should be established in person before an RT meeting. This relationship created security, which was confirmed by informants in the category, er study .This component emphasizes a holistic view of the person or patient that is more than the illness or disease that the person is diagnosed with ,26. It iproximity and distance with technology. Lavoie et al [In this study, participants felt closeness via the screen and described an experience of personal contact in the category, ie et al  believe ie et al . In the ie et al , some paTherefore, the caregiver needs to know the person’s digital literacy and ability to communicate via ICT and whether the person has any disabilities such as visual, hearing, or cognitive impairments or those that require or which a physical meeting.shared power and responsibility, indicates that the patient needs to be an active part in their care, and the care delivered should be individualized and based on the person’s own needs, wishes, and values [The component, d values .participation in care, and the subcategory, being prepared. However, new ways of meeting could also be demanding for some people; thus, they forgot to ask questions [quality of and familiarity with technology facilitating the meeting, and participants described feelings of not being in control. Some informants felt comfortable because they were using this technology in everyday life, and others felt uncomfortable at the beginning of the meeting; however, later in the conversation, they stated that “...there was nothing strange about that.” The staff are also responsible for ensuring that the patient can use the technology before they organize an RT meeting, and they are also responsible for being able to handle the technical equipment themselves. Informants in this study commented that in some meetings, the physician was uncomfortable with the technology, and in another RT meeting, the person saw just half of the physician’s face.There was a willingness among the patients in this study to be prepared for the RT meeting, which could be to write down questions in advance and could be seen in the category, uestions , which aThis component highlights the importance of the individual being active in their own care. The staff needs to provide patients with information, support, and resources that make it possible for them to be able to make their own decisions .equitable care, where patients were surprised that the digital meeting gave the same results as a face-to-face meeting, which is also described in other studies [good accessibility. If the person can be in their own context where they feel safe, it is a way of empowering the individual, and the RT meeting can be as effective as a physical meeting [In this study some participants felt that the care via RT was equivalent to a physical meeting described in subcategory,  studies ,20. Part studies  thought  meeting .participation in care. Another study showed that some participants had a wish for video meetings in the future, to avoid unnecessary trips to the hospital, especially because the informants became old and for other reasons [The informants wanted to continue to meet via RT and saw opportunities to develop digital meetings further, where even mild ailments could be treated, something that could be seen in the category,  reasons . Allowin reasons .The trust and respect component involves recognition of the person as a unique individual with their own values, preferences, lived experiences, and needs. Health care practitioners need to consider the person’s individualized needs and incorporate them into their care .cherishing personal integrity. For the patients who participated in an RT meeting, it was important to trust their physician, and to have a mutual respect to emphasize privacy. Patients in this study highlighted the importance of the room for digital meetings being in such a way that no other people were present except for those who were to attend the meeting, which was also found to be important in another systematic review [In this study, the RT meetings can be equated with a physical meeting with a physician or other health care professional, where confidentiality and personal integrity are important. The informants in this study pointed out the importance of not having other people in the same room or people passing by the room in the category, c review . New demThis component about communication between the person, their family, and health professionals is important to discuss and deliver understandable and correct information about the person’s care .Communication is the most salient difference from face-to-face meeting because it is mediated through digital devices in RT. Our results showed that RT affects the person before, during, and after the meeting. This means that the other PCC components need to be taken into consideration when planning for an RT meeting.Before the RT meeting, it is important to establish a therapeutic relationship, get to know the person, and empower the person as mentioned previously. This concerns the person’s vision, hearing, cognition, and IT literacy. Some participants in this study had hearing problems and difficulty in perceiving what was being communicated, which was experienced as insecurity. People with hearing loss may misunderstand advice or instructions, which could be prevented by using a headset or if the physician, a nurse, or an accompanying person could be present at the meeting and explain what is said . The facquality of and familiarity with technology facilitating the meeting, which was also confirmed by Johansson et al [Communication and information about how the technique works and how the meeting will be conducted are at least as important as communicating about the patient’s illness and health care for an optimal RT meeting. Some participants felt it unusual to communicate via a video screen for the first time, and some were used to it as shown in on et al .Moreover, in this study, the staff connected the device for some patients and started the meeting, which normalized the meeting and made the patients more comfortable; this is also noted in the study by Currie et al . PatientTo master care via RT, health care professionals need to acquire knowledge and understanding about how digital technology affects the interaction between people. Technology has an important supporting function in the meeting between health care professionals and patients, but there is also a risk of the technology contributing to frustration and alienation .proximity and distance with technology.Finally, it may be noted that an RT meeting may not be suitable for some people, as explained in the category, During the RT meeting, it is important to share power and responsibility by ensuring technical quality and control conditions both at the caregiver’s site and at the person’s site, which may affect trust and respect.For a person to be perceived as more sympathetic and present in a digital meeting, they should look at the camera and not at the face on the screen. The camera should also be placed at the minimum eye level; however, the distance may be less decisive .distance and could not be spontaneous in the conversation; for the latter, it was difficult because they could not see body language and facial expressions, which made them feel distant in proximity and distance with technology.In this study, it was easy for some informants to communicate in the RT meeting when the sound was good, whereas others experienced a feeling of support or disturbed by technology. An informant, for example, saw only half of the face and thought it was embarrassing to tell the physician. Similar incidents were reported by participants from another study when they could not see the computer screen because it was placed incorrectly and did not understand that they could ask for the screen to be placed differently [Technology could also be disruptive as shown in the subcategory, ferently . If the trust and respect.To limit the experience of distance and isolation, the health care service can appoint a person to support the patient during the visit . If the Other authors believe that mobile ICT should be used with caution and that health care professionals need to understand older users, and the lack of knowledge about modern technology can affect the person’s attitude toward the new technical solutions ,44.After the RT meeting, it is important for the patient to feel safe, with no need to worry about the equipment. In this study, a patient “felt awfully bad...” about the fate of the equipment if it was still turned on—in the subcategory, want more information. As described in the category, participate in care, it is necessary to know the end of your responsibility when the meeting is over.Only 8 people participated in this study, which can be a weakness, but there was a diversity of sex and age represented. There were also difficulties in including more patients, as there were few patients who have had an RT meeting. However, in the remote regions and SPRs, it is a proportionally good representation because there are few people living in this area. In the last interviews, no new information emerged, which could be a sign of saturation . In thisMethods and Results sections for a comprehensive understanding of the patients’ experiences of RT meetings and to increase the trustworthiness of the results. Our interpretation is that the categories cover the data well and can be confirmed by the quotes, thus increasing the credibility of the study.Each person’s experience is unique, and there is no perfect truth to be found, but common patterns and individual differences have been reproduced and described. The results are comparable with those of previous studies with other patient experiences in digital meetings, which also confirms the transferability of the results from this study. Qualitative analysis was used because the purpose was to describe experiences of a phenomenon, a new method for meeting, and a qualitative method is therefore suitable . The anaFuture studies are needed to identify additional factors that affect telemedicine acceptance, such as human-technology interaction, the organization of the health care system, and social and cultural human factors. Information and education about how digital services work in practice are especially needed for patients and professionals who lack technical skills.Patients felt that close relatives were a support, and it is important to interview relatives’ experience of RT and the staff’s perceptions. More systematic studies are needed about how people experience a digital meeting and for whom this way of meeting is suitable depending on individual conditions, resources, what disease or diseases and symptoms the person is affected by, and what the cultural context means for the willingness to seek care. For some individuals, the best way to connect to the physician may be from home. In the future, RT, in addition to patients not having to travel long distances and saving time, may also result in an economic benefit for people, communities, and health care systems as care becomes close and more accessible. Thus, there is also a great need for health economic evaluations of digital meetings in health care. It is an interesting area of research regarding what savings can be made in terms of climate impact and sustainable development when travel is reduced.Finally, as technical development is exponentially fast and both professionals and older people will probably have better skills, high digital literacy, and more experiences with various platforms, the results of this study may not be repeatable over time; however, other factors may turn out to be as important for optimal RT meetings.participation and relationships are important for good and close care via RT. To improve the quality of an RT meeting, PCC can be applied but needs to be extended to ePCC, especially the communication component as the most salient difference from a face-to-face meeting.This study has shown that Before an RT meeting it is crucial to do the following:therapeutic relationship, get to know the person, and empower the person, preferably at a previous physical meeting.Establish a Decide whether the meeting should be held in person or via RT based on the person’s preferences, abilities, and social and practical needs.If conducting an RT meeting, get to know the person’s vision, hearing, and cognitive abilities and digital literacy and take measures, if necessary.Acquire knowledge and understanding of how digital technology works and how to manage it among the health care staff.During the RT meeting it is vital to do the following:Ensure technical quality and control conditions both at the caregiver’s site and at the person’s site.Ensure that the meeting rooms are designed in a safe and secure manner and that privacy and confidentiality are ensured.Place the camera at the minimum eye level.Look at the camera and not at the face on the screen.After the RT meeting, it is important to do the following:Ensure that the patient feels safe and that they do not need to worry about the equipment.RT meetings need to be created with various actors within the care organization based on a person-centered approach, where the patient is a cocreator of good and close care in the future.


The involvement of the antitumor immune response and the dose/fractionation/volume factors as well as the therapeutic sequence in the case of new multimodal treatments including immune checkpoint inhibitors (ICIs) could be included as an additional term in the quadratic linear formalism especially for hypo-fractionation regimens. This term should take into account the dual immunomodulatory effect (immunosuppressant and stimulator of antitumor immunity) of radiotherapy, which varies from case to case and can bring benefit or a detrimental effect.Altered fractionation concepts and especially moderate hypo-fractionation are evaluated as alternatives to standard treatment for head and neck squamous cell carcinoma (HNSCC), associated with or not concurrent with or sequential to chemotherapy. The calculation of the iso-equivalent dose regimens has as its starting point the linear quadratic (LQ) formalism traditionally based on the “4Rs” of radiobiology. The higher rates of therapeutic failure after radiotherapy of HNSCC are associated with the heterogeneity of radio-sensibility. The identification of genetic signatures and radio-resistance scores aims to improve the therapeutic ratio of radiotherapy and to conceptualize personalized fractionation schemes. The new data regarding the involvement of the sixth “R” of radiobiology in HNSCC, especially for the HPV-driven subtype, but also for the “immune active” minority of HPV-negative HNSCCs, bring to the fore a multifactorial variation of the  Head and neck cancers are generally aggressive tumors that are resistant to non-surgical multimodal treatment. The concepts of altered fractionation  for curative intent in association with pharmacological agents—among which we mention the anti-alkylant cisplatin; cetuximab, a molecular therapy target anti-epidermal growth factor receptor (EGFR) monoclonal antibody; or nimorazole, a radio-sensitizer nitrosamine—aim to activate a synergistic effect and increase the response rates to multimodal treatment. The identification of a subtype of squamous cell carcinoma of the head and neck (HNSCC) associated with the human papillomavirus (HPV) with a different treatment response pattern and also the advances in radiobiology that extend beyond the traditional “4R”  concept—the newer “2Rs” (intrinsic radio-sensitivity and reactivation of the antitumor immune response) in the current “6R” concept—bring new challenges in the application of the linear quadratic (LQ) model for the calculation of iso-equivalent fractionation schemes. We propose to summarize the concepts that will be the basis for the reconsideration of the LQ formalism for the category of “active immune class” of HNSCC regardless of HPV status ,2,3  mean a predominance of the linear component. A rough approximation proposes the value α/β = 10 for the tumor and acute effects and α/β = 3 for healthy tissues and late effects [The quadratic linear formalism proposes a relationship between cell survival and delivered dose in radiotherapy, offering an ideal model for equating the biological effect of the fractionation regimes. However, the variation in the intrinsic and extrinsic radio-sensitivity of tumors as well as of healthy tissues makes this model an ideal and imperfect model. Potential challenges including the use of this model at very low and very high dose per fraction and possible confounding factors must be evaluated before applying this model in clinical routines. A low f α/β 6–1 Gy mean  effects .In HNSCC, a dose of 70 Gy in 33–35 fractions delivered on gross disease (primary tumor and involved lymph nodes) is considered necessary in definitive settings, and 66Gy in 33 fractions is usually preferred as adjuvant treatment. Noninvolved lymph node irradiation is also administered in dose regimens of 45 to 60 Gy in a standard fractionation regimen, with 50Gy and 60Gy dose levels being the most frequently proposed for low risk and high risk of tumor involvement, respectively ,9. CurreWe performed a search in the PubMed/MEDLINE database using the keywords radiotherapy, radio-chemotherapy, HPV, head and neck, HNSCC, hypo-fractionated, quadratic linear model, immunotherapy, and immune checkpoint inhibitors.The higher rates of therapeutic failure and the limited number of responses to immune checkpoint inhibitor (ICI) therapy justify the design of a genetic prognostic index in order to predict the response to immunotherapy in HNSCC. The purpose of the immune-related gene prognostic index (IRGPI) is to identify the immune and molecular characteristics of HNSCC that will influence ICI therapy response rates. In all, 22 genes related to antitumor immunity were evaluated, and 3 genes  were chosen for the construction of the IRGPI. The IRGPI value was inversely correlated with overall survival (OS). Reduced values of the IRGPI have been associated with DNA damage repair mechanisms. Higher levels of CD8+ and CD4+ T lymphocytes and M1 macrophages and lower expression of TP53 seem to be associated with a favorable response to ICIs. The aggressive phenotype is associated with higher rates of TP53 and PIK3CA mutations and also with the infiltration of B cells and M0 and M2 macrophages .The subtype of head and neck cancers associated with human papillomavirus (HPV) infection is currently considered an independent subtype of HNSCC, but more recently, HPV-positive head and neck cancers could be divided into HPV-DNA-positive tumors and HPV-expressing RNA and proteins. Even though the two subtypes are reported to be different, there are many similarities between HPV-DNA-positive HNSCC and HNSCC associated with smoking. With an increasing incidence, the so-called “HPV-driven” subtypes represent up to 25% of HNSCCs, but oropharynx squamous cell carcinomas (OPCCs)—the oropharynx is the most frequent location of this subtype—benefit from a new staging in the eighth-edition TNM classification. The incidence of the HPV-driven subtype varies from 20% to 60%, with the countries of Western Europe and North America having increased incidences. Both the prognosis and the response to treatment seem to be superior in this subcategory of HNSCC. Among the particularities of HPV-driven HNSCC, especially for OPSCCs, there is a frequent involvement of the lymph nodes in young, nonsmoking patients, but certain socio-sexual conditions make the appearance of this subtype at older ages possible. The increased sensitivity to irradiation and the favorable prognosis justified the initiation of de-escalation trials. Considering the differences in TNM staging compared to the subtype related to a long history of smoking and chronic alcohol consumption, identification tests for the HPV-driven subtype should be performed in all cases of OPSCC according to the recommendations of the College of American Pathologists ,15,16. FTorres-Roca et al. proposed a radiation sensitivity index (RSI), in order to modulate treatment for a subgroup of 40–45% of rectal cancer cases considered not respondent to neo-adjuvant chemo-radiotherapy. The authors also noted a 20% complete pathological response, considering the critical role of radio-sensitivity in the tumor control of locally advanced rectal adenocarcinoma . ExtendiStarting from this concept of different radio-sensitivities, Foy et al. aimed to evaluate the molecular factors associated with these differences in radio-sensitivity in HPV-HNSCC patients. The authors proposed a RadR radio-resistance score based on a training set and a validation set that included HPV-negative HNSCCs from The Cancer Genome Atlas HNSCC cell lines with known radio-sensibility levels ,19,20. PThe effects of HPV on the repair of radiation-induced cellular damage are controversial. In the case of double-strand breaks (DSBs) there is a deficit in repairing the lesions; however, for single-strand break (SSB) lesions, there is a possible repair mechanism involving the E6 oncoprotein. XRCC1, DNA polymerase β, PNKP and PARP-1, all factors involved in SSB repair, appear to be upregulated in HPV-driven HNSCC. A defect in homologous recombination (HR) that also affects the expression of some factors involved in DSB repair promotes radio-sensitivity. However, the lack of a favorable effect of poly (ADP-ribose) polymerase (PARP) inhibition on HPV-driven HNSCC suggests the minimal involvement of the HR mechanism in radio-sensitivity. Non-homologous end joining (NHEJ), HR, mismatch repair, and the involvement of the E6 and E7 oncoproteins all have an overwhelming role in the high radio-sensitivity of HPV+ HNSCC. In addition, the radio-sensitizing role of the ataxia telangiectasia gene (ATM) is considered minimal in the case of HPV-driven HNSCC compared to the subtype of HNSCC related to smoking. Köcher et al. mentions that a lack of effectiveness in the ATM-orchestrated DNA damage response contributes to the DNA repair defect in HPV + HNSCC. TP53 mutation is frequently associated with radio resistance, but HPV-driven HNSCCs are associated with wild-type TP53. Consequently, it is unlikely that in this case radio-sensitivity is dependent on this pathway, so strategies to increase radio-sensitivity by using the inhibition of proteasomal degradation cannot be applied to HPV+ HNSCCs. The hypoxia-related proteins CAIX or HIF-1α are considered biomarkers of radio resistance in HPV+ HNSCC. CAIX or microvascular density does not seem to be correlated with the HPV status of HNSCC, and in the case of HIF-1α, the correlation with HPV+ OPSCC and involvement in radio-sensitivity has not yet been elucidated ,27,28,29Analyzing the tumor metabolism, there are major differences between smoking-related HNSCC tumor cells and HPV-driven HNSCC cells. In the case of HPV-HNSCC the Warburg effect, an increased glucose uptake associated with the fermentation of glucose to lactate, is the predominant metabolic pathway; however, HPV+ cancers produce energy using mitochondrial oxidative phosphorylation. The strategy of radio sensitization using the biguanide metformin, a glycolytic inhibitor, or the association of both agents thus proves useless in the radio sensitization of the HPV+ subtype of HNSCC ,31. Simiα/β ratios values and the radio-sensitivity variation during radiotherapy treatment are arguments against the de-escalation of radiation treatment based only on HPV status. The estimation of α/β ratios highlights variations from 1.01 to 40.68 Gy: there are cell lines that act as late-responding tissues at α/β ratios < 6 but also cell lines that behave as acute-responding tissues at α/β ratios between 8.19 and 34.1 Gy. Heterogeneity in radio-sensitivity was confirmed by van Leeuwen and colleagues, who estimated in a systematic review α/β ratios varying from 8.36 Gy to 30.0 Gy. In this context, the individual determination of radio-sensitivity could be essential for improving treatment performance. In the case of HPV-positive cell lines, a resistance to cisplatin is highlighted, and the response to cetuximab and radiotherapy does not seem to differ compared to the HPV subtype according to the reports of Nagel et al. [Analyzing six HNSCC cell lines of which three were HPV negative and three were HPV positive, Reid et al. evaluated the radio-sensitivity after repeated irradiation simulating fractionated radiotherapy. Even if the HPV-positive cell lines demonstrated a superior radio-sensitivity, repeated fractionations resulted in radio-sensitivity changes in both subtypes of cell lines. This variation in radio-sensitivity during treatment justifies the difficulties in implementing de-escalation strategies. The radiobiological heterogeneity of HPV-driven HNSCCs regarding l et al. ,24,25. El et al. ,36,37. Tl et al. ,39.Traditionally, it is considered that anticancer treatments act through the effect of DNA damage in three phases: physical, chemical, and cellular. Fractional irradiation is supported by the classic concept including the “4Rs” of radiobiology and the counteracting the mechanisms of radio-resistance—the repair of radiation-induced injuries; the recovery of cells for their arrest in radiosensitive phases including mitosis and the increase in the chance of arresting the cells coming out of the S phase, considered radio-resistance; cellular repopulation between fractions; and re-oxygenation—with the aim of increasing radio-sensitivity. The concept of intrinsic radio-sensitivity introduces a new, decisive factor of treatment response. The high rates of G2/M arrest, the shortage of stem cells, and a less hypoxic microenvironment are mechanisms involved in determining the radio-sensitivity of HPV-driven HNSCC. The DNA lesions produced by radio-chemotherapy generate the release of neo-antigens and tumor antigens and generate the effect of “in situ vaccination” ,38,39.The sixth “R”, the reactivation of the immune response becomes all the more important since immunotherapy with immune checkpoint inhibitors has demonstrated a synergistic potential with radiotherapy. The effect of “in situ vaccination” and the systemic effect induced by “abscopal” radiotherapy are determined by the immune modulation induced by irradiation. Fractionation, dose, and timing are three essential factors that define the immunosuppression/immunopotentiation balance induced by irradiation. The modulation of the tumor microenvironment (TME) by irradiation is also considered a cause of the radio-sensitivity variation. The TME modulation by irradiation includes the stimulation of some immune mechanisms that act directly or indirectly at the tumor level by releasing cytokines, chemokines, and enzymes. The different radio-resistances of some components of the TME and their different effects (immunosuppressor or stimulator of antitumor immunity) are the basis of the opposite effects of irradiation at the level of the TME. Among the factors associated with immunosuppression, we mention MDSCs, Tregs, and natural killer cells (NKs), and CD8+ T lymphocytes are stimulators of immune-mediated tumor evasion. NKs are considered among the most radiosensitive immune cells, and cancer-associated fibroblasts (CAFs) and dendritic cells (DCs) are considered much more radio-resistant. Furthermore, Tregs are considered more radio-resistant than other types of T lymphocytes including the CD8+ subtype ,42,43,44Targeting the cyclic GMP–AMP synthase/stimulator of interferon genes (cGAS/STING) pathway is also considered a future strategy in immune-modulated radio-sensitization . A findiThe increase in antigen presentation and recognition by the host’s immune system, phenomena linked by cell death related to irradiation, seem to have a decisive role in the induction of an antitumor immune response. The inflammation associated with irradiation and the release of cytokines IL-6 and IL-8 and tumor necrosis factor (TNF)-α are involved in the activation of the immune system. Damage-associated molecular patterns (DAMPs) are endogenous molecules released by dying cells that initiate a reaction of the host immune system. Their release is activated by radiotherapy, chemotherapy, and also by oncolytic viruses. An association between the viral neoantigen and DAMPs is considered essential to generate an immune response that defeats the tumor’s immune-escape mechanisms ,51,52.high vs. TILSlow HPV+ cancers and similar OS rates at 3 years (51%) for HPV− HNSCC clearly demonstrate the association of the immune-mediated response with the subtype HPV+. Infiltration of the stroma with CD8+ T lymphocytes is considered by Oguejiofor et al. as a positive prognostic factor in HPV-driven HNSCC. CD4, but not FoxP3 T regulatory cells, was identified in HNSCC HPV+ stromal infiltrates. A CD8+ T cell cutoff level > 24% is identified by Wansom and collaborators as a favorable prognostic factor. CD3+ and CD8+ lymphocytes infiltrated in the tumor border core included in an immune score are considered more significant biomarkers for treatment de-escalation strategies than p16 status. Disproportional enrichment of FoxP3+ CD4+ regulatory T is mentioned by Park et al. as a decisive factor in the creation of an immune-suppressive TME in HNSCC. The authors mentioned the involvement of this Treg in the possible unfavorable response to immunotherapy. However, the HPV-driven HNSCC cases are notable for a high proportion of both Treg and CD8+. Surprisingly, although they are considered immunosuppressive, increased levels of FoxP3+ T cells were correlated with better disease-free survival (DSF) and OS. The highest median Treg/CD8+ T cell ratio and high levels of CD8+ were identified by Mandal et al. in HNSCC, regardless of HPV status. A higher level of NK cell infiltration in HNSCC is a positive prognostic factor, but the genetic signature of smoking is associated with a poor immune TME and a worse prognosis [CD39 and LAG3, PD1, TIGIT, and TIM3, considered T-cell exhaustion markers, are identified in a large proportion of HPV+ HNSCC cases but not in the HPV− subtype. The presence of T-cell exhaustion markers is considered a biomarker of a T-cell-inflamed tumor phenotype correlated with a favorable prognosis. The difference between two subtypes of HPV+ OPSCCs classified according to tumor-infiltrating lymphocytes (TILs) demonstrates the biomarker value of this parameter of cellular immunity in HPV+ HNSCC as well. Overall survival (OS) rates at 3 years of 94.72% and 56%, respectively, for TILSrognosis ,55,56,57The increased response rate to pembrolizumab treatment in cases of HPV+ HNSCC programmed cell death Protein 1 (PD-1)–positive patients, 25% vs. only 14% for HPV-HNSCC cases, also suggests the involvement of HPV status in the response to a possible association of PD-1 inhibitors and radiotherapy. Higher levels of myeloid-derived suppressor cells (MDSCs) are considered factors associated with immunosuppression in HNSCC. Regardless of HPV status, higher levels of MDSCs are associated with advanced stages (III–IV). Jayaraman et al. mentioned the potential of transforming a tumor suppressor in association with TGF-β1 (TGFβ-MDSC) in a stimulation of the immune-mediated rejection of the tumor. The upregulation of FAS potentiated by radiotherapy in combination with intratumoral injection of TGFβ-MDSC could generate a long-lasting tumor immune response with potential benefit to survival ,59.The classification proposed by Keck et al. defined five subtypes of HNSCCs regardless of HPV status and named the phenotype of HNSCC rich in CD8+ “inflamed/mesenchymal”. Among HPV+ cancers, 63% are part of the “active immune class”. M1 tumor-associated macrophages (TAMs), antigen-presenting cells (APCs), and the large number of effector cells could be considered defining elements of this immune-related tumor subtype. The “exhausted tumor subtype” is associated with TGFβ and Wnt signaling (involved in stem cells and differentiation ability with consequences in the unfavorable prognosis). The “exhausted” percentage is considered much higher in HPV-HNSCC, with only 13% being in the HPV+ group. It is considered that the majority (67%) of HPV+ HNSCCs are associated with the immunologically active group, and only 5% are in the “exhausted” group ,60,61,62α/β = 10Gy and α/β = 12Gy [A regimen dedicated to older and frail patients proposed by the American Society for Radiation Oncology–European Society Radiation Oncology (ASTRO-ESTRO) consensus proposed hypo-fractionated radiotherapy (HFRT) as an alternative regimen to the standard protocol (68–70 Gy in 34–35 daily fractions). Two other regimens  were compared with the standard regimen, and the results at 2 years were comparable between the three study groups. A dose per fraction of 2.4 Gy/fraction is the maximum at which the option of administering concurrent chemotherapy is considered possible. In the evaluation of doses, an equivalent dose in 2 Gy fractions (EQD2) and a biologically effective dose (BED) were evaluated for each regimen using β = 12Gy ,64.Huang et al. recommended to implement as standard a hypo-fractionated regimen with 2.4Gy/fraction for HPV+ T1-T3N0-N2c according to TNM-7 staging HNSCCs, HPV–T1-T2N0 HNSCCs, and also for selected cases of stage III HNSCCs in a pandemic context, which requires reducing the time spent by patients in the radiotherapy department .Hypo-fractionated irradiation by the IMRT technique in total doses ≥ 50 Gy in 20 fractions for HNC was evaluated on a group of 56 patients. The results highlighted the absence of grade 4 and 5 acute and late toxicities and 79% and 25% rates of grade 2 and 3 acute and late toxicity, respectively. With a median OS of 46 months, the loco-regional control rate at 2 years was 87%. It should also be mentioned that the patients did not require a feeding tube or tracheostomy. Definitive or postoperative hypo-fractionated radiotherapy is considered to be well tolerated and without risks of unacceptable toxicity . Re-irraThe role of HPV status in cell death and in the expression of immune checkpoints was evaluated in four HNSCC cell lines using two irradiation regimens: a 5 × 3.0 Gy hypo-fractionated irradiation protocol and a 1 × 19.3 Gy single-dose irradiation protocol quantified with flow cytometry and polymerase chain reaction (PCR) shortly after irradiation. The hypo-fractionated regimen favored apoptosis in all cell lines regardless of HPV status, but necrosis was identified only in HPV-positive cell lines. In general, irradiation influenced immune-stimulatory checkpoints less than immunosuppressive ones, including PD-1 and PD-2, with the hypo-fractionation sequence generally favoring the surface expression of immune checkpoints regardless of HPV status. However, in the case of ICOS-L, the 5 × 3.0 Gy regimen induced a higher expression for HPV-positive cells. The authors conclude that HPV expression cannot be used as a single biomarker in anticipating a possible response to immunotherapy, but the expression of checkpoints after irradiation could be used for precision immunotherapy . The faiConsidering these data, we propose an adaptation of the LQ model in which we add a term “equivalent to an additional dose” that can be lost or added as biological efficacy depending on the modulation induced on the immune response by irradiation (Equation (2)).Di = ti × sbi × r × i;i—a term dependent on the moment of initiation of immunotherapy. In principle, this term could have higher values for the time interval of 24 h–10 days after the completion of radiotherapy;ti—a term dependent on the tumor’s immune phenotype;sbr—the proposed fractionation regime;i—a term dependent on the type of immunotherapy.All the terms proposed could also have negative values that can cause a loss of the effectiveness of the synergistic treatment depending on the tilt of this scale in favor of immunosuppression or the antitumor immune effect. It should also be mentioned that, if more than one term has negative values, the general effect will be an amplified detrimental one and the second negative term must be included in the absolute size.α/β ratio. The involvement of the antitumor immune response and the dose/fractionation/volume factors as well as the therapeutic sequence in the case of multimodal treatments must be included as an additional term in the quadratic linear formalism for the adaptation of the LQ model in altered fractionation regimens and especially in hypo-fractionation settings.The new data regarding the involvement of the sixth “R” of radiobiology in HNSCC, especially for the HPV-driven subtype, but also for the “immune active” minority of HPV-negative HNSCC, bring to the fore a multifactorial variation of the


Low- and middle-income countries (LMICs) account for nearly 85% of the global cervical cancer burden, yet have the least access to high-performance screening. International guidelines recommend human papillomavirus testing (HPV) as primary screening, yet implementation is inhibited by the cost of HPV testing. Atila AmpFire® HPV Assay (AmpFire) is both affordable and easy to use, and offers individual genotyping. The objective of this study was to compare the performance of the AmpFire HPV assay to the Xpert® HPV assay in detection of both HPV and clinically significant cervical disease.We utilized stored cervical specimens from a prospective cohort study of women living with human immunodeficiency virus (HIV) in Botswana conducted from May to July 2018. Positive and negative percent agreement was calculated for the AmpFire and Xpert assays, as was detection of high-grade cervical dysplasia.63 stored cervical specimens had detectable DNA after thawing and were included in the analysis. The positive percent agreement was 91.2% (95%CI 76.3–98.1) and negative percent agreement was 79.3% (95% CI 60.3–92.0). Six cases positive by AmpFire but negative by Xpert were HPV genotypes 35, 52 (n = 2), 58, 68, and co-infection with HPV 45 and 68. Both Xpert and AmpFire assays detected HPV in all 10 samples of women who had high-grade cervical dysplasia.The AmpFire HPV assay demonstrated excellent analytic performance in both detection of HPV and clinically significant cervical disease. AmpFire HPV is a promising option to increase access to affordable, type-specific HPV screening for cervical cancer in LMICs. Primary human papillomavirus (HPV) testing for HPV-associated cancer screening has become standard in high-income countries but is not widely available in low- and middle-income countries (LMICs) –3. It isThe greatest barrier to HPV screening in LMICs is the cost of the World Health Organization-prequalified HPV assays , 10. A wWe recently reported that high-risk HPV testing combined with colposcopy has the highest sensitivity and positive predictive value for detecting high-grade cervical dysplasia in women living with HIV in Botswana . While BAtila AmpFire® Multiplex HPV Assay (AmpFire) produced by Atila BioSystems  is more affordable ($9 for 15 high-risk genotyping) than most available HPV assays and thus has the potential to increase access to HPV testing in LMICs –22. AmpFWe utilized 67 stored cervical swab specimens from a prospective cohort study of women living with HIV in Botswana conducted from May to July of 2018. The study investigated the performance of primary high-risk HPV testing followed by triage evaluation with cytology, visual inspection with acetic acid and colposcopy to diagnose pre-invasive cervical disease in women with living with HIV in Botswana. Histopathology outcomes were available for all HPV specimens which were positive by original Xpert testing .In the study, provider-collected samples were taken from the cervix for HPV testing and cervical cytology using a Cervex-brush®. HPV specimens were then placed in a PreservCyt® transport medium and taken to the Botswana Harvard Health Partnership Laboratory for HPV testing as previously described . All parHPV genotyping was performed in the original study using the Xpert assay according to the instruction manual. The Xpert assay was used to qualitatively genotype the 14 high-risk HPV genotypes, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The results were reported as HPV 16, HPV 18/45, and other high-risk HPV genotypes. The input volume of cervical cell suspension for this assay was 1 mL per sample.Stored specimens were thawed and prepared for assay processing in March 2021. One milliliter of each specimen was briefly vortexed and transferred to a sterile 1.5 mL eppendorf tube. The tubes were then centrifuged for 30 min at a maximum speed followed by a complete supernatant removal and subsequently 30 μL of the lysis buffer was added and then thoroughly vortexed to resuspend the cell pellet. The whole content from each sample tube was transferred into a 0.2 mL polymerase chain reaction (PCR) tube. Incubation of the PCR tubes was then performed at 95 °C for 10 min in a thermocycler.https://atilabiosystems.com/multiplex-high-risk-hpv-by-fluorescent-detection/). This assay detects 15 individual high-risk HPV genotypes in a single tube, including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68 (the same types as the Xpert HPV assay plus HPV 53). AmpFire uses the cyanine5 (CY5) fluorophore for HPV 16, the carboxyrhodamine (ROX) fluorophore for HPV18 and other high-risk HPV genotypes using the fluoresce in amidites (FAM) fluorophore, all combined with an internal control using the HEX fluorophore. The Real-Time PCR was programmed using Bioer Real-Time PCR system for an isothermal reaction setting of 60 °C while taking fluorescence readings at the FAM/HEX/CY5/ROX channels once every minute for a total of 60 min. The thermocycling software system automatically reported the results of the cycle threshold values for each amplification curve in all fluorescence channels.Samples were then genotyped using the AmpFire by fluorescent detection according to the instruction manual , negative percent agreement (both results were negative), and discordant if the results of each assay were different. The 95% CIs of the positive and negative percent agreements were calculated. P-values were also calculated, and 67 stored cervical samples were available for validation. When Ampfire was run on thawed specimens, 4 (6%) samples had invalid controls, leaving 63 samples with detectable DNA that was genotyped and included in the analysis. In the original study, Xpert detected HPV in 34 samples while 29 samples tested negative. AmpFire detected HPV in 37 of the 63 samples, classified into 13 of the 15 high-risk HPV genotypes detected by AmpFire , 58, 68, and co-infection with HPV 45 and 68. Additional notes on type-specific discordance are found in Table The results of the HPV assays were compared to the histopathologic diagnosis in the original study Table . Among tThe AmpFire HPV assay demonstrated excellent analytic performance in detecting high-risk HPV genotypes, demonstrating 85.7% overall agreement with the Xpert assay. The AmpFire assay also performed equally well to Xpert HPV in identifying women with clinically significant cervical disease (CIN2+), thus meeting an essential requirement of a high-performance screening test. Our study also demonstrated the benefit of AmpFire in detecting individual HPV genotypes, as 13 of 15 high-risk HPV individual genotypes were detected in this small sample. Individual genotyping offers the possibility of HPV genotype restriction triage for positive HPV results, which is of increasing interest in cervical screening research , 24. FinThese results show that an affordable and accurate HPV assay with individual HPV genotyping is within reach of the global market. Currently WHO recommends primary cervical screening with HPV assays with particular regulatory approvals . Our datThe high rate of agreement between AmpFire and Xpert was most likely due to the similar real time fluorescence detection methodology and the same high-risk HPV genotypes detected by the two assays (aside from HPV 53 which is not included in the Xpert assay). Our finding is in keeping with prior studies evaluating AmpFire compared to WHO prequalified HPV tests in various specimen types. AmpFire successfully detected 15 high-risk HPV genotypes from cervical, vulvar and oropharynx tissue in paraffin embedded blocks with > 95% agreement with Linear Array HPV DNA genotyping tests , 27. VagIt is not clear what accounts for the differences in detection of HPV between assays. The use of different materials for the detection process could be a factor to consider. AmpFire produces cell lysates using the kit extraction lysis buffer, whereas Xpert performs cell lysis and purification within the cartridge, ensuring high quality HPV DNA before PCR amplification. Another possible factor is the open reading frames targeted by the Xpert assay which are E6/E7 whereas the AmpFire assay targets E1 and/or L1. Process monitoring to further optimize performance is warranted.The AmpFire assay is well-suited for HPV detection in most resource limited settings aiming to implement HPV testing. The ability to detect HPV DNA in samples that had been frozen for three years in a practical setting is reassuring; samples can be collected, stored, and tested at a later time when stored in the correct conditions for prolonged periods. Data from other studies support the stability of HPV DNA on dry swabs across unregulated temperatures for at least a month which increases the feasibility of conducting population-based screening with decentralized self-collected vaginal HPV samples that could be stored in variable conditions until transport and testing are possible in a referral laboratory , 30. OthThis study has several limitations. Our study used a small sample of stored specimens due to feasibility constraints. Additionally, we only have histopathology data on women with positive HPV results, limiting our ability to evaluate the sensitivity and specificity of both assays in detecting CIN2 + in the entire sampled population. Another limitation is the lack of gold standard used as a comparator. However, the Xpert assay is WHO pre-qualified, and in validation of other infectious disease tests it has generally been acceptable to evaluate the interchangeability of a test with a clinically utilized comparator without altering diagnostic accuracy in cases where a gold standard is not available . Given tA larger study with a true gold standard  is necessary to evaluate the performance of AmpFire in the detection of high-grade cervical dysplasia. This study is currently underway by our research group in Botswana and will allow validation of the accuracy of AmpFire in diagnosing CIN2+ in 3000 women. The study will also include histopathologic assessment of a proportion of the women who test negative with AmpFire, such that test performance parameters  can be evaluated.The AmpFire HPV assay demonstrated excellent performance compared to a clinically utilized alternative and is a promising option to increase access to affordable, type-specific HPV screening for cervical cancer in LMICs. Because of its simplicity, low cost, and ease of use, AmpFire can be easily implemented in facilities that currently have no HPV testing. Expedited approval is essential to rapidly scale-up affordable HPV primary screening for cervical cancer.