Anxiety, fatigue and depression are common neurological manifestations after COVID-19. So far, post-COVID complications were treated by rehabilitation, oxygen therapy and immunotherapy. Effects of neurofeedback on post-COVID complications and their potential interrelatedness have not been studied yet. In this pilot study, we investigated the effectiveness of neurofeedback (Othmer method) for treatment of fatigue, anxiety, and depression after COVID-19.10 participants met inclusion criteria for having positive anamnesis of at least one of the following complications following COVID-19: fatigue, anxiety, and depression which were measured by questionnaires. ANOVA was used for calculating differences in questionnaire score before and after neurofeedback. Pearson’s correlation coefficient was used to calculate correlations between anxiety, depression and fatigue.After five neurofeedback sessions, there came to significant reduction of severity of post-COVID anxiety and depression persisting for at least one month. Effect of neurofeedback on fatigue was insignificant. Severity of anxiety, fatigue and depression as well as reductions in depression and fatigue were positively correlated with each other.These findings showed effectiveness neurofeedback for reducing anxiety and depression after COVID-19 and for studying correlations between neurological complications after COVID-19. However, since our pilot clinical trial was open-label, it is hard to differentiate between neurofeedback-specific and unspecific effects on our participants. Future randomized controlled trials with more robust sample are necessary to investigate feasibility of neurofeedback for post-COVID neurological complications. The study has identification number trial ID ISRCTN49037874 in ISRCTN register of clinical trials (Retrospectively registered). Complications after COVID-19 occur in approximately 8–47.5% of COVID-19 survivors . Post-COBiofeedback (BFB) is a type of neuro-modulation method that is based on providing auditory and/or visual feedback to the participant based on changes in the activity of the participant’s selected biosignal modality –20. ModaIn this pilot study, we decided to investigate the effect of neurofeedback therapy on the following selected neurological complications after COVID-19: fatigue, anxiety and depression. Electrode locations used for NFB included the right and left temporal lobes (T3 and T4 according to the 10–20 system). The temporal lobes were chosen for the following reasons: first, abnormalities in the structure and/or activity levels in these brain regions in COVID-19 survivors who suffered complications after COVID-19 –35 and f5 sessions of NFB will significantly reduce fatigue, anxiety, and depression after COVID-19.NFB-induced significant improvement in the above post-COVID symptoms will be still evident one week after NFB.NFB-induced significant improvement in the above post-COVID symptoms will persist for one month after NFB.Severity of fatigue, anxiety and depression will be positively correlated with each other.mprovements in fatigue, anxiety and depression will be positively correlated with each other.The following criteria were required for inclusion in this study: age at least 18 years , a positive history of Sars-CoV-2 infection confirmed by a positive antigen/RT-PCR/antibody test, and at least one of the following symptoms: fatigue, depression, and anxiety that were not present prior to Sars-CoV-2 infection. The specific symptoms should have been present or persisted for at least 3 months after confirmed Sars-CoV-2 infection and should not have been attributable to any other neurological disease prior to COVID-19. Participants should have been free of health problems prior to Sars-CoV-2 infection and medication-free or medication-stabilized in dose and type of drug for at least 3 months prior to the beginning of the study. As this is a pilot study, no specific age group was required.In total, 17 participants were enrolled in the study. 6 of them did not meet inclusion criteria and/or they decided not to participate in the experiment. 1 participant has completed 4 NFB sessions and she dropped the experiment due to medical reasons. In total, 10 participants  met inclusion criteria and completed all 5 NFB sessions and fill medical questionnaires and therefore they were included in the statistical analysis. https://www.isrctn.com/ISRCTN49037874). The study has been approved by Ethic Committee of Third Faculty of Medicine, Charles University in Prague, Czechia and was conducted in accordance with the Code of Ethics of the World Medical. Association (Declaration of Helsinki for Human Experiments). The reason for retrospective registration of the study to clinical trial register is that at the time of approval of the study by Ethic Committee, the study was not considered as clinical trial. The authors confirm that all ongoing and related trials for this drug/intervention are registered. All participants signed and submitted informed consent (written form) during the first session prior to the whole experimental procedure. Protocol of the study  as well as estimated time schedule for data collection are included in Participants have been invited to participate in this study, provided that they met inclusion criteria. Recruitment of the participants started on 2021-07-16 and ended on 2021-11-02. Participants were recruited online among COVID-19 survivors by means of online posting of recruitment leaflets in student facebook groups for students from Charles University in Prague, Czechia as well as in facebook online groups dedicated to people suffering from self-referred post-COVID complications. Experiments took place in Department of Medical Biophysics and Medical Informatics in Charles University in Prague, Czechia. Medical questionnaires were filled and submitted online 1–3 days prior to the first experimental session, then immediately after finishing all 5 NFB sessions, then one week after the termination of the experiment and finally month after termination of NFB experiment. Follow-up  period ranged from 2021-08-11 and ended on 2021-12-22. The study was conducted in within-subject design. Pre-NFB and post-NFB data were compared within the same participant. In the first NFB session, informed consent has been signed. Then the short anamnestic semi-structured interview, related to details of dynamics of acute COVID-19 and post-COVID-19 symptoms, was given. Then the participants got familiarized with the NFB procedure. None of the participants had undergone NFB therapy prior to this experiment. The study has identification number trial ID ISRCTN49037874 in ISRCTN register of clinical trials . Ground electrode was put at right earlobe. Electrodes were filled with Ten 20 Past  used for making electrodes to be fixed at the scalps of participants. Conductive electrode gel was used to keep impedance as low as possible . NFB feedbacks included visual as well as auditory modalities.For visual feedbacks, watching the computer screen was required. On the computer screen, there was graph of ongoing EEG activity of the underlying NFB-rewarded bandwidth. When NFB-rewarded selected EEG activity kept below reward threshold, online EEG graph was grey. As soon as amplitude of EEG activity was at least as high as reward threshold value, EEG graph got blue. Reward threshold was adjusted manually by certified neurofeedback therapist to keep frequency of feedbacks as constant as possible. There was the effort to adjust reward threshold to ensure both ‘‘grey” intervals of no feedbacks and ‘‘blue” intervals associated with NFB-feedbacks are present in order to make brain able to differentiate between the states of being and states of not being rewarded by NFB device. For auditory feedback, the participants were given ‘‘Quiet Bong” sound which was selected from sound options of our NFB software .For indicating the severity of pre-NFB and post-NFB post-COVID-19 fatigue, anxiety and depression, the following standardized medical questionnaires were used: Fatigue Assessment Scale for measuring the level of fatigue, Beck Anxiety Inventory for measuring the level of anxiety and Beck Depression Inventory (version 2) for measuring the level of depression.For measuring the level of fatigue, Fatigue Assessment Scale was used. Fatigue Assessment Scale includes 10 questions related to the subjectively perceived level of fatigue. For each of these 10 questions, there exist 5 possible answers from which just one is supposed to be chosen. These 5 answers are the following: never, rarely, sometimes, often and always. Answer “never” is scored by 1 point, answer ‘‘rarely” is scored by 2 points, answer ‘‘sometimes” is scored by 3 points, the answer ‘‘often” is scored by 4 points and the answer ‘‘always” is scored by 5 points. This scoring algorithm is applied for the questions, 1,2,3,5,6,7,8 and 9. For the questions 4 and 10, inverse scoring of the answer is used. Score less than 22 indicate no fatigue; score higher than 22 indicate fatigue . For beiFor measuring the level of anxiety, Beck Anxiety Inventory was used. Beck Anxiety Inventory includes 20 questions regarding the severity of occurrence of a various anxiety symptom within the period of the last month. For each question, there exist 4 possible answers: The first answer is scored by 0 points and states that particular anxiety symptom is not present. The second possible answer is scored by 1 point and states that the particular anxiety symptom was mildly unpleasant. The third possible answer is scored by 2 points and states that the particular anxiety symptom was moderately unpleasant. The third possible answer is scored by 3 points and states that the particular anxiety symptom was perceived as a severe and very disturbing. For indicating the level of anxiety, total score has been calculated for all 20 questions. 0–21 points indicate low anxiety level. 22–35 points indicate moderate anxiety level. Score higher than 36 points indicate high level of anxiety . In ordeTo measure the level of depression, Beck Depression Inventory (Second version) was used. Beck Depression Inventory includes 21 questions. For each question, there exist 4 possible answers from which one answer is supposed to be chosen. The answers are scored by 0–4 points. Total score obtained from answering all 21 questions indicates the level of depression. 0–10 points indicate normal state. 11–16 score indicate mild mood disturbance. 17–30 points indicate borderline clinical depression. 20–30 points indicate moderate depression. 31–40 points indicate severe depression and score higher than 40 points indicate extreme depression . To be iThe study was conducted in within-subject (repeated measures) design. The score of Fatigue Assessment Scale, Beck Depression Inventory and Beck Anxiety Inventory was calculated. As all these observed quantitative variables fulfil the normal distribution conditions as revealed by Kolmogovov-Smirnov test, the means and standard deviations were presented. For comparison among pre-NFB and post-NFB data  the repeated measures ANOVA with multiple comparisons with Bonferroni test was used. To find the relationship between fatigue, anxiety and depression, Pearson’s correlation coefficients were calculated. Pearson’s correlation coefficients were calculated also to find the associations between improvement of scores for fatigue, depression and anxiety measured by Fatigue Assessment Scale, Beck Anxiety Inventory and Beck Depression Inventory. Descriptive statistical values such as mean value, minimum, maximum and standard deviation are presented in Table 1 in For statistical analysis STATISTICA version 14.0.0.15 (TIBCO Software Inc.) was used. The required sample size for all statistically significant correlation coefficients was calculated using G*Power software version 3.1. All tests were considered to be statistically significant at the level of p<0.05.Age of the participants ranged from 19 to 46 years (median = 21). 8 participants had positive confirmation by Sars-CoV-2 infection done by nasopharyngeal RT-PCR test. 1 participant had positive nasopharyngeal antigen test and since he was symptomatic at the time of being positively tested  he went into quarantine without the confirmation RT-PCR test. 1 participant was positively tested for IgG antibodies done by ELISA method. The participant decided to take IgG testes due to be suspected to recover from symptomatic COVID-19 after epidemiological relevant contact with person with confirmed Sars-CoV-2 infection. In relation to acute COVID-19 symptoms (and suspected COVID-19 in case of 1 participant with positive IgG test), distribution of acute COVID-19 symptoms was as follows: headaches (70% of participants), fatigue (80% of participants), elevated temperature (30% of participants), fever (20% of participants), dyspnoea (20% of participants), chest pain (20% of participants), olfactory impairment (10% of participants), gustatory impairment (30% of participants), cough (30% of participants) and sore throat (30% of participants). None of the participants was hospitalized due to COVID-19. In relation to the participants having positive nasopharyngeal swab confirming Sars-CoV-2 infection, the median period between their acute Sars-CoV-2 infection and enrolment into our study due to persisting post-COVID-19 problems, ranged from 3–19 months (median = 12 months). The participant, who was tested positive for IgG antibody, was enrolled in our study 6 months after her positive IgG test due to persisting neurological symptoms. In relation to fatigue, depression and anxiety, which were target post-COVID-19 complications of our interest, there was the following distribution within the participants: fatigue (90% of participants), anxiety (80% of participants) and depression (60% of participants). In relation to observed post-COVID-19 complications of our interest, according to semi-structured entering interview as well as data from standardized medical questionnaires  for measuring the level of depression, there was the occurrence of single symptom in 1 participant, occurrence of 2 simultaneous symptoms in 3 participants, and occurrence of 3 simultaneous symptoms in 6 participants.According to data from Fatigue Assessment Scale, 9 from 10 participants (N = 9) met inclusion criteria for fatigue . In comparison to pre-NFB data from Fatigue Assessment Scale, there came to insignificant reduction of score of Fatigue Assessment Scale immediately after 5 NFB sessions (p = 0.083583). Also, relative to pre-NFB data, insignificant reduction of score of Fatigue Assessment Scale was revealed 1 week after termination of NFB (p = 0.122952) as well as 1 month after termination of NFB (p = 0.088454). Raw data, used for this analysis, are included in Table 2 in th) NFB session due to the characteristics of that particular questionnaire-assessment of 2 weeks is too short period for Beck Anxiety Inventory [According to data from Beck Anxiety Inventory 8 from 10 participants (N = 8) met inclusion criteria for anxiety (at least moderate level of anxiety) . In comparison to pre-NFB data from Beck Anxiety Inventory, there came to significant reduction of score of Beck Anxiety Inventory 1 week after 5 NFB sessions (p = 0. 014910) as well as 1 month after termination of NFB 0.0088334). Beck Anxiety Inventory was not administrated to participants immediately after termination of the last (5334. BeckAccording to data from Beck Anxiety Depression Inventory (2.version) 6 from 10 participants (N = 6) met inclusion criteria for suffering from depression (at least borderline level of depression) . In comparison to pre-NFB data from Beck Depression Inventory, there came to significant reduction of score of Beck Depression Inventory immediately after 5 NFB sessions (p = 0.018763). Also, relative to pre-NFB data, significant reduction of score of Beck Depression Inventory was present 1 week after termination of NFB (p = 0.018763) as well as 1 month after termination of NFB (0.027231). Raw data, used for this analysis, are included in Table 4 in In this analysis, data of all 10 participants (N = 10) were calculated. Raw data, used for this analysis, are included in Table 5 in Graphical demonstrations of positive correlations between fatigue, depression and anxiety before, immediately, one week and one month after NFB can be seen in Figs 1–10 in In this analysis, data of all 10 participants (N = 10) were calculated. Raw data, used for this analysis, are included in Table 5 in Graphical demonstrations of positive correlations between reductions in fatigue, depression and anxiety before, immediately, one week and one month after NFB can be seen in Fig 11–17 in In accordance with our criteria of IOF (Section: 3.6 Neurofeedback) for all participants, target EEG NFB-rewarded frequency has been individualized and adjusted within 5 NFB sessions with regard to the particular participant. Regarding intra-session experimental period potential unwanted side-effects of NFB revealed via subjective reports of increased fatigue, and/or muscle tension and/or occurrence of headaches were temporarily present as follows: in all 10 participants in the first experimental session, in the second NFB session in 5 participants, in the third NFB session in 2 participants and in fourth and fifth session for 1 participant. Decreasing number of participants experiencing these unwanted symptoms with progressing session number is likely to speak in favour of the effect of adjustment of NFB-rewarded EEG frequency as aforementioned unwanted symptoms were associated only with NFB training of some particular EEG frequencies whereas during training of other EEG frequencies, such unwanted symptoms were not present. Based on subjective reports of the participants, if these unwanted symptoms occurred, they did not have a longer persistence than for several hours. However, in order to be able to differentiate between NFB-related specific effects, nocebo effect and others, inclusion of control group receiving sham NFB-related feedback as well as of control group receiving no NFB-related feedback should be included.Based on Beck Depression Inventory (version 2), 5 NFB sessions completed within 2 weeks resulted in a significant reduction in depression immediately after NFB, Such reduction of depressive symptoms persisted one week as well as one month after termination of NFB. Our results are in line with the outcomes of other studies reporting reduction in levels of depression after NFB –59. TherCompared to baseline condition (prior to NFB sessions), there was significant reduction of anxiety level one week as well as one month after completion of all 5 NFB sessions. In comparison to data one week after NFB (p = 0.014910), the levels of reduction of anxiety were significantly greater 1 month after NFB (p = 0.008334). Apart from possible improvement of anxiety caused by NFB, this trend might be also possibly attributable to natural time-dependent recovery from post-COVID condition as post-COVID anxiety was demonstrated to improve with time –64 thougTo the best of our knowledge, this is the first study demonstrating beneficial influence of Othmer method NFB in temporal lobes on anxiety. So far the following NFB modalities were used for the investigation of their effects on anxiety: fMRI-based NFB , infraloIn contrast to anxiety and depression, no significant pre-to-post NFB difference was observed for the levels of fatigue measured by Fatigue Assessment Scale. Although, there are some studies reporting significant reductions in fatigue following NFB, compared to our study, much greater number of NFB sessions was used in other studies ranging between 20–40 NFB sessions , 73–75. Regarding the possible inter-relatedness of post-COVID fatigue, anxiety and depression, significant positive correlation between depression and fatigue has been found before as well as immediately after NFB intervention which persisted one week and one month after NFB. These findings seem to be in line with other study discovering positive correlation between post-COVID fatigue and depression . and betFinally, we investigated whether there will be inter-correlated improvement of multiple post-COVID neurological symptoms after NFB. Score from Beck Depression Inventory and Fatigue Assessment Scale revealed significant inter-correlated improvement of fatigue and depression, which was present immediately, one week and one month after NFB. Our outcomes are in line with evidences that NFB can simultaneously improve fatigue, anxiety and depression . Also, oFinally, it is necessary to mention potential limitations and strengths of Othmer method of training IOF as well as its documented feasibility in the treatment of other neurological conditions. So far, Othmer method of training IOF has been found to be effective in treating various conditions such as epileptic seizures , pain 8, impairmWe feel there are some general limitations that need to be mentioned. First, we used small sample of the participants. Furthermore, there was quite a wide age-range of participant which also might have caused some unsystematic variations to the results. In addition, there might have been some information noise caused by recruitment of the participants based on their subjective self-reference of post-COVID complications. Secondly, we did not include control group, so it is hard to differentiate between influences of true effects of NFB and other factors. Third, the exact aetiology of post-COVID manifestations is unknown due to the absence of entering medical examination. There might have been also some noise in interpretation of our results by discussing other studies investigating effects of brain non-invasive stimulation as they differed in factors such as number of intervention sessions, modalities of non-invasive stimulation, and types of questionnaires, study protocols, and target non-clinical or clinical populations. Last but not least, the absence of use of objective measurement of neurophysiological activity limits our ability to determine the relation between neural correlates of post-COVID complications and NFB-related processes.In this open-label pilot study, we investigated the effect of 5 NFB sessions exploiting Othmer method of individual IOF on post-COVID fatigue, anxiety and depression. We are coming with the following findings: According to Beck Depression Inventory (2. version) and Beck Anxiety Inventory, NFB significantly reduced anxiety and depression. There was found positive correlation between depression and fatigue, anxiety and fatigue, and between depression and anxiety. There was observed inter-correlated improvement of fatigue and depression. In discussion, we focused on interpretation of our findings. According to these findings, we propose NFB is feasible for treatment of post-COVID complications as well as for studying their inter-relatedness.In spite of aforementioned limitations, we feel that the findings of our pilot study are relevant and worth-studying and we hope that they will be useful for the future research. Future randomized controlled trials with robust sample would be necessary to investigate feasibility of neurofeedback therapy for COVID-19 complications.S1 Protocol(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


Ectopic pregnancy (EP) is one of the major causes of maternal mortalityduring the first trimester of pregnancy.Four treatment methods for EP including single-dose methotrexate (SD-MTX),double-dose methotrexate, expectant and surgical management wereconsidered. In this cross-sectional study, the clinical characteristics of 365 womenaged 15-44 yr who had been diagnosed with EP were reviewed from March 2017to March 2019 in hospitals affiliated to Shiraz University of MedicalSciences, Shiraz, Iran. Receiver operating characteristics curves wereplotted to determine the cut-off points for size of ectopic mass andbeta-human chorionic gonadotropin (β-hCG) that suitably discriminatedbetween double-dose methotrexate and surgery management. The most common site of EP was adnexa. According to the receiver operatingcharacteristics analysis, surgery was the best plan for the women with anectopic mass Surgery is proposed as the best option for the cases with large ectopic massor high β-hCG level. SD-MTX had a higher success rate and shorter hospitalstay than the double-dose protocol, and so was found to be an efficient andsafe alternative. Further randomized clinical trials with larger samplesizes are recommended to validate the current results.  Ectopic pregnancy (EP) is defined as implantation of a blastocyst outside the uterinecavity (1). It is a major reason for maternal morbidity and mortality during thefirst trimester (2). The incidence is about 2% of all pregnancies (3). Although theetiology of EP is not identified clearly, several risk factors such as tubalinfection, previous tubal surgery and previous EP increase the probability of thisoccurrence (4). Usually, an EP is located in one of the fallopian tubes and requiresemergent treatment; as it gets large, it can cause tubal rupture due to heavyinternal bleeding and hypovolemic shock (5). Today, the options for the treatment ofEP include surgery, medical treatment and expectant management (6). Surgerytreatment includes salpingectomy, salpingotomy and salpingostomy, which can beperformed by laparotomy or laparoscopy (7). Laparoscopy has significant advantagessuch as shorter hospital stays, quicker recovery and lower cost than laparotomy.Sometimes laparotomy is preferred because of easier access to tissue inhemodynamically unstable cases (8).In women who are hemodynamically stable, without any sign of rupture of EP, systemicadministration of intramuscular methotrexate (MTX) is acceptable by single-dose(SD), double-dose (DD) or multiple dose protocols (9). MTX is contraindicated withrenal, hepatic, gastrointestinal or haemopoietic disorders (10). Weekly beta-humanchorionic gonadotropin (β-hCG) monitoring follow-up is recommended until levelsdecline below 15 mIU/ml. If medical treatment is deemed unsuccessful, the patientswill need surgical treatment (11).It is known that some cases of tubal EP with no significant symptoms resolvespontaneously. Therefore, in selected women who are clinically stable with anundiagnosed EP location in ultrasound and a β-hCG less than 1000 mIU/ml, expectantmanagement is a valuable option of treatment.As there is no consensus on the best management for EP, we aimed to compare thedifferent protocols for treatment of EP in terms of the gestational age, size ofectopic mass, duration of hospital stay, and history of pelvic surgery, pelvicinfection, infertility, abortion and EP, as well as site of EP and β-hCG level. Inorder to discriminate properly between double-dose methotrexate (DD-MTX) and surgerymanagement, we plotted receiver operating characteristics (ROC) curves to determinecut-off points for β-hCG levels and size of ectopic mass.In this cross-sectional study, the data of 365 women aged between 15-44 yr withthe diagnosis of EP were reviewed between March 2017 and March 2019 in hospitalsaffiliated to Shiraz University of Medical Sciences, Shiraz, Iran.Women aged 15-44 yr who were diagnosed with EP were included. EP was diagnosed ifthe serum β-hCG level had a slower doubling time on serial measurement and thegestational sac was not seen in the uterine cavity by transvaginal sonography.The subjects who refused to complete their treatment in the current center orhad incomplete historical data in the hospital records were excluded.Data on participants' demographic, medical and clinical characteristics .2, injected intramuscularly; Ebewe company, Austria) on dayone, or days one and four, respectively. If the β-hCG level declined Surgery was the first plan for the cases with: 1) severe free fluid in theabdominal pelvic cavity and who were unstable hemodynamically; 2) severeabdominal pain; 3) based on participant's preference; or 4) contraindication formedical treatment. The remaining cases were treated by SD- or DD-MTX . All ofthe enrolled participants signed a written informed consent form after completeexplanation of the study design.t test. Chi-square test was performed todetermine whether there were any statistically significant differences betweenthe descriptive data. Quantitative data were expressed as mean The Statistical Package for the Social Sciences (SPSS) version 22 was used toanalyze the data. One-way analysis of variance (ANOVA) was used to statisticallycompare the means of the four treatment groups. Tukey's test was used for apairwise comparison of all means. The difference between two groups was analyzedby independent sample Among the 365 women with EP, 87 cases were treated by SD-MTX, 43 by DD-MTX, 154 bysurgery, and 81 by expectant management.According to our results, the most common site of EP was right and left adnexa andthere were no significant differences between different treatments. The women whounderwent surgery had the largest ectopic mass, the highest β-hCG level, and theshortest hospital stay (p Surgery had the highest success rate (99.35%) and DD-MTX had the lowest (72.09%)(Table I). Among the surgery group, the treatment failed for only one case who hadlaparoscopy-salpingostomy.The ROC analysis is presented in figure 1. The areas under the curve for the size ofectopic mass and β-hCG level were statistically significant . Only SD-MTX group represented a significant difference in β-hCG level betweensuccessful and failed treatment (p = 0.02).The current study was performed to compare different EP treatments including surgery,medical treatment and expectant management. EP treatment depends on the hemodynamicstability of patients, β-hCG levels, pregnancy planning, the size of the EP and thepresence of fetal cardiac activity (12). Specialists have agreed that medicaltherapy is not recommended if the initial serum β-hCG values are The SD-MTX method is the most commonly used protocol for cases with EP. The successrate of this protocol has been reported in previous studies to range between 52% and94% . In our study, the success of SD-MTX was 80.46%. Although there are nospecific criteria used to recommend SD-MTX, the treatment success may be affected byβ-hCG levels, positive fetal cardiac cavity, presence of a yolk sac, and the size ofEP (16). It has been observed that the treatment success of the SD protocoldecreases when the initial serum β-hCG values are Surgery is approved as the first option for women who present with signs of tubalrupture and probable intra-abdominal bleeding (19). We found that the women who wereselected for surgery had significantly larger ectopic masses and shorter hospitalstays. According to the results of this study, we recommend surgery for cases withan ectopic mass size Our findings are compatible with previous studies which reported that a high failurerate of medical therapy occurred if the size of the EP mass was In this study, among the management groups, surgery had a 99.35% success rate and sowas the most effective method, and DD-MTX, with a 72.09% success rate, was theleast. Previous studies have found a success rate from 72-87% for SD- and DD-MTXtreatment . Some studies have reported that the success rates of DD andmultiple dose MTX regimens are higher than that of SD (24); and Song et al., in aprospective study, showed that the SD-MTX method, with the option of second doseadministration for the cases with treatment failure, could be an appropriate medicaltreatment of EP for eligible women (23). In our study, women who were given SD-MTXand DD-MTX treatment did not significantly differ in their size of ectopic mass,β-hCG level or the treatment success; furthermore, the hospital stays in the SD-MTXgroup were significantly shorter than in the DD-MTX group. We conclude that SD-MTXis preferable to DD-MTX for eligible cases.The limitation of this study was that characteristics like former EP in the samelocation, child-bearing characteristics and others that may influence thespecialist's opinion on treatment method were not examined. It is recommended tocategorize the patients according to different criteria in further studies.Based on our results, surgery is suggested as the best option for cases with a sizeof ectopic mass The authors declare that there is no conflict of interest.


While the definition of the LymphoSIGHT platform (Sequenta/Adaptive) as the standard method derives from its large use and validation in clinical studies on the prognostic value of NGS-based MRD, other commercially available options exist. Recently, the LymphoTrack assay has been evaluated in MM, demonstrating a sensitivity level of 10−5, hence qualifying as an alternative effective tool for MRD monitoring in MM. Here, we will review state-of-the-art methods for MRD assessment by NGS. We will summarize how MRD testing supports clinical trials as a useful tool in dynamic risk-adapted therapy. Finally, we will also discuss future promise and challenges of NGS-based MRD determination for clinical decision-making. In addition, we will present our real-life single-center experience with the commercially available NGS strategy LymphoTrack-MiSeq. Even with the limitation of a limited number of patients, our results confirm the LymphoTrack-MiSeq platform as a cost-effective, readily available, and standardized workflow with a sensitivity of 10−5. Our real-life data also confirm that achieving MRD negativity is an important prognostic factor in MM.Assessment of minimal residual disease (MRD) is becoming a standard diagnostic tool for curable hematological malignancies such as chronic and acute myeloid leukemia. Multiple myeloma (MM) remains an incurable disease, as a major portion of patients even in complete response eventually relapse, suggesting that residual disease remains. Over the past decade, the treatment landscape of MM has radically changed with the introduction of new effective drugs and the availability of immunotherapy, including targeted antibodies and adoptive cell therapy. Therefore, conventional serological and morphological techniques have become suboptimal for the evaluation of depth of response. Recently, the International Myeloma Working Group (IMWG) introduced the definition of MRD negativity as the absence of clonal Plasma cells (PC) with a minimum sensitivity of <10 The treatment landscape for multiple myeloma (MM) has been radically changed during the past decade by the introduction of new drugs with different mechanisms of action, which has led to a significant improvement of survival .−3  that is defined by less than 5% plasma cells in the bone marrow (BM) regardless of their clonal nature and by the absence of monoclonal proteins in serum or urine by immunofixation or disappearance of soft tissue plasmacytomas. In 2006, the criteria of stringent complete response (sCR) were introduced  that cannot be detected with standard disease evaluation methods . Consequ-5 sensitivity), by either NGS or NGF  can have a complementary role to marrow MRD assessment. The absence of metabolically active areas after treatment had already been correlated with improved clinical outcomes. Moreover, it proved to be an independent outcome predictor . For exaLiquid biopsy has also been proposed as an alternative approach for tracking MRD after treatment based on the hypothesis that a peripheral blood sample would be able to reflect BM and/or EMD status. Current approaches for liquid biopsy testing are mostly based on circulating tumor DNA (ctDNA). While one study  reportedCurrently, MRD has been incorporated in numerous clinical trials with different aims, as follows: 1) as a prognostic factor; 2) as a surrogate end to evaluate treatment efficacy and compare different treatment approaches; 3) as an adapted treatment strategy according to MRD status and its follow-up especially in maintenance duration and early salvage approach.The association between depth of response and long-term outcome is a current topic of debate in MM. CR has been generally considered as the best possible response after treatment and an indicator of improved outcome. The value of sCR compared to CR is still controversial, with only one out of four studies reporting a better prognosis after autologous stem cell transplantation (ASCT) when sCR rather than CR was achieved –22.On the opposite, MRD is emerging as a more sensitive assessment of depth of response and a more powerful prognostic tool, with many studies demonstrating that undetectable MRD is associated with improved progression-free survival (PFS) and overall survival (OS) –31.-4), cytogenetic risk, method of MRD assessment, and depth of clinical response at the time of MRD measurement  and relapsed or refractory multiple myeloma (RRMM)  that demonstrates the high efficacy of daratumumab-based regimens in improving MRD negativity rates and reducing the risk of disease progression or death. In all of these studies, MRD was assessed by NGS using the clonoSEQ assay  with a sensitivity of 10a (RRMM) .Another example supporting the value of MRD in prolonged survival is the phase III PETHEMA/GEM2012MENOS65 trial. MRD was assessed by NGF in BM samples in NDMM after bortezomib-lenalidomide and dexamethasone (RVD) induction, autotransplant, and two RVD consolidation cycles in order to modify accordingly the maintenance therapy. Importantly, with a median follow-up of 40 months, patients with undetectable MRD after consolidation showed very low risk of disease progression (7%), with a 3-year survival rate reaching 90%. Attaining undetectable MRD also overcame poor prognostic features at diagnosis, including high-risk cytogenetics, thus confirming MRD as the strong prognostic factor for MM .Several other studies support these conclusions and confirm their value in different patient populations at different time points and with several MRD assessment methods.In conclusion, MRD negativity is associated with improved survival independently of the disease status (newly diagnosed or relapsed disease), cytogenetic risk, MRD sensitivity level, method used for MRD assessment, and the level of the clinical response at the time of MRD evaluation negativity. Moreover, MRD negativity is the most relevant predictor of clinical outcome compared with other prognostic factors , 34–38.−5 using as the reference methods  allows highly sensitive discrimination between polyclonal and clonal plasma cells based on their immunophenotypic characteristics . MFC detects cell surface markers using fluorescently labeled antibodies. Canonical markers include CD138, CD38, CD45, CD56, CD19, and cytoplasmic κ and λ immunoglobulin light chain, while other markers include CD20, CD27, CD28, CD81, CD117, and CD200. In order to overcome the limitations of conventional MFC, highly sensitive NGF MRD approaches have been developed in the last years by EuroFlow. Current NGF methods rely on a more efficient sample preparation protocol that allows acquisition of >10 million BM cells (eight-color two-tube panel) and on innovative automatic data analysis software tools that have resulted in increased sensitivity of 10-6 , 39, 40.NGS consists of the molecular assessment of immunoglobulin gene rearrangements, and it allows to detect MM MRD in the BM. The development of NGS methods for MRD assessment has focused mainly on the third complementarity determining region (CDR3), a sequence of 30–70 nucleotides, at the intersection of variable (V), diversity (D), and joining (J) immunoglobulin gene segments . During Once amplified, the immunoglobulin gene DNA is sequenced and the frequencies of the different clonotypes are determined. To avoid disproportional amplification of the immunoglobulin heavy chain gene (IGH) rearrangements and immunoglobulin light chain gene (IGK) rearrangements, the extensive sets of primers need to be attuned and validated to guarantee correct and proportional amplification of each target rearrangement compared with many rearrangements derived from normal B cells .These gene rearrangements must be studied at diagnosis in a baseline sample; baseline clonality detection rates vary between 80% and 97% in different studies using different NGS platforms , 39, 43.Extensive SHM can interfere with PCR primer annealing, resulting in less effective amplification of V(D)J rearranged sequence and making the clonal sequence indistinguishable from the background of normal polyclonal B cells. When using primers that anneal inside the IGHV exon , the myeloma-associated rearrangement may be missed, making impossible to determine clonality. This could be avoided by using primers that bind in the leader region targeting the leader sequences upstream of the VH region avoiding amplification bias due to SHM. However, these approaches lack validation data for MRD tracking –46.The NGF and NGS technology platforms have different advantages and limitations; −6) BM cells if 10 million cells can be acquired; NGS has an analytical sensitivity of 10−6 once a DNA input from 3 million BM cells is granted. Therefore, NGS requires a smaller specimen volume than NGF. Moreover, NGS can be applied retrospectively on stored material including not only cryopreserved cells but also archival BM slides, while NGF requires a fresh sample that has to be processed within 24 h from aspiration.NGF has an analytical sensitivity of 2 tumor cells in 1 million  than NGF (24–48 h).A novel, not yet clinically approved, NGS approach, the LymphoTrack-MiSeq platform, potentially overcomes this pitfall because it could potentially be performed at individual institutions, with a higher degree of patient applicability. Currently, the turnaround time of the LymphoTrack-MiSeq platform for fresh samples is about 5 days .NGS is still an expensive technique, with the cost per sample about twice or thrice that of NGF; however, prices vary for different platforms, and the LymphoTrack-MiSeq platform is among the least expensive and can be competitive with NGF. Moreover, NGS costs can be reduced by testing multiple samples at the same time.−5 . Notably, the sensitivity threshold was claimed to be of at least 10−5 , 31 or 1−5  but has the advantage of not requiring patient-specific primers , reaching a sensitivity of 10-6. This follow-up study demonstrated that the ability to measure deeper response provides superior outcome. With 10−6 sensitivity, MRD negativity was a strong prognostic biomarker of PFS and OS. This approach demonstrated a higher level of discrimination than had previously been achieved with the less sensitive MFC technique , MRD was originally evaluated by first-generation flow cytometry (seven-color MFC), showing that patients who were MRD-negative with a sensitivity of 10echnique , 53, 54.−5) and by NGF in subgroup patients. MFC and NGS evaluations with high sensitivity (sensitivity 10−5–10−6) were available only in ≥CR patients, while MRD was analyzed both by NGS and second-generation MFC in patients achieving VGPR or better. In 30% of cases, a discordance between NGS and second-generation MFC was detected: in all of the discordant cases but one, MRD positivity was missed by MFC. However, when NGF was compared with NGS in the subgroup of CR patients, results were highly concordant with the two techniques (83%)  .−5), regardless of response in patients achieving CR or more, indicating that both techniques performed similarly in evaluating MRD  was observed using the same sensitivity threshold  and NGS (LymphoTrack) methods for MRD detection. NGS showed good concordance with NGF, with only 15 out of the 106 patients studied having contradictory results. Most discordant cases had MRD levels below 10rocedure .In conclusion, the NGS approach is a powerful tool for MRD detection, considering the key role of the achievement of MRD negativity in the clinical management of MM patients. During the last few years, several NGS platforms for MRD detection in MM were tested but there are no data that compare the different platforms yet.−5 of a new commercial NGS panel.We present our single-center experience with the LymphoTrack-MiSeq platform for the diagnosis and MRD detection in a selected cohort of MM patients. Our aim was to test the applicability and verify a sensitivity of at least 10In 2018, we retrospectively selected 28 consecutive transplant-eligible newly diagnosed MM patients. We selected 52 samples, consisting of 28 diagnoses and 24 follow-up specimens at day +100 following ASCT. BM aspirates were collected at diagnosis and subsequently at follow-up from our Institutional Biobank. All patients included in the present study provided a written informed consent to have their biological samples stored and characterized.Genomic DNA (gDNA) was isolated from BM aspirates with the automated Maxwell 16 system using 16 LEV Blood DNA kit according to the manufacturer’s instructions . gDNA was isolated from total white blood cells (WBC) after lysis of red blood cells. In some cases, DNA was extracted immediately after sample processing according to Maxwell manufacturer’s protocol. In other cases, cells were resuspended with a solution of 10% Dimethyl Sulfoxide (DMSO) and 90% fetal bovine serum (FBS) and stored in liquid nitrogen. After the extraction, gDNA was conserved at -20°C. For long-term storage (over 1 year), DNA was conserved at –80°C.After the extraction, gDNA purity was checked using the Nanodrop spectrophotometer  considering absorbance ratio at both 260/280 and 230/260 nm. In order to verify gDNA quality, we analyzed its size distribution electrophoresis on 1% agarose gel. Samples with smears below 2.5 kb were classified as degraded and were excluded from further analysis. DNA concentration was measured by the Qubit dsDNA HS assay kit and Qubit 2.0 fluorometer .InVivoScribe Technologies, San Diego, CA, USA). Briefly, LymphoTrack Dx IGH  and IGK Assays include a single multiplex master mix that targets one of the conserved IGH framework regions  within the VH and JH regions and Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements, respectively. Each master mix contains primers with Illumina adapters and up to 24 different indices that allow library preparation in one-step PCR reaction. Amplicons were purified using AMPure XP beads  and quantified with KAPA Library Quantification Kit for Illumina platforms . Here, 4 nM of each library was pooled, denatured, and diluted to 12–20 pM. Sequencing was performed in the MiSeq platform  using v3 (600 cycles) reagent kits. FASTQ files generated from sequencing were analyzed using the LymphoTrack-Miseq and LymphoTrack MRD Software .Library preparation was performed using the LymphoTrack Dx IGH  and IGK assays according to the manufacturer’s recommendation . Importantly, 1,000 ng per replicate was loaded for all of the MRD-negative samples , ensuring 10-5 sensitivity. Manufacturer’s brochure was updated in 2021 and now reports a minimum requirement of 2,000 ng total gDNA analyzed and a total read depth of 44 million reads to reach 10-5 sensitivity with 95% confidence of a true MRD negativity. This real-life experience reveals how standardization and optimization of NGS techniques are still ongoing and crucial.For the 28 samples of newly diagnosed patients, clonality was assessed by both LymphoTrack IGH  and IGK assays. Each MRD sample was analyzed in triplicate, and for each replicate, 500–1,500 ng of DNA was used as input ; for each replicate, we obtained at least 1,080,000 total reads. Experiment setting up was performed using Lymphotrack MRD software according to the manufacturer brochure available at the time (2020) that indicated a minimum of 1,000 ng per replicate to reach 10We retrospectively selected 28 consecutive transplant-eligible newly diagnosed MM patients . All patients received VTd induction  and tandem ASCT. No maintenance therapy was used. Clinical variables of our cohort are described in We evaluated clonal rearrangements of IGH  and IGK target region by the LymphoTrack-MiSeq platform on BM samples at two time points: at diagnosis and at day 100 following first ASCT. At diagnosis, 26 samples (26/28) showed monoclonality and 2 samples (2/28) polyclonality. Interestingly, these two patients had EMD at PET/CT evaluation. In the evaluation of the single targets (IgH and IgK), the results highlighted the presence of monoclonality for the IgH loci in 7 samples (7/26) and for the IgK loci in 4 samples (4/26) while 15 samples (15/26) in both of them. At day 100 after ASCT I consider only 24 patients who had evaluable MDR. We evaluated MRD at day 100 after first ASCT: 2 patient samples were not evaluable, and MRD was positive in 18 patients (18/24) and negative in 6 (6/24) patients. In the evaluation of the single targets (IgH and IgK) at day 100 post first ASCT, the results highlighted the presence of monoclonality for the IgH loci in 7 samples (7/24) and for the IgK loci in 4 samples (4/24), in 7 samples (7/24) for both of them. Such variations are in line with disease heterogeneity in which the dominant clone is eradicated during treatment, but resistant and quiescent subclones persist and inevitably proliferate, resulting in relapsing disease.The response to treatment was evaluated according to IMWG criteria at different time points: after induction  and at day 100 following first ASCT . Among patients who reached sCR at day 100 following first ASCT, five patients were MRD-negative and six patients were MRD-positive; a single MRD-negative patient was in CR and 10 patients in VGPR were MRD-positive and one patient was MRD-negative.At a median follow-up of 81 months (range: 25–149 months), 15 out of 24 MRD evaluated patients relapsed during follow-up, 2 in the MRD-negative group (2/6) and 13 in the MRD-positive group (13/18). The time to next treatment was 87 months for MRD-negative patients (range 35–140 months), while the median time to next treatment was significantly low in the MRD-positive patients: 44 months .At the last follow-up, five patients in the MRD-positive group had died (5/28), one due to infection and four for progressive disease. In the MRD-negative group, all patients are alive. Among the MRD-negative patients, four out of six are maintaining complete remission until the last control.The OS of all our cohorts from diagnosis was 82 months (range: 25–150 months), with 23 patients still alive. The OS was 90 months for MRD-negative patients (range 63–140), while the median OS in the MRD-positive group was 71 months   and at 2 years (35.5% vs. 18.8%)  in transplant-eligible patients with newly diagnosed MM. The MRD was assessed by MFC during induction/consolidation and additionally by NGS during maintenance, both with the 10. 18.8%) –58.-4–10-5 sensitivity), after a median follow-up of 75 months, 5-year PFS was 66% in MRD-negative patients vs. 31% in MRD-positive patients, and 5-year OS was 86% vs. 69%, respectively. In the 1-year maintenance MRD population, 42% of MRD-positive patients at pre-maintenance became MRD-negative after lenalidomide exposure. The authors concluded that MRD by MFC is a strong prognostic factor and that lenalidomide maintenance can improve MRD negativity rate and therefore prolong OS (An MRD correlative study conducted in a randomized phase 3 study (EMN02 HOVON95) supported the role of lenalidomide maintenance in patients with newly diagnosed MM after different induction strategies. In patients achieving a CR before maintenance BM samples were processed, applying EuroFlow-based MFC protocols  rate in the absence ASCT. MRD negativity was achieved in 71% of patients. Median time to MRD negativity was 6 cycles. At 11 months of the median follow-up, the 1-year PFS rate and the OS rate were 98% and 100%, respectively. This rate of MRD negativity allowed the authors to underline the efficacy in response rates and PFS of the new combination therapy based on carfilzomib-lenalidomide-dexamethasone-daratumumab  in patients with newly diagnosed MM. The primary end point was the MRD .Phase II/III clinical trials can explore treatment intensification or deintensification according to MRD status after treatment at different time points or during maintenance. MRD in the different studies is valuated with NGS  or NGF (threshold of 10-5) or high (MRD >10-5) risk. Standard-risk patients are randomized to six additional cycles of Isa-KRD or ASCT followed by two cycles of Isa-KRD, while high-risk patients are randomly assigned to ASCT followed by two cycles of Isa-KRD or tandem ASCT. Also, maintenance is informed by post-induction MRD status, as standard-risk patients receive lenalidomide for 3 years and high-risk patients receive iberdomide and isatuximab for 3 years  (NCT04934475) is an interventional and prospective phase III randomized trial. After an induction regimen consisting of isatuximab, carfilzomib, lenalidomide, and dexamethasone (Isa-KRD), MRD status is determined by NGS and used to classify patients as standard (MRD <10 3 years . Results-5) was used to inform therapy at two time points. First, Dara-KRd consolidation was interrupted in patients with two consecutive MRD-negative assessments. Second, patients who completed consolidation therapy and remained MRD-positive received lenalidomide maintenance. This study demonstrated how MRD response-adapted consolidation leads to higher rates of MRD negativity a higher PFS in NDMM, offering an alternative strategy to indefinite maintenance  is a multicenter, single-arm, phase II study in which NDMM patients received induction with daratumumab, carfilzomib, lenalidomide, and dexamethasone (Dara-KRd) followed by autologous transplant and Dara-KRd consolidation. MRD evaluation by ClonoSEQ NGS MRD <100-5 was uIn the recruiting phase 3 DRAMMATIC (NCT04071457) study, after induction and ASCT, patients are randomized between lenalidomide and lenalidomide with daratumumab/rHuPH20. After 2 years of maintenance, MRD is assessed to guide further therapy. MRD-positive patients will continue with the assigned treatment. MRD-negative patients will be further randomized to continue/discontinue the assigned treatment.-5, Euroflow NGF) post consolidation are randomized to receive second-line treatment at MRD reappearance (arm A) or at progressive disease as defined by the IMWG criteria (arm B) in order to evaluate whether treating MRD relapse after first-line treatment prolongs PFS and OS for myeloma patients vs. treating relapse after progressive disease.In the recruiting phase 2–3 study REMNANT (NCT04513639), newly diagnosed patients are treated with standard induction (RVD), ASCT (single or tandem), and consolidation. Patients who reach MRD negativity  will test MRD status after induction therapy with DRd combination : MRD-positive patients will receive consolidation therapy with DRVd , with the aim of testing if MRD-adapted consolidation will result in more patients achieving MRD negativity.In the phase 3 AURIGA (NCT03901963) study, patients who remain MRD-positive after frontline ASCT are randomized to daratumumab-lenalidomide or lenalidomide alone in order to evaluate the intensification of maintenance therapy with daratumumab. NGS is used to assess the conversion rate to MRD negativity during maintenance.A phase 2 recruiting study, DART4MM (NCT03992170), is now evaluating the effect of daratumumab administration in patients who test MRD-positive by NGF after any therapy . If patients will be still MRD-positive after 6 months of therapy, treatment will be continued up to 2 years. If MRD negativity will be reached, daratumumab will be suspended.All these trials are examples of how the spectrum of uses of MRD in the future will expand and become increasingly crucial in everyday choices.MRD detection is widely used in several hematological malignancies , both for risk assessment and therapeutic choices. In MM, the prognostic value of MRD has been amply demonstrated, but its role in therapeutic decision is still a matter of debate , 60–67. A crucial discussion regards the identification of optimal time points of MRD testing. Now, no standard rule exists, and the matter is further complicated by the possible additional value of repeated MRD measurement over time rather than a single determination at the first achievement of CR.Many studies are exploring the role of MRD testing in defining the timing of ASCT. In particular, the question is whether patients who achieve deep MRD-negative responses after induction therapy should harvest stem cells but delay ASCT until salvage therapy after disease progression , 55, 63.Another possible application of MRD is in the definition of the optimal length of maintenance therapy following ASCT. Current data suggest an advantage of continuous maintenance therapy –72, but Similar questions exist also for the non-transplant-eligible patients, where the positive impact of continuous treatment on OS is not well demonstrated , 73, 74.Similar to other hematological malignancies, MRD could also be used to guide retreatment. The current paradigm for relapsing patients is a relative delay of retreatment initiation until appearance of CRAB  features , 71. ThoIn this work, we summarize and comment on the status of MRD testing in MM. In particular, our review retraces the recent and ongoing clinical trials that demonstrate or explore the prognostic role of the different MRD testing techniques. We also summarize the current efforts to determine the power of MRD as a tool in dynamic risk-adapted therapy.We also test the potential applicability and usefulness of a new commercial NGS panel, the LymphoTrack-MiSeq NGS methodology, for the diagnosis and MRD detection in our court of MM patients. Our work highlights the advantages of NGS over flow cytometry for MRD testing. One potential perceived disadvantage of the NGS test is that it is performed in a central laboratory, and results require more time as compared with in-house tests. Our single-center pilot experience supports the potential applicability of a commercial NGS panel and the value of MRD determination by the LymphoTrack-MiSeq platform as a cost-effective, readily available, and sensitive workflow. Even if the limit of our analysis is the low number of patients and the sensitivity level of the method adopted (acceptable in 2018), our results are in line with all available studies that confirm that achieving MRD negativity is an important prognostic factor in MM.While the high efficacy of new treatment strategies requires highly sensitive tools to monitor disease status and inform clinical decisions, the role of MRD testing in MM is currently limited to clinical trials. With the increasing validation of its prognostic role, MRD testing will move from passive  to active  and will leave the clinical trial setting to be included in standard care. For these reasons, NGS standardization is of extreme importance for the clinical applicability of MRD detection. While NGS can have discrepant results because of interlaboratory differences, variable performance of the available technologies, and site-specific sample collection procedures, strict standardization will ensure consistent and comparable results. Ongoing efforts are aimed at reaching uniform timing of BM collection and sample processing and at defining the amount of DNA input, number of replicates, number of sequencing reads, and sequence similarity thresholds for determining clonotypes. With the aim of implementing MRD in MM everyday patients’ care, careful standardization, consistent sensitivity, reproducibility, and affordability of NGS-MRD assays will be fundamental , 75, 76.FV, EA, CT, TP, MM designed the study. collected study data. EA, SeM, CT performed laboratory analysis. FV, EA, TP, FF wrote the manuscript draft. MM, MTLP, FC edited the manuscript draft and did subsequent revision. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Postacute sequelae of SARS-CoV-2 infection, commonly known as long COVID, is estimated to affect 10% to 80% of COVID-19 survivors. We examined the prevalence and predictors of long COVID from a sample of 1,338 COVID-19 cases among university members in Washington, DC, USA, during July 2021‒March 2022. Cases were followed up after 30 days of the initial positive result with confidential electronic surveys including questions about long COVID. The prevalence of long COVID was 36%. Long COVID was more prevalent among those who had underlying conditions, who were not fully vaccinated, who were female, who were former/current smokers, who experienced acute COVID-19 symptoms, who reported higher symptom counts, who sought medical care, or who received antibody treatment. Understanding long COVID among university members is imperative to support persons who have ongoing symptoms and to strengthen existing services or make referrals to other services, such as mental health, exercise programs, or long-term health studies. Only antigen tests approved for emergency use under the Food and Drug Administration emergency use authorization were accepted , which is responsible for campus-related COVID-19 case management .During July 2021‒March 2022, all 4,800 positive COVID-19 test results reported during August 2020‒February 2022 were followed up with confidential electronic surveys sent to each patient at least 28 days after their initial positive result that included questions about long COVID. Those data were merged with the COVID-19 case investigation data.>2 COVID-19 diagnoses during August 2020‒February 2022; those persons were only included once in the long COVID follow-up data collection, resulting in a total of 4,657 persons who were COVID-19 positive during the study period. The follow-up survey had a response rate of 32% . We observed major differences in age, university affiliation, underlying conditions, and vaccination status at the time of test between follow-up survey respondents and nonrespondents  and dates of vaccinations and boosters as a condition of employment and access to campus. This information was used to determine vaccine status on the date of first positive COVID-19 test. The case investigation interviews also collected data about whether medical care was sought, hospitalizations, and administration of monoclonal antibodies.Over the course of the study period, COVID-19 vaccine availability and recommendations shifted dramatically. In December 2020, vaccines were first available but only for select groups such as healthcare workers, the elderly, and certain other susceptible populations. During March‒April 2021, vaccines were made available to all adults (>16 years of age) across all US states. In June 2021, George Washington University mandated all members of the campus community to be up to date .We described continuous variables by using medians and interquartile ranges (IQRs) and categorical variables by using frequencies and percentages. We compared characteristics of survey respondents by using χAll university community members provided informed consent to participate in the George Washington University COVID-19 surveillance program. The George Washington University Institutional Review Board concluded that these were non‒research-related activities.Overall, the median age of respondents was 23 (IQR 21–32) years, and the median symptom count was 4 (IQR 1–6) . More thMost respondents had no underlying conditions (75.2%), never smoked (83.0%), had acute COVID-19 symptoms (79.1%), did not seek medical care at the time of their first positive COVID-19 result (96.6%), and did not receive monoclonal antibody treatment (94.5%) . ApproxiNearly 36% of survey respondents reported experiencing symptoms of long COVID . RespondUnadjusted associations between characteristics of survey respondents and long COVID status showed that sex, race/ethnicity, underlying conditions, smoking status, vaccination status, any symptoms, symptom type, symptom count, seeking out medical care, and receiving antibody treatment were strongly associated with long COVID . Multiva––>1 long COVID symptom  for those who have long COVID that affects their ability to engage in university activities such as classes and work. In addition, universities might benefit from adopting preventive resources for their populations, as well as extended pandemic leave, given the considerable long-lasting effects of long COVID.Future research avenues should consider following up with long COVID survivors/patients to assess long-term or long-lasting symptoms. Such analysis could explore the consequences of long COVID for 5‒10 years after the initial infection, especially to gain a better understanding of its effect on young, healthy populations. Follow-up could also occur with older populations to assess whether symptoms progress into retirement age and to determine the cost of long-term care resulting from long COVID. Furthermore, research should continue to examine the effect vaccine booster doses have on long COVID symptoms. Such research is vital to clarifying long-term effects of long COVID and how universities can support those dealing with long COVID to promote health and wellness across campus communities.Additional information on postacute sequelae of SARS-CoV-2 in university setting.COVID-19 follow-up survey for postacute sequelae of SARS-CoV-2 in university setting.


Immunotherapy has become the central pillar of cancer therapy. Immune checkpoint inhibitors (ICIs), a major category of tumor immunotherapy, reactivate preexisting anticancer immunity. Initially, ICIs were approved only for advanced and metastatic cancers in the salvage setting after or concurrent with chemotherapy at a response rate of around 20–30% with a few exceptions. With significant progress over the decade, advances in immunotherapy have led to numerous clinical trials investigating ICIs as neoadjuvant and/or adjuvant therapies for resectable solid tumors. The promising results of these trials have led to the United States Food and Drug Administration (FDA) approvals of ICIs as neoadjuvant or adjuvant therapies for non-small cell lung cancer, melanoma, triple-negative breast cancer, and bladder cancer, and the list continues to grow. This therapy represents a paradigm shift in cancer treatment, as many early-stage cancer patients could be cured with the introduction of immunotherapy in the early stages of cancer. Therefore, this topic became one of the main themes at the 2021 China Cancer Immunotherapy Workshop co-organized by the Chinese American Hematologist and Oncologist Network, the China National Medical Products Administration and the Tsinghua University School of Medicine. This review article summarizes the current landscape of ICI-based immunotherapy, emphasizing the new clinical developments of ICIs as curative neoadjuvant and adjuvant therapies for early-stage disease. The past decade has witnessed the emergence of systemic and personalized therapies in cancer treatment. After the approval of the first immune checkpoint inhibitor (ICI), ipilimumab, targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4), initial investigations with ICIs were focused on salvage therapy for metastatic cancers. As a salvage therapy, ICIs have demonstrated objective responses that, compared with conventional chemotherapy, are often durable. Gradually, ICIs have heralded a new era in treating advanced and metastatic cancers, moving from the last resort to first-line therapy Fig. . HistoriThis review article summarizes the recent developments in cancer immunotherapy with ICIs, including those discussed at the 2021 China Cancer Immunotherapy Workshop. This annual workshop has been co-organized by the Chinese American Hematologist and Oncologist Network (CAHON), the China National Medical Products Administration (NMPA) and Tsinghua University since 2015 –9. ThrouThere are major developments for immunotherapy in both small cell lung cancer (SCLC) and NSCLC. In NSCLC, immunotherapy has been approved as neoadjuvant therapy and maintenance therapy for nonmetastatic disease, in addition to its use as a single agent or combination first-line therapy for metastatic NSCLC. At the 2021 China Cancer Immunotherapy Workshop, Patrick Forde, MD from Johns Hopkins University, presented a comprehensive review on the development of neoadjuvant immunotherapy in lung cancer.NSCLC has witnessed the paradigm shift of ICIs from salvage therapy for metastatic cancers to neoadjuvant/adjuvant therapy for early-stage cancers. For patients with advanced NSCLC, first-line systemic treatment generally consists of targeted therapy, immunotherapy, cytotoxic chemotherapy, or a chemo-immunotherapy combination depending on the tumor's expression of programmed death-ligand 1 (PD-L1), histology type (squamous versus nonsquamous) and the presence of driver mutations, such as epidermal growth factor receptor (EGFR) activation mutation and anaplastic lymphoma kinase (ALK) translocation. In case there is no specific driver mutation, ICIs are commonly used as single agents for those NSCLCs with PD-L1 expression of over 50% and with little cancer-related symptoms. So far, three ICIs have been approved by the United States (U.S.) FDA as single agents for the treatment of advanced NSCLC: two anti-PD1 (Programmed Cell Death Protein 1) antibodies, pembrolizumab based on the KEYNOTE-024 trial , 11 and Despite multimodality treatment, the outcome of unresectable stage III NSCLC remains poor, with a five-year survival rate of approximately only 15% . As ICIsThe paradigm change for NSCLC is to administer immunotherapy for resectable tumors with curative intent. The CheckMate 816 trial evaluated neoadjuvant therapy with nivolumab plus platinum-doublet chemotherapy in 358 patients newly diagnosed with resectable stage IB to IIIA NSCLC. No sensitizing EGFR or ALK mutations were allowed. Patients were stratified by cancer stage, PD-L1 status, and sex. Patients were randomly assigned 2:1 to receive nivolumab at 360 mg every three weeks plus chemotherapy for three cycles vs. the same chemotherapy schedule. Then, patients underwent radiological staging and surgery within six weeks of neoadjuvant therapy. They had the option of adjuvant therapy with or without radiation therapy. An exploratory arm of nivolumab plus ipilimumab was closed early. The primary endpoint was pathological complete response (pCR) by blinded independent review, defined as no viable residual tumor in the resected primary tumor and lymph nodes after surgery. Neoadjuvant nivolumab plus platinum-doublet chemotherapy significantly improved pCR rates compared with chemotherapy alone . The magnitude of pCR benefit with nivolumab was similar between patients with stage IB and IIIA disease with squamous and nonsquamous histologies, regardless of PD-L1 status and tumor mutational burden. The major pathological response rate among patients who underwent surgery was 46.8% in the nivolumab-containing arm vs. 12.7% in the chemotherapy alone arm. The radiographic objective response rate based on scans was 54% with nivolumab plus chemotherapy vs. 37% with chemotherapy alone. At a minimum follow-up of 21 months, the coprimary endpoint of median event-free survival (EFS) was 31.6 months for the nivolumab plus chemotherapy group and 20.8 months for the chemotherapy alone group. This corresponded to a significant reduction in the risk for disease progression, recurrence, or death of 37% in favor of nivolumab plus chemotherapy .More recently, the Phase II NEOpredict-Lung trial was presented at the European Society for Medical Oncology (ESMO) Congress 2022. In this randomized, multicentric Phase II study, 60 patients with NSCLC stage IB to IIIA were randomized to 4 weeks of anti-PD1 antibody nivolumab (arm A) or nivolumab plus anti-LAG-3 (Lymphocyte activation gene-3) antibody relatlimab (arm B) before curative surgery. R0 resection was performed in 98% of patients. Radiological response rates were 11% (arm A) and 27% (arm B) per RECIST and complete or major histopathological response rates were 28% (arm A) and 32% (arm B), suggesting further clinical trials of this combination are warranted [IMpower010 was a phase III study evaluating adjuvant atezolizumab  or best supportive care (observation and regular scans for disease recurrence) after adjuvant platinum-based chemotherapy (one to four cycles) in resected stage IB–IIIA NSCLC. After a median follow-up of 32.2 months, atezolizumab treatment improved disease-free survival compared with best supportive care in 495 patients in the stage II–IIIA population whose tumors expressed PD-L1 on  ≥ 1% of tumor cells  and in all patients in the stage II–IIIA population . In the intention-to-treat population, the HR for disease-free survival was 0.81 . IMpower010 showed a disease-free survival benefit with atezolizumab versus best supportive care after adjuvant chemotherapy in patients with resected stage II–IIIA NSCLC, with pronounced benefit in the subgroup whose tumors expressed PD-L1 on 1% or more of the tumor cells and no new safety issues. Based on this trial, the US FDA approved one year of atezolizumab therapy after adjuvant chemotherapy for stage II-III NSCLC with PD-L1 > 1% .As both neoadjuvant and adjuvant ICIs are available for resectable stage II-IIIA NSCLC, further study is needed to compare these two approaches to determine which yields better clinical benefit. Ongoing phase III studies will determine the role of adjuvant ICIs in patients who received neoadjuvant ICI-chemotherapy Table .Table 1OSCLC is a poorly differentiated neuroendocrine tumor, representing approximately 15% of lung cancers. Even though the Tumor, Node, Metastasis (TNM) staging classification is recommended, patients are often divided into limited-stage (LS) versus extensive-stage (ES) disease. LS-SCLC is limited to the ipsilateral hemithorax and regional lymph nodes and can be confined to one safe radiotherapy field. ES-SCLC has spread beyond this and often has distant metastases.For LS-SCLC, surgery for stage I and radiation for stages II and III, supplemented with systemic chemotherapy, are preferred. Prophylactic cranial irradiation (PCI) following chemotherapy is the standard of care for patients who achieve a complete or good partial response following treatment. It has been shown to increase OS in patients with LS-SCLC. No ICI, however, has been approved for LS-SCLC to date.For ES-SCLC without symptomatic brain metastasis, the first-line therapy is platinum-etoposide chemotherapy paired with an anti-PD-L1 antibody, followed by maintenance immunotherapy with an ICI until progression. Two humanized monoclonal anti- PD-L1 antibodies, atezolizumab and durvalumab, have been approved for the treatment of ES-SCLC in combination with etoposide-platinum for induction and maintenance therapy based on the Impower133 and CASPIAN trials, respectively , 30. EveTreatment options and recommendations are very personalized in breast cancer and depend on several factors. The decision for upfront therapy is not only determined by the stage, age and menopausal status, but is also guided by the genomic markers and expression status of hormonal receptor and human epidermal growth receptor 2 (HER2). Currently, ICIs have been approved for triple-negative breast cancer (TNBC). Before starting treatment for TNBC, genomic testing for germline breast cancer genes (BRCA) and PD-L1 expression should be conducted. For symptomatic or rapidly progressive TNBC, patients should be treated with combination therapy to achieve a fast and higher response rate. For TNBC in the absence of symptoms or rapidly progressive disease, sequential single-agent chemotherapy is preferable, and the addition of an ICI is preferable in cases of positive PD-L1 expression. To date, the combination of pembrolizumab and chemotherapy has gained FDA approval for patients with locally recurrent, inoperable or metastatic TNBC with a PD-L1 combined positive score (CPS)  ≥ 10 based on the KEYNOTE-355 trial .In order to improve the cure rate of early-stage TNBC, several clinical trials have been conducted to determine the efficacy of the addition of an ICI to neoadjuvant chemotherapy in TNBC –34. OnlyAmong the major genitourinary malignancies, prostate and testicular cancers have few mutations and are usually considered minimally immunogenic and less responsive to immunotherapy with ICIs. At the 2021 China Cancer Immunotherapy Workshop, Chong-xian Pan, MD, PhD, MS, from Harvard Medical School reviewed the recent advances in immunotherapy for bladder and kidney cancers.Bladder cancer has a high tumor mutation burden and, hence, is considered to be more immunogenic. From 2016 to 2017, five ICIs were approved for the treatment of advanced bladder cancer for carboplatin-ineligible patients or as salvage therapy after disease progression within 12 months of platinum-containing chemotherapy –43, evenThe standard of care for NMIBC in high-risk patients is transurethral resection of bladder tumor (TURBT) followed by intravesical instillation of a therapeutic agent, usually Bacillus Calmette-Guerin (BCG). This treatment, however, is associated with a 10-year cancer recurrence of 74% and cancer progression can be seen in up to one-third of patients . Upon diFor locally advanced bladder cancer, neoadjuvant cisplatin-based chemotherapy followed by radical cystectomy, with its associated urinary diversion, is the standard of care , 47. ForMany neoadjuvant immunotherapy-based clinical trials are currently underway for this disease group, either as a single agent, such as atezolizumab in the ABACUS trial  and pembKidney cancer is another highly immunogenic genitourinary cancer. No other cancer has more first-line immunotherapy combinations approved than kidney tumor. Historically, before ICIs, interleukin-2 (IL-2) and interferon were used to treat renal cancer and melanoma. IL-2 is highly toxic with most patients requiring critical care, and an objective response is observed in approximately 15% of patients, with some achieving long-term remission . The resFor localized kidney cancer, definitive surgery resection is curative and, therefore, the preferred treatment for patients with stage I and II disease (limited to the kidney), stage III disease  and even in patients with limited metastasis. No neoadjuvant therapy is currently indicated.The U.S. FDA has approved adjuvant therapy with pembrolizumab for patients at intermediate-high to high risk of cancer recurrence following nephrectomy, or after nephrectomy and resection of oligometastatic lesions. This was based on the double-blinded placebo-controlled phase III KEYNOTE-564 trial. Pembrolizumab improved the 24-month DFS compared with placebo in the entire study population . The estimated 24-month OS of 96.6% versus 93.5% of the placebo  also showed statistical significance, but long-term follow-up is in progress . All patEven though adjuvant pembrolizumab therapy improves clinical outcomes, two other adjuvant trials failed to show clinical benefit. In the IMmotion010 trial with atezolizumab, 778 patients were randomized to one year of adjuvant atezolizumab or placebo treatment after nephrectomy. At a median follow-up of 44.7 months, no significant improvement of investigator-assessed disease-free survival was observed: 57.2 months for atezolizumab versus 49.5 months in the placebo group  . SimilarThe first ICI therapy approved by the FDA for cancer treatment is ipilimumab for advanced melanoma in 2011, which opened a new chapter of cancer therapy. During the early era of immunotherapy with ICIs, several phase III clinical trials showed that the single agent ipilimumab significantly improves OS , 69. HowOf the ICI-based regimens approved for metastatic and recurrent melanoma, the combination therapy with nivolumab and ipilimumab , 73 is pBecause of the high success of ICI in advanced melanoma, tremendous interest has been shifted to early-stage melanoma in adjuvant and neoadjuvant settings. Long before ICI was studied, immunotherapy with interferon was extensively studied both in advanced and localized melanoma. However, secondary to the efficacy and safety profiles, drug availability, and other factors, most clinicians recommend single-agent immunotherapy with nivolumab or pembrolizumab. Nevertheless, it is anticipated that combination immunotherapy will be examined in the neoadjuvant setting for high-risk localized melanoma.Interferon alfa-2b now mainly remains a historical adjuvant therapy after more effective and less toxic ICIs have been approved. Based on the Eastern Cooperative Oncology Group 1684 and 1694 trials as well as meta-analysis of trials with various doses and schedules, interferon alfa-2b improves OS compared to the control –80. HoweIn the CheckMate 238 trial, 906 patients with stage IIIB, IIIC or IV melanoma who had complete resection of melanoma were randomized to receive one year of adjuvant nivolumab or ipilimumab (10 mg/kg every three weeks for four cycles followed by every 12 weeks for one year). At a median follow-up of 51 months, the 4-year recurrence-free survival was 51.7% with nivolumab compared with 41.2% with ipilimumab  , 83. FivIn the double-blind, placebo-controlled phase III European Organization for Research and Treatment of Cancer (EORTC) 1325/KEYNOTE-054 trial, 1019 patients with completely resected stage III melanoma were randomized to one year of adjuvant pembrolizumab or placebo –86. WithThe phase III EORTC18071 trial that led to the U.S. FDA approval of ipilimumab as an adjuvant therapy at 10 mg/kg every three weeks for four doses followed by every three months for up to three years. In that trial, 951 patients were randomized to ipilimumab or placebo. With a median follow-up of 5.3 years, the 5-year recurrence-free survival was 40.8% with ipilimumab compared with 30.3% with placebo  , 88. In The benefit of ICIs has been further extended to high-risk resected melanoma without lymph node involvement. This group of patients mainly have melanoma with deep local disease (> 4 mm) or with relatively superficial disease (2–4 mm) with ulceration but without lymph node metastasis. In this scenario, adjuvant pembrolizumab is commonly recommended based on the KEYNOTE-716 trial, although surveillance and enrollment in a clinical trial are reasonable alternatives. In this double-blinded, placebo-controlled phase III trial, 976 patients with stage IIB and IIC melanoma were randomized to pembrolizumab every three weeks for 17 cycles or a placebo. PFS was the primary endpoint. At a median follow-up of 21 months, pembrolizumab therapy significantly prolonged PFS, with an 18-month PFS of 86% versus 77% in the placebo group  .Built on the success of adjuvant immunotherapy in melanoma, neoadjuvant ICI-based therapy is actively being explored. In the randomized Phase II SWOG 1801 trial, patients with Stage IIIB-IV cutaneous, acral or mucosal melanoma were randomized to surgery followed by pembrolizumab adjuvant therapy for 18 cycles  or neoadjuvant pembrolizumab for three cycles, followed by surgery and 15 cycles of adjuvant pembrolizumab (neoadjuvant arm). Patients in the neoadjuvant arm had significant improvement in event-free survival, the study primary endpoint  . In addiGastric cancer, including gastroesophageal junction (GEJ) cancer, is the fourth leading cause of cancer-related deaths worldwide. Outcomes remain poor with standard-of-care fluoropyrimidine and platinum-based chemotherapy in unresectable diseases , 97. CurCurrently, the role of ICI as an adjuvant therapy has been substantiated for squamous cell esophageal cancer. Preoperative chemoradiation has been a standard of care for this patient population. Complete pathological response to preoperative chemoradiation is a well-established prognostic marker for superior outcomes following surgical resection, whereas residual diseases are associated with worse outcomes. Nivolumab demonstrated its significant clinical benefit in the adjuvant setting in the global randomized double-blind placebo-controlled phase III CheckMate 577 study of patients with stage II/III disease following definitive treatment with chemoradiation and surgical resection with residual disease . In thisThese encouraging results from the utilization of immunotherapy as the first-line therapy for adenocarcinoma of the stomach, esophagus, and GEJ have led to the development of immunotherapy as neoadjuvant therapy for this patient population. Approximately 30% of this patient population has resectable adenocarcinoma at the time of diagnosis; the five-year survival is estimated at 45% following local definitive therapy if it is diagnosed at an advanced clinical stage . The current standard of care for this locally advanced resectable gastric cancer is the perioperative chemotherapy combination of fluorouracil /leucovorin, oxaliplatin, and docetaxel, which offers a median overall survival of 50 months . SeveralColorectal cancer is the third leading cause of cancer death in both men and women in the U.S. and most parts of the world . CurrentIn addition, there is significant interest in testing ICIs as neoadjuvant therapy in combination with either radiation alone or with chemoradiation for MSI-H rectal cancer. Such efforts may lead to a paradigm change as patients with a complete clinical response to neoadjuvant therapy have been selected for watchful waiting without surgical resection. If immunotherapy or a combination of immunotherapy with chemoradiation leads to a higher complete response rate in MSI-H rectal cancer, more patients would be spared from morbid surgical resection. Strikingly, a recent study of 14 patients with locally advanced MSI-H rectal cancer reported that all 14 patients had a complete response to a six-month course of neoadjuvant anti-PD-1 antibody treatment, sparing them not only from surgical resection but also chemotherapy and radiotherapy .However, ICI for the majority of CRC patients without MSI-H/dMMR does not seem to be beneficial .Hepatocellular carcinoma (HCC) is considered relatively chemorefractory, and conventional cytotoxic chemotherapy has become less popular in treating HCC. Since 2008, targeted therapy and immunotherapy have gained increasing popularity, but with limited efficacy. ICI-based combination therapies are now the first-line treatment for locally advanced and metastatic HCC , and tyrFor advanced HCC with a Child‒Pugh class A on the liver function scale, the combination of atezolizumab and bevacizumab was approved by the U.S. FDA, based on the Phase III IMBrave 150 trial . At a meFor localized HCC with sufficient liver function, surgical resection with curative intent is commonly performed, and no other neoadjuvant or adjuvant therapies are widely accepted. Adjuvant antiviral therapy improves treatment outcomes for hepatitis B-related HCC and is recommended for those patients with an active viral infection. Chemotherapy has failed to demonstrate a significant role as adjuvant therapy for localized HCC. Targeted therapy primarily has a cytostatic antitumor effect and is hence unlikely to be an effective adjuvant therapy. The long-lasting effect of ICI has been observed in clinical trials, thus making ICI an ideal adjuvant therapy in HCC. At the 2021 China Cancer Immunotherapy Workshop, Richard Finn, MD, from the University of California, Los Angeles, provided updates on the recent developments in immunotherapy for HCC. The phase III KEYNOTE-937 (NCT03867084) clinical trial randomized 950 patients to compare adjuvant pembrolizumab with placebo in Child‒Pugh A HCC with a complete radiographic response after surgery or local ablation. Recurrence-free survival (RFS) and OS were the primary endpoints . A few oHNSCC includes cancers of the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx. For early-stage, localized and locoregionally advanced HNSCC, the treatment intent is curative. For locally advanced HNSCC, combined therapeutic modalities are commonly adopted to achieve better control, improved function preservation, superior long-term survival and can be curative in a small percentage of patients. Depending on the tumor size and location, the patient’s general health and comorbidities, and local expertise, patients can undergo surgery followed by postoperative radiation with or without chemotherapy, induction chemotherapy followed by surgery or radiation, or sequential or concurrent chemoradiotherapy without surgery. Surgery is the main therapy for oral cavity cancer supplemented with radiation with or without chemotherapy. For large cancers at other locations, function-preserving approaches, mainly radiation-based combined therapeutic modalities, are adopted.Curative treatments, including surgery and chemoradiation, can result in high morbidity. ICIs are tested in the neoadjuvant setting in an attempt to downstage the disease for a less morbid surgical resection. Multiple phase II neoadjuvant studies have been completed, and most trials have demonstrated a better pathological treatment response (pTR) with ICI treatment as well as the association of pTR with better survival outcomes following surgery. Uppaluri et al. (2020), reported a single-arm study of 36 patients with locally advanced, resectable HNSCC, most of whom had oral cavity cancers . There wImmunotherapy has been approved for several gynecological malignancies, a highly diversified disease group, mostly for recurrent or advanced disease. Endometrial carcinoma is the second most common malignancy associated with MSI-H or dMMR after colorectal cancer. The anti-PD-1 antibody dostarlimab is now approved by the U.S. FDA as a second-line therapy, following platinum-based chemotherapy, for patients with dMMR/MSI-H endometrial cancers .For recurrent and metastatic cervical cancer, the activity of immunotherapy was demonstrated in the Phase III KEYNOTE-826 trial. Compared with chemotherapy with or without bevacizumab, the addition of pembrolizumab as the first-line treatment significantly improved PFS from 8.2 months to 10.4 months  and 2-year OS from 41.7% to 53.0%  . Based oDirect evidence supporting the role of ICIs for treating cervical and endometrial cancers in the adjuvant or neoadjuvant setting is still lacking, but there is a strong rationale for testing ICIs in clinical trials. Currently, four phase II clinical trials of neoadjuvant therapy with an anti-PD-1 antibody in combination with paclitaxel and platinum-based chemotherapy for locally advanced cervical cancer , one clinical trial of adjuvant therapy with anti-PD-1 antibody sintilimab in combination with the standard of care chemoradiation for resected cervical cancer (NCT04918628), and one clinical trial of adjuvant therapy with pembrolizumab plus chemotherapy with or without radiation for resected endometrial cancer (NCT04634877) are ongoing.Ovarian cancer is relatively sensitive to chemotherapy and resists to ICI treatment. The role of immunotherapy in metastatic ovarian cancer still needs to be determined.Immunotherapy with ICIs has revolutionized the treatment of various cancers, initially for more advanced disease and, more recently, for early-stage tumors. With numerous ongoing clinical trials, we expect to see more immunotherapy approvals for early-stage cancers. Similar to those with advanced cancer, only a minority of patients benefit from ICIs as a monotherapy. One approach to counteract this predicament is the biomarker-based selection of patients who are more likely to respond to ICIs, as seen in lung cancer with high PD-L1 expression, cancers with mismatch repair deficiency or cases with a high tumor mutation burden , 120. Ho