This meta-epidemiological study aimed to assess methodological quality of a sample of contemporary non-randomised clinical studies of clinical interventions.n = 2); minimisation of bias in study design and data collection (n = 11); use of appropriate methods to create comparable groups (n = 6); appropriate adjustment of observed effects (n = 5); validation of observed effects (n = 9); and authors interpretations (n = 2).This was a cross-sectional study of observational studies published between January 1, 2012 and December 31, 2018. Studies were identified in PubMed using search terms ‘association’, ‘observational,’ ‘non-randomised’ ‘comparative effectiveness’ within titles or abstracts. Each study was appraised against 35 quality criteria by two authors independently, with each criterion rated fully, partially or not satisfied. These quality criteria were grouped into 6 categories: justification for observational design  were published in two US general medical journals. No study fully satisfied all applicable criteria; the mean (+/−SD) proportion of applicable criteria fully satisfied across all studies was 72% (+/− 10%). The categories of quality criteria demonstrating the lowest proportions of fully satisfied criteria were measures used to adjust observed effects  and validate observed effects . Criteria associated with ≤50% of full satisfaction across studies, where applicable, comprised: imputation methods to account for missing data (50%); justification for not performing an RCT (42%); interaction analyses in identifying independent prognostic factors potentially influencing intervention effects (42%); use of statistical correction to minimise type 1 error in multiple outcome analyses (33%); clinically significant effect sizes (30%); residual bias analyses for unmeasured or unknown confounders (14%); and falsification tests for residual confounding (8%). The proportions of fully satisfied criteria did not change over time.Recently published observational studies fail to fully satisfy more than one in four quality criteria. Criteria that were not or only partially satisfied were identified which serve as remediable targets for researchers and journal editors.The online version contains supplementary material available at 10.1186/s12874-022-01797-1. The growth of electronic medical records and other ‘real-world’ digitised sources of clinical data has led to a proliferation of observational studies of the effectiveness of clinical interventions. While the scientific standard for assessing intervention efficacy remains randomised controlled trials (RCTs), well-designed observational studies have been used to elucidate potential harms, and expand the evidence base in situations where existing RCTs have limited generalisability because of selective patient enrolment or outcome reporting, or new RCTs are logistically very difficult to perform . The maiNevertheless, reviews of observational studies suggest that they often report effects and generate inferences similar to those of RCTs studying the same therapy and involving similar populations and outcome measures –7. AdvanHowever, the level of trustworthiness of observational studies remains controversial. We hypothesised that, due to advances in observational research, such studies are becoming more rigorous and valid. The aim of this meta-epidemiological study was to assess the methodological quality of a sample of recently reported non-randomised clinical studies of commonly used clinical interventions, and ascertain if quality is improving over time.In reporting this study, we applied the guidelines for meta-epidemiological methodology research proposed by Murad and Young . No a prA backward search from December 31, 2018 to January 1, 2012 was performed using PubMed with no language filters to identify observational studies of therapeutic interventions. Search terms comprised ‘association’, ‘observational,’ or ‘non-randomised’ or ‘comparative effectiveness’ within titles or abstracts. We included studies which: involved clinician-mediated therapeutic interventions administered directly to adult patients; reported comparison of two concurrent therapeutic interventions which could include ‘usual care’; and whose outcomes included patient-important sentinel events  rather than solely disease or symptom control measures.We excluded studies that: 1) featured case control comparisons, historical controls only, single arm cohorts, adjunct therapies, diagnostic tests (with no associated therapeutic intervention) or cost-effectiveness analyses (with no separate comparative effectiveness data); 2) compared a single intervention group with a placebo group; 3) comprised RCTs, or reviews and meta-analyses of either RCTs or observational studies; 4) involved paediatric, anaesthetic or psychiatric interventions or patients; 5) analysed therapies which were highly specialised, or not in common use ; 6) assessed effects of system-related innovations rather than direct clinical care (eg funding or governance structures); 7) studied non-medical interventions ; 8) studied exposures, risk factors or prognostic factors that may influence therapeutic effectiveness but did not involve head to head comparisons of two interventions (eg effects of dose changes or co-interventions); or 9) were descriptive studies with no reporting of outcome measures. One author (SG) performed the search and initial study selection, with subsequent independent review by the second author (IAS).From each selected study we extracted the following data: study title, journal, and date of publication; rationale stated in the introduction for choosing an observational study design; existence of a pre-specified study protocol; patient selection criteria; methods of data collection from primary sources; reference to validation checks for coded administrative data or longitudinal data linkage processes; methods for minimising recording bias (in administrative data), recall bias, social desirability bias, and surveillance bias ; methods for assessing clinical outcomes; choice of predictor variables; population characteristics and statistical methods used for balancing populations; imputation methods used for missing data; subgroup analyses and interaction testing for identifying independent prognostic variables; use of unplanned post-hoc analyses; statistical methods used for adjusting for multiple outcome analyses, clustering effects (in multicentre studies) and time-dependent bias; effect size and confidence intervals; sensitivity analyses for unmeasured confounders; stated intervention mechanism of action, temporal relation between intervention and outcomes, and dose-response relationships; any falsification tests performed; comparisons with results of other similar studies; and statements about study limitations and implications for clinical practice.n = 2); minimisation of bias in study design and data collection (n = 11); use of appropriate methods to create comparable groups (n = 6); appropriate adjustment of observed effects (n = 5); validation of observed effects (n = 9); and authors interpretations (n = 2).Both authors independently read the full text articles of included studies and applied to each study a list of 35 quality criteria which were, with some modification, based on those the authors have previously published Table  17] and and17] aFor each study, the extent to which each criterion was satisfied were categorised as fully satisfied (Y) – all elements met; partially satisfied (P) – some elements met; or not satisfied (N) – no elements met; or not applicable (NA) if that criterion was not relevant to the study design, analytic methods or outcome measures. Inter-rater agreement between authors for criterion categorisation was initially 95.2% and consensus was reached on all criteria after discussion.For each study, we calculated the proportion of all applicable quality criteria categorised as fully, partially or not satisfied. For each criterion applicable at the level of individual studies, we calculated the proportion of studies which fell into each category of satisfaction. We calculated the proportion of criteria which were fully, partially or not satisfied by all studies for which criteria were applicable. Trend analysis assessed whether the proportion of applicable criteria that were fully, partially or not satisfied changed over time for studies published between 2012 and 2018. All analyses were performed using Excel functions or Graph Pad software..The literature search identified 1076 articles from which 50 unique studies met selection criteria –67 of wh2The proportions of applicable criteria which were fully, partially or not satisfied for each study are depicted in Fig. At the level of individual studies, the proportions of all criteria fully or partially satisfied ranged between 60.7% 17 of 28 criteria) and 96.6% (28 of 29 criteria) and the proportions of all criteria that were not satisfied ranged from 3.4% (1 of 29 criteria) to 39.3% (11 of 28 criteria). Only two studies had more than 80% of applicable criteria fully satisfied  was not applicable to any study as informal patient self-report was not used as a data source.At the level of individual criteria, the proportions of studies in which a specific criterion was fully, partially and not satisfied, or was not applicable, are depicted in Fig. 2Across all studies, criteria associated with high levels (≥80%) of full satisfaction (where applicable) comprised: appropriate statistical methods (most commonly propensity-based methods) used to ensure balanced groups (100%); absence of social desirability bias as all studies either used validated, externally administered questionnaires or did not rely on patient self-reported symptoms or function as their primary end-points (100%); coherence of results with other studies of similar interventions (100%); temporal cause-effect relationships (98%); prospective, validated data collection (97%); plausibility of results (96%); absence of surveillance bias in clinical registry data (95%); formulation of pre-specified study protocol (94%); consistency of results to similar studies of same interventions (88%); clear statements on how prognostic variables were selected and measured (86%); data from the majority of the population sample being used in analyses (86%); absence of overstatement of study conclusions (84%); independent blind assessment of outcomes (84%); and adequate matching of patient populations being compared (80%).Criteria associated with intermediate (51 to 79%) levels of full satisfaction comprised: absence of recording bias in administrative datasets (79%); presence of dose-response relationships (75%); absence of unplanned post-hoc analyses (76%); statistical exclusion of potentially beneficial effect in studies with conclusions of no effect or harm (76%); adequate accounting for selection bias in patient recruitment (74%); and representativeness of the study population (74%).Criteria associated with low (≤50%) levels of full satisfaction comprised: imputation or other processes to account for missing data or drop-outs (50%); justification for not performing an RCT (42%); interaction analyses in identifying independent prognostic factors that may have influenced intervention effects (42%); use of statistical correction methods to minimise type 1 error arising from multiple analyses of several different outcome measures (33%); clinically significant effect sizes (30%); residual bias analyses that accounted for unmeasured or unknown confounders (14%); and falsification tests for residual confounding (8%).The proportions of all applicable criteria that were fully, partially or not satisfied showed no appreciable change over time Fig. .Fig. 3TrTo our knowledge, this is one of only a few studies to apply a comprehensive list of criteria for assessing the methodological rigour of a cohort of contemporary observational studies of commonly used therapeutic interventions in adult patients reported in high-impact general medicine journals. Overall, there was a high level of adherence to criteria related to study protocol pre-specification, sufficiently sized and representative population samples, prospective collection of validated and objective data with minimisation of various forms of ascertainment and measurement bias, appropriate statistical methods, avoidance of post-hoc analyses, testing for causality, and impartial interpretation of overall study results. These criteria are central to most critical appraisal guides and reporting guidelines for observational studies, are well known to researchers, and hence will likely attract a high level of adherence.However, there is room for improvement. On average, each study failed to satisfy at least one in four quality criteria which were applicable to that study. The most frequent omission was failing to conduct a falsification (or specificity of effect) test for studies which reported intervention benefits. This test demonstrates whether a benefit is seen for outcomes that can be plausibly attributed to the intervention , but no change for a separate outcome most unlikely to be affected by the intervention , whereas if a benefit is seen for both outcomes, then the intervention is probably not the causative factor but some other confounding factor that affects both outcomes . Second A further concern is that despite the promulgation of reporting guidelines for non-randomised studies and the development of statistical methods for gauging the level of sensitivity of results to residual bias, our trend analysis indicates little improvement in methodological quality of studies published between 2012 and 2018. Overall, deficits in statistical analytic methods featured more prominently than deficits in study design and conduct. In particular, the absence of falsification tests, E-value quantification, subgroup analyses using tests for interaction, and adjustment for missing data and multiple comparisons limited the ability of many studies to account for residual confounding in their results.Our study has several limitations. First, despite excellent agreement between authors in categorising levels of criterion satisfaction, this task involves subjective and potentially biased judgement. However, this problem is common to most quality assessment tools . Second,Contemporary observational studies published in two high impact journals show limitations that warrant remedial attention from researchers, journal editors and peer reviewers. Reporting guidelines for such studies should promulgate the need for falsification testing, quantification of E values, effects sizes that denote less vulnerability to residual confounding, appropriate statistical adjustment for multiple outcome analyses, statistical interaction tests for identifying important predictors of intervention effects, complete patient follow-up, and justification for choosing to undertake an observational study rather than an RCT.Additional file 1. Study characteristics.


This cohort study evaluates stroke and major bleeding rates before and after switching from warfarin to a direct oral anticoagulant (DOAC) in patients grouped by pre-switch time-in-therapeutic range guideline thresholds. Medical societies have provided some guidance when considering switching to a DOAC based on time in therapeutic range (TTR): the European Society of Cardiology (TTR <70%), the American College of Chest Physicians (TTR <65%), and the American College of Cardiology (TTR <58%).3 Our objective was to use the Michigan Anticoagulation Quality Improvement Initiative clinical registry to evaluate stroke and major bleeding rates before and after switching from warfarin to a DOAC in patients grouped by pre-switch TTR guideline thresholds.Direct oral anticoagulants (DOACs), including apixaban, dabigatran, edoxaban, and rivaroxaban, are recommended instead of warfarin in patients newly diagnosed with nonvalvular atrial fibrillation (NVAF).4 Funded abstractors at 6 participating anticoagulation clinics with institutional review board approval enter patient data into the registry. Our study cohort included patients initiated on warfarin for NVAF who were later switched to a DOAC between 2010 and 2021. Patients were excluded if NVAF was not the only indication for anticoagulation or if they did not have at least 6 months of follow-up after switching to a DOAC. The outcomes assessed were ischemic stroke and major bleeding based on International Society of Thrombosis and Hemostasis criteria.5 This report follows the STROBE reporting guideline. This study received a waiver of participant consent from the institutional review board because of minimal risk to participants.The registry is part of a Blue Cross Blue Shield of Michigan–sponsored quality improvement collaborative.6 and patients were grouped based on guideline TTR thresholds. Each group’s outcome rates (number per 100 patient-years) while taking warfarin were compared with rates after switching to a DOAC. Rates of major bleeding were adjusted by a modified HAS-BLED  score (as continuous variable-removing the labile INR) while rates of ischemic stroke were adjusted by CHA2DS2-VASc score (as continuous variable) and compared using a mixed model with patients as random effect.5TTR was calculated using the Rosendaal method,2 test or Fisher exact test. Two-sided P < .05 was considered significant for all comparisons. All statistical analyses were performed using SAS version 9.4 (SAS Institute) and R version 3.6.3 .Categorical variables were assessed using χ2, multiply by 0.0167])  meeting inclusion criteria switched to a DOAC. At the time of switch, the mean (SD) age was 73.3 (10.7) years, 231 (44.1%) were female, and 168 (35.2%) had at least moderate chronic kidney disease (creatinine clearance < 60 mL/min [to convert to mL/s/m0.0167]) . ApixabaIn all but the group with TTR less than 65%, the rate of major bleeding was significantly higher after switching . The groIn this cohort study, patients had similar or higher rates of major bleeds and similar rates of ischemic stroke after switching to a DOAC, regardless of TTR threshold. This suggests that commonly cited TTR thresholds may not predict patients likely to benefit from a switch to DOAC therapy. The higher rates of bleeding on a DOAC is contrary to trial findings, possibly due to our patients being higher risk and having a different response to treatment. Limitations of this study include the potential for unmeasured confounding and attrition bias. Further research is needed to identify the best criteria for identifying NVAF candidates to switch from warfarin to a DOAC.


Human papillomavirus associated oropharyngeal squamous cell carcinoma (HPVOPSCC) usually affects a younger patient population. As such, the risk for long term toxicity associated with therapy is an important consideration. Multiple trials focused on de-escalation of therapy to preserve survival outcomes while minimizing treatment toxicity are currently in progress, however the question of which patients are ideal candidates for de-escalation remains unanswered. Circulating tumor DNA (cfHPVDNA) has emerged as a means of monitoring disease in patients with HPVOPSCC. Undetectable postoperative cfHPVDNA levels portend a better prognosis and by extension, may identify ideal candidates for de-escalation therapy. We propose an overview and rationale for a new institutional clinical trial protocol focusing on the use of cfHPVDNA to risk stratify patients for adjuvant therapy. We hypothesize that many surgical patients currently receiving radiation therapy may be clinically observed without adjuvant therapy.Patients with measurable cfHPVDNA and clinically resectable HPVOPSCC will undergo TORS resection of tumors and neck dissection. Patients with undetectable cfHPVDNA at 3 weeks post-op will be allocated to low or high-risk treatment protocol groups. The low risk group consists of patients with <4 positive lymph nodes, ≤2 mm extranodal extension (ENE), and perineural invasion (PNI) or lymphovascular invasion (LVI) alone. The high-risk group is made up of patients with ≥4 positive lymph nodes, gross ENE, positive margins, N2c disease and/or the combination of both PNI and LVI. The low-risk group will be allocated to an observation arm, while the high-risk group will receive 46 Gy of adjuvant radiotherapy and weekly cisplatin therapy. The primary outcome of interest is 2-year disease recurrence with secondary outcomes of 2-year disease free survival, locoregional control, overall survival, and quality of life measures. A sample of 126 patients in the low-risk group and 73 patients in the high-risk group will be required to evaluate non-inferiority to the standard of care.This study will provide much needed recurrence and survival data for patients that undergo primary TORS followed by observation or de-escalated adjuvant therapy. Additionally, it will help delineate the role of cfHPVDNA in the risk stratification of patients that undergo treatment de-intensification. HPV-related oropharyngeal squamous cell carcinoma (HPVOPSCC) now accounts for over 80% of oropharyngeal carcinoma seen in the United States and an increasing fraction of these malignancies in Europe –3. The dGiven the excellent prognosis of HPV-related oropharyngeal cancers, emphasis has shifted to reducing the morbidity associated with therapy while maintaining survival. Many of the functional limitations incurred by CRT are related to long term toxicities. One retrospective analysis of three RTOG trials suggested that the rate of severe late toxicities in patients receiving CRT is 35% for patients with oropharyngeal cancer . AnotherProponents of transoral robotic surgery (TORS) for oropharynx cancer contend that it offers improved functional outcomes when compared to non-surgical treatment with radiation therapy with or without chemotherapy. Multiple studies report favorable swallowing outcomes using TORS for resection of oropharyngeal cancers –18. AddiFinally, the retrospective oncologic outcomes from TORS surgery for oropharyngeal cancer have demonstrated favorable results. Several studies have reported local failure rates which varied between 0-3% with median follow-up rates ranging from 18 months to two years , 22. RegThese findings compared favorably to existing reports of oropharyngeal cancer treated with CRT –27. TherIn general, patients with HPVOPSCC are young and will live for prolonged periods. They are at high-risk for long-term toxicity and mortality from therapy . While tThere are currently few trials examining the role of de-escalation using surgery alone in early T-stage HPV-related disease or reduced RT in intermediate risk patients. New surgical techniques have broadened the range of patients capable of achieving a complete resection and the functional outcomes in such patients. Furthermore, the sensitivity of HPVOPSCC to chemotherapy and RT raise the possibility that delayed or salvage treatment in early-stage patients would be highly effective, would result in similar survival outcomes and could be applied to a much smaller population than current standards call for. Looked at from a different perspective, the need for post-operative RT in this younger, HPV+ and more functional population have not been validated in clinical trials to date.The recently published non-randomized phase II Sinai Robotic Surgery (SIRS) trial by our group (NCT02072148) demonstrated the highly favorable oncologic outcomes with de-intensified risk stratified postoperative adjuvant treatment following TORS . Here, wCell-free HPV DNA (cfHPVDNA) has emerged as a promising biomarker for HPVOPSCC . DynamicThere is also emerging evidence that this prognostic information can be used to risk stratify patients for personalized therapy in other head and neck cancers. In a modern cohort treated with intensity modulated radiation therapy, patients with nasopharyngeal carcinoma with high Epstein-Barr virus (EBV) cfEBV DNA levels displayed superior overall survival and distant metastasis-free survival with the addition of concurrent chemotherapy while patients with lower EBV cfEBV DNA levels did not derive apparent benefits from chemotherapy . SimilarMultiple trials are underway to evaluate the role of de-intensification of therapy, including varied approach such as decreasing radiation dose, limiting chemotherapy dose, and upfront surgery followed by risk-stratified adjuvant therapy . Amidst The SIRS 2.0 trial (NCT05419089) is a non-randomized Phase II non-inferiority trial approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai Figure 1To test the non-inferiority of 2-year recurrence rates in patients with HR-HPV and p16+-related OPSCC treated with surgery alone or surgery with de-intensified adjuvant therapy when compared to current regimens involving standardized post-operative radiotherapy +/- chemotherapy. The secondary objectives include determination of 2-year DFS, LRC, OS, and quality of life outcomes for patients treated with surgery alone and surgery with de-intensified adjuvant therapy.● The proportion of early stage HPVOPSCC patients treated with surgery alone having undetectable cfHPVDNA after surgery showing LRR at 2 years (Group 1)● The proportion of HPVOPSCC patients treated with surgery and de-intensified adjuvant protocol having undetectable cfHPVDNA after surgery showing LRR at 2 years (Group 2)● PFS at 2 years is defined as the proportion of patients without events (recurrence or death) at 2 years after surgery ● DFS at 2 years is defined as the proportion of patients without events  at 2 years after surgery .● OS at 2 years is defined as the proportion of patients alive at 2 years after surgery ● The score of QOL questionnaires .● PFS at 2 years is defined as the proportion of patients without events (recurrence or death) at 2 years after adjuvant CRT ● DFS at 2 years is defined as the proportion of patients without events  at 2 years after adjuvant CRT .● OS at 2 years is defined as the proportion of patients alive at 2 years after adjuvant CRT ● The score of QOL questionnaires after adjuvant CRT ● The frequency, percentage, and measured values of biomarkers present in patients with locoregional and distant failure.● Histologically or cytologically confirmed and identified resectable primary OPSCC with p16+ IHC● Pre-surgery detectable (≥10 copies/ml) baseline cfHPVDNA using NavDX assay . Undetectable cfHPVDNA at 3 weeks postoperatively.th edition early and intermediate stage disease  (non-matted) disease without evidence of distant metastases or gross extranodal extension.● AJCC 7● Age ≥ 18 years● No previous surgery, radiation therapy, or chemotherapy for head and neck cancer  is allowed at time of study entry.● Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.● No active tobacco use  and no cumulative smoking history of > 20 pack years.● Ability to understand and willingness to sign a written informed consent document.9/l, Platelet count ≥ 100 x 109/l, Hemoglobin ≥ 10 g/dl (may achieve by transfusion), Renal function: ≥ 60 ml/min  or surgically unresectable disease or disease that cannot be fully resected, unequivocal radiographic extranodal extension, supraclavicular or matted metastatic disease, >3 radiographic pathologic cervical nodes.● Advanced nodal stage  within 7 days of registration● PET/CT or high-resolution CT or MRI of the neck within 28 days before registration.● Clinical TNM staging● Examination under anesthesia within 8 weeks or at time of surgeryThe following evaluations are required prior to adjuvant treatment group assignment:● Pathologic evaluation of surgical specimens including p16 IHC and HR-HPV PCR, margin status in mm, PNI, LVI, ENE defined as ≤ or > 2mm, number of cervical metastatic nodes, location of involved nodes, and pAJCC staging.● 3-week postoperative cfHPVDNA testingEligible patients will undergo TORS resection of the tumor with negative intraoperative frozen section margins. Ipsilateral levels II-IV selective neck dissection will be routinely performed. Contralateral neck dissection, additional neck dissection levels , and appropriate reconstruction will be performed at the discretion of the operating surgeon. Surgery is to proceed until negative final frozen section margins are obtained.Patients will be assigned to the low-risk group 1 if they meet the following criteria:● Complete resection to negative frozen section margins (pT1-2)● ≤ 3 nodes, ≤ 2 mm extranodal extension (ENE), no supraclavicular nodes● HR-HPV positive● Undetectable cfHPVDNA at 3 weeks post-operativelyPatients assigned to the low-risk group 1 will treated and monitored as outlined below:● No adjuvant therapy● Clinical evaluations every 3 months the first year● PET-CT at 3 months post-surgery.● CT neck and chest at 6 months.● PET-CT at 1 year and 2 years post-operatively unless clinically indicated.● Imaging to be supplemented with HPV cfHPVDNA testing 12 weeks after surgery and every 3 months for the first two years after surgery.● If cfHPVDNA test is positive, patient will have repeat testing 4 weeks later. If test continues to be positive, this finding will mandate an enhanced PET-CT. For patients with two consecutive positive cfHPVDNA tests post-operatively and no obvious primary identified on clinical examination and imaging, both enhanced PET-CT and clinical examination will be performed every 3 months until the emergence of a defined site of recurrence. If no obvious recurrence after 6 months of routine testing and examination, timing of further clinical evaluation and imaging will be relegated to investigator’s choice.Patients will be assigned to the high-risk group 2 if they meet the following criteria:● The presence of any of the following: 4 positive nodes, gross ENE, final positive margins, or bilateral neck disease● High-risk PNI/LVI (defined as PNI and LVI in combination or either factor in the presence of 3 or more positive nodes)● Undetectable cfHPVDNA at 3 weeks post-operativelyPatients assigned to the high-risk group 2 will treated and monitored as outlined below:2)● Postoperative chemoradiation , which is also called intensity modulated proton therapy (IMPT) techniques depending on the facility’s experience and equipment.All patients will receive daily radiation treatment with either intensity-modulated radiotherapy (IMRT) or proton beam therapy. Photon beams of ≥4 MV are required. Patients not receiving proton therapy are required to receive Intensity Modulated Radiotherapy (IMRT) is required for all cases. IMRT A thermoplastic head mask (or similar immobilization device) is required for IMRT or proton beam. A treatment planning CT scan is mandatory for defining target volumes, however IV contrast is not required. Volumes will be contoured on each relevant CT slice (≤0.3 cm thickness).Since radiotherapy will only be administered postoperatively, there should not be gross tumor volume (GTV). Clinical target volume (CTV) is generated to account for microscopic disease. CTV46 will include the primary tumor bed and/or at-risk lymph node regions in the neck (excluded bone and air). The planning target volume (PTV) includes a 0.5 cm expansion margin that accounts for daily setup variation. For proton planning, the worst case CTV dose distribution corresponding to a setup error of 3 mm and 3% range uncertainty will be used for dose optimization, evaluation and dose reporting in place of PTV.Target dose constraints and avoidance structures are shown in All plans must be normalized such that 95% of the PTV is covered with the prescription dose. No more than 20% of the PTV may receive > 110% of the prescription dose. No more than 1% of the PTV may receive < 93% of the prescription dose. No more than 1cc of tissue outside the PTV may receive > 110% of the prescription dose.For proton planning, each beam has an individual and unique expansion from the CTV especially along the beam direction. When the robust optimization is employed, the optimization target volume is determined from CTV based on beam arrangement and will take into account proton range uncertainty as well. For dose reporting, CTV+5mm can be utilized as the PTV; however, for proton plans the plan should always be optimized to the CTV.The following quality assurance measures will be implemented to ensure effective treatment and patient safety.● On the first day of treatment each patient will have electronic portal images taken that localize the isocenter placement. For proton plans, quality assurance is performed by delivering the plan onto a phantom and measuring the dose using an ion chamber array or similar device.● Every week, verification portal images are taken and approved by a radiation oncologist prior to continuation of treatment. For PBRT patients, weekly verification CT or conebeam CT scans are required and any plan adaptations will be determined accordingly.● Each treatment plan will be reviewed by an non-treating radiation oncologist prior to therapy to assure appropriate fields and doses.2/week for a total dose of 200 mg/M2. Patients will be prehydrated with 1 liter normal saline before and after. The prehydration liter will include a 5HT3 antagonist as part of the antiemetic regimen. 20 Meq KCl and 2 gm Magnesium sulfate will be administered in the post hydration liter. In the event of toxicity to cisplatin, carboplatin may be substituted as weekly chemotherapy with AUC of 2.0.Patients in Group 2 will also receive weekly cisplatin at a dose of 40mg/MParticipants will be followed for 5 years after completion of the study treatment or until death.Group 1 and Group 2 patients will have the following recurrence monitoring protocol:● Clinical evaluations every 3 months the first year● PET-CT at 3 months post-surgery.● CT neck and chest at 6 months.● PET-CT at 1 year and 2 years post-operatively unless clinically indicated.● Imaging to be supplemented with cfHPVDNA testing 12 weeks after surgery for Group 1 and 6 and 12 weeks after surgery for Group 2. Participants will also get cfHPVDNA testing every 3 months for the first two years after surgery.● If cfHPVDNA test is positive, patient will have repeat testing 4 weeks later. If test continues to be positive, this finding will mandate an enhanced PET-CT. For patients with two consecutive positive cfHPVDNA tests post-operatively and no obvious primary identified on clinical examination and imaging, both enhanced PET-CT and clinical examination will be performed every 3 months until the emergence of a defined site of recurrence. If no obvious recurrence after 6 months of routine testing and examination, timing of further clinical evaluation and imaging will be relegated to investigator’s choice.Survival and recurrence data will be tracked for five years from surgery. Quality of life measures including the European Organization for Research and Treatment of Cancer Core measure (EORTC QLQ-C30) and the head and neck-specific measurement tool EORTC QLQ H&N35, M.D. Anderson Symptom Inventory – Head and Neck (MDASI-HN), and MDADI-HN dysphagia and xerostomia questions will be assessed at the 3-, 6-, and 12-month time points and then yearly for 5 years. Toxicity evaluation for both arms will be assessed starting from the time of surgery.Sample sizes for the two study arms were calculated separately. For the low-risk group, assuming a historical recurrence proportion of 15%, a non-inferiority margin of 11%, one interim analysis, and 10% dropout rate, a total sample of 126 patients will be enrolled to provide power of 83.3% and one-sided alpha of 0.025. For the high-risk group, assuming the same recurrence proportion, a non-inferiority margin of 15%, one interim analysis, and 30% dropout rate, a sample of 73 patients will be enrolled to provide 82.5% power and one-sided alpha of 0.05%. An interim analysis will be performed after 57 and 26 patients are recruited and followed for 2 years for the low-risk and high-risk groups, respectively. We expect to accrue 5 patients per month for both treatment arms with 80% allocating to the low-risk group.Participant characteristics will be summarized using descriptive statistics. The analysis for the primary and secondary endpoints will be performed for the low- and high-risk groups separately using intention-to-treat (ITT) and per-protocol cohorts, respectively . The primary endpoint of proportion of recurrence for the low- and high-risk groups will be reported with corresponding 97.5% and 95% upper confidence limits. The Kaplan-Meier (KM) method will be used to estimate locoregional control (LRC), disease-free survival (DFS), and overall survival (OS). Patients without the designated event will be censored at the time of the last available follow-up record or the study analysis cutoff time point. Quality of life outcomes will be compiled and recorded for each of the outlined time points and linear mixed-effect models will be used to test changes of the scores over time. Biomarker outcomes will be reported descriptively and will be summarized by patient outcomes.HPV-related oropharyngeal cancers have demonstrated a significantly better prognosis compared to non-HPV-related cancers. Given the excellent prognosis, focus has shifted to reducing the morbidity associated with therapy while maintaining survival. In the original Sinai Robotic Surgery trial, patients treated with surgery alone maintained a >90% progression free survival at a median of 43 month follow up. In this study we plan to evaluate the non-inferiority of 2-year locoregional recurrence rates as well as survival outcomes for low-risk patients treated with TORS alone and high-risk patients treated with TORS and reduced dose adjuvant chemoradiotherapy with undetectable postoperative cfHPVDNA. The objective of this study is to determine whether or not comparable survival outcomes can be achieved in patients treated with de-escalated adjuvant therapy. Additionally, we plan to evaluate the impact of the proposed de-escalation protocol on quality of life outcomes.et al. demonstrated that surgery alone can reduce cfHPVDNA to undetectable levels in 59% of patients . Each of these studies will add to the current body of literature, particularly with regard to standard radiation dosing, however there is a growing number of de-escalation studies currently in progress.The survival differences observed in HPVOPSCC when compared to EROPC motivated the pursuit of de-escalated therapy for these typically more radiosensitive tumors. At present, there are few studies listed on clinicaltrials.gov employing TORS as part of a de-escalation protocol. In addition to our own study, there are two other ongoing single arm prospective studies, NCT04502407 and NCT03729518, recruiting patients to receive reduced dose adjuvant (chemo)radiotherapy after transoral robotic surgical resections with a focus on survival and quality of life outcomes. In contrast, NCT04572100 and NCT03107182 employ neoadjuvant therapy regimens to determine radiologic tumor response before assigning subjects to receive TORS vs (chemo)radiotherapy. Interestingly, NCT04572100 also evaluates the cfHPVDNA changes that occur in response to therapy. Similar to our study, NCT05387915 is enrolling patients with undetectable cfHPVDNA and intermediate pathologic risk factors to receive three weeks of adjuvant radiotherapy after TORS. In contrast to our protocol, the is no observation arm and this study primarily focuses on the evaluation of swallow function after de-intensified therapy, with secondary survival and recurrence outcomes. While this does not represent a comprehensive list of ongoing trials, these protocols highlight the transition occurring in the treatment of HPVOPSCC, with de-escalated therapy using a surgical approach becoming a more common focus of research.While HPV-related cancers generally confer a favorable prognosis, outliers frequently remain particularly memorable in our minds. In these individuals, the pathologic features and clinical risk factors may not identify these more insidious tumors. As such, discovery of new biomarkers for risk stratification is critical in order to continue tailoring individualized treatment plans. This study represents one of the first prospective clinical trials integrating circulating tumor biomarkers into a de-escalation study for HPVOPSCC. We believe this not only benefits enrolled patients from a morbidity and quality of life standpoint, but also serves to push our field forward toward integration of biomarkers into the treatment decision process.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Review Board of the Mount Sinai School of Medicine. The patients/participants provided their written informed consent to participate in this study.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.


Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are widely used for the treatment of EGFR mutation positive advanced nonsmall cell lung cancer (NSCLC); however, acquired resistance is known to develop during these treatments. Among these mechanisms, histological transformation is seldom encountered. Although platinum based chemotherapy has been reported to be effective in the treatment of patients with small cell lung cancer transformation, there is a lack of information on the treatment of patients with squamous cell carcinoma (SQ) transformation.An 80-year-old nonsmoking woman was referred to our hospital because of an abnormal shadow on her chest radiograph. Diagnostic bronchoscopy was performed and pathological examination revealed adenocarcinoma. Mutation analysis of the EGFR gene revealed deletion of E746-A750 in exon 19. She refused both surgical treatment and radiation therapy, and preferred periodic radiologic follow-up. Unfortunately, approximately a year and a half after the initial diagnosis, the primary lesion enlarged, and many pleural nodules were newly detected .Based on EGFR mutation analysis, a reduced dose of daily erlotinib was prescribed, which achieved a partial response and 34 months of progression-free survival (PFS). A repeated biopsy with an endobronchial cryoprobe was performed on the enlarged primary lesion. Pathological examination revealed SQ harboring an identical EGFR mutation with a secondary EGFR T790M mutation. Osimertinib 80 mg once a day was started as second line therapy, which resulted in 8 months of PFS and 15 months of survival.The literature review and our report suggest that osimertinib is a promising treatment for NSCLC regardless of histology if T790M is present as an acquired mutation. Two main types of resistance mechanisms to EGFR mutated NSCLC are “on target” and “off target” resistance. The former is the secondary alteration in the target oncogene, including either a second site mutation that promotes TKI resistance or the amplification or loss of the targeted oncogene. The EGFR T790M mutation is found in >50% of patients with acquired resistance to early generation EGFR TKIs,,4 which occurs at a conserved “gatekeeper” threonine residue within the ATP binding pocket. EGFR T790M mutation is sensitive to osimertinib as a second line therapy. The latter is a tumor cell alteration occurring in proteins other than the targeted oncoprotein, which includes downstream signaling pathways and parallel bypass signaling pathways.Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely used in EGFR mutation positive advanced nonsmall cell lung cancer (NSCLC). Tumors are known to acquire resistance to EGFR TKIs as first line treatment. In such circumstances, tumor rebiopsy is recommended as a second line therapy, especially after the failure of first or second generation EGFR TKIs.,6 In such cases, cytotoxic drugs are commonly used according to the histologic findings. However, transformation to squamous cell carcinoma (SQ) is rare, and a standard therapy has not yet been established. We herein report an EGFR T790M mutation positive lung adenocarcinoma that showed histologic transformation to SQ after erlotinib treatment; thus demonstrating the efficacy of osimertinib. The pharmacokinetics of osimertinib were also investigated in this patient. The purpose of this report is to propose a method for the clinical management of T790M mutation positive SQ transformation from EGFR-mutated lung adenocarcinoma through our patient.Histologic transformation is a type of resistance mechanism that is not a feature of “on target” nor “off target” mechanisms. It can alter tumor cell susceptibility to the inhibition of target oncoproteins. Small cell lung cancer (SCLC) transformation is seen in approximately 3% to 14% of NSCLC cases after first line EGFR TKI treatment. which achieved a confirmed partial response  of the chest revealed a nodular shadow in the right lower lung (S8) measuring 26 mm, accompanied by pleural indentation. Diagnostic bronchoscopy was performed using endobronchial ultrasonography with a guide sheath (EBUS-GS) transbronchial biopsy (TBB). Pathological examination revealed adenocarcinoma with napsin A, and the patient tested positive for thyroid transcription factor 1 (TTF-1) and negative for p40  revealed that tumor cells harbored an identical EGFR mutation (deletion E746-A750 in exon 19) with a secondary EGFR T790M mutation. According to the EGFR mutation analysis, osimertinib 80 mg once a day was started as second line therapy, which resulted in a partial response in 2 months  showed EGFR T790M mutation. Eventually, the patient survived for 15 months from the initiation of osimeritinib treatment. This showed the importance of both rebiopsy and treatment selection based on gene profile.Although transformation from adenocarcinoma to SQ is uncommon, more cases are being reported.–31 a total of 27 cases of SQ transformation in EGFR mutated lung adenocarcinoma after treatment with EGFR TKIs have been reported. The ages of these patients were 40 to 80 years (median age was 62 years), and 18 patients (66.7%) were female. All patients had EGFR mutations, 15 (55.6%) patients had an exon 19 deletion; 10 (37.0%) patients had an L858R point mutation; and 2 (7.4%) patients had other minor mutations. The T790M mutation was detected in only 1 case (3.7%) at the first diagnosis. These characteristics were not significantly different from those of known treatment naïve EGFR mutated NSCLC without SQ transformation in previous reports.,33 Moreover, the time from EGFR-TKI initiation to SQ transformation was 4 to 116 months , indicating a trend toward a slightly longer treatment period than that known for EGFR-mutated NSCLC.As shown in Table  Osimertinib use after SQ transformation was observed in 40.7% of the 11 patients. The median survival time was estimated to be at least 8 months. In addition, the median survival time was at least 6 months in patients without osimertinib, suggesting that the effect of osimertinib is likely underestimated. These findings suggest that, unlike small cell carcinoma transformation, driver-mutation specific treatment strategies may be beneficial for survival in SQ transformation.The positive rate of T790M during the treatment of 27 cases was 59.3%, which was similar to that in a previous report. In this patient, prolonged high drug concentrations could induce gastrointestinal adverse events such as diarrhea and anorexia. This suggests that dose reduction and individualization are required for osimertinib treatment.Moreover, we measured the steady state trough concentration of osimertinib when it was prescribed at 40 mg every other day Table . Its conThere are some considerable limitations in this report. Firstly, in our patient, we should consider the possibility that the tumor contained an adenosquamous component from the beginning. The possibility of SQ transformation is more likely than the possibility of SQ selection from adenosquamous epithelium because the time from EGFR-TKI initiation to SQ appearance was relatively long and tissue of SQ transformed part detected T790M by NGS. Second, the treatment of SQ transformation is still controversial. The preceding research included patients in whom osimertinib did not always show a response despite the presence of the T790M mutation. Third, publication bias should be taken into account, as there may be published cases where the drug has shown more benefit. Clinical studies are needed to prove the efficacy of osimertinib in patients with SQ transformation and T790M.This case report and literature review highlighted the importance of confirming the genetic profile of EGFR mutated lung adenocarcinoma showing SQ transformation as resistance to EGFR TKI, and treatment with individualized EGFR TKI doses. We propose osimertinib is eligible for the treatment of SQ transformation from EGFR-mutated lung adenocarcinoma with T790M mutation.www.editage.com) for English language editing.We would like to thank Editage (Conceptualization: Yusaku Kusaba, Yuichiro TakedaData curation: Yusaku Kusaba, Sakurako AbeWriting - original draft: Yusaku Kusaba, Yuichiro TakedaWriting - review & editing: Sakurako Abe, Akinari Tsukada, Go Naka.


This study was aimed to evaluate the fit of occlusal veneer restoration for twoCAD/CAM materials with different cement space settings, using microCT scans.Sixty resin dies were made and divided into two groups (n=30) according to thematerials, (I): Hybrid all-ceramic, and (II): zirconia-reinforced lithiumsilicate glass-ceramic. Each group was subdivided into three subgroups (n=10)according to the cement space parameters . Occlusal veneersfor the six subgroups were milled. A circle with 20 different sections wasplaced at the center of every scanned specimen to measure four differentlocations . Datawere analyzed using two-way ANOVA at a 0.05 level of significance. There was nostatistically significant effect of material type on the mean values of internaland marginal gaps for the three cement space parameters (P>0.05). There wereno statistically significant differences in the occlusal and axial gap betweenthe cement space parameters, furthermore, there were statistically significantdifferences in marginal gap distances and absolute marginal discrepancies(P>0.05). Hybrid all-ceramic showed smaller marginal and internaldiscrepancies than zirconia-reinforced lithium silicate glass-ceramic withoutstatistically significant differences, and, for both materials, 50 µm cementspace significantly improved the marginal fit and absolute marginaldiscrepancy. Crown restorations created with (CAD/ CAM)technologies, on the other hand, normally have their spacer thickness specifiedduring the software's design phase. However, the most appropriate spacer thicknesssetting for proper occlusal veneer adaptation is uncertain This study was directed to assess the marginal and internal adaptation for occlusalveneer restoration using two types of CAD/CAM materials (zirconia-reinforced lithiumsilicate glass-ceramic and polymer-infiltrated ceramic network) with three differentcement space settings , using microCT scans. The nullhypotheses to be tested were that there are no differences in the marginal andinternal adaptations between A typodont mandibular right first molar  wasprepared for occlusal veneers by a medium grit tapered diamond bur with arounded end, using a milling machine . Thetooth was prepared as follows: 1.5 mm height extending from the margin to theocclusal plane, 1 mm circumferential rounded chamfer, and rounded Axio-gingivalangle with 1.0 mm curvature radius ,60 resin master dies were made by replicating the prepared tooth using the CerecinLab 3D system . The dies were randomlyand equally assigned to two groups (n=30) according to the materials, group (I):zirconia-reinforced lithium silicate glass-ceramic , and group (II): Hybrid all-ceramic material . Then, e,,,The master dies were homogenously sprayed using fluorinated hydrocarbon pigmentsuspension , then digital impressions ofall master dies were made using the Cerec scanner . The same occlusal veneer design with identical externalcontours for all groups was made. A simulated cement space of 25 µm strap of 0.5µm above the finish lines was designed, then, additional cement spaces of 30 µm,40 µm, and 50 µm were set forming the following three tested subgroups for eachtype of material, a (25-30), B (25-40), and C (25-50) ,After designing each type of occlusal veneer material, the data was sent to theCerec in Lab milling unit for occlusal veneers milling. After the milling process, occlusal veneers forthe vita suprinity samples were crystallized in program at the P310 furnace. No adjustments weremade to the ceramic veneers before marginal and internal adaptation measurementsTo ensure standardization of the sample’s position during scanning, one masterdies with its corresponding occlusal veneer placed in a mold with dimensions of4X3X3 cm filled with light body polyvinyl siloxane  from its buccal side until mesial and distal dimples arecovered, then, a stainless steel wire (0.1mm) was embedded parallel to thelongitudinal axis of the mesial side (used as a fixed starting reference point),The occlusal veneers were seated onto their corresponding dies without any lutingmedium [1] Occlusal gap: the mean of the three occlusal values: After dividing thelength of the occlusal surface into four equal parts, one measurement at themiddle and one at the middle of each half were taken.[2] Axial gap: The mean of two right and left axial values: in the middle ofaxial walls.[3] Marginal gap: The mean of two right and left marginal values at the middle ofthe finish line.,[4] Absolute marginal discrepancy value (AMD): The mean of two right and leftdistances between the margin of occlusal veneer and die margin ,Images with a resolution of 9.77 μm /pixel were acquired from 20 slices and wereused to measure four sites per slice  with a total of 80 measuring values for each tooth ,,The digital data were developed using reconstruction software , and converted into a tagged image file format (TIFF) forthe subsequent segmentation . Using (trabecular thickness) algorithm option by the image processingand analyzing software ; the absolute marginal discrepancy (AMD),marginal and internal adaptation have been measured . The sample mean and standard deviation variancewas 84.19 ± 13.69; accordingly, a sample size of 30 per group (10 per cementspace parameter) was considered satisfactory to obtain a type I error for powermore than 0.99 of alpha of 0.05.Statistical analyses were performed with statistical software . The Shapiro-Wilk test of normality confirmed thatthe data were normally distributed (P>.05).A Two-way ANOVA test has been used to identify the main effect of material typesand cement space settings on each site of measurement  followed by One - way ANOVA with post hoctests (LSD) for each effective variable  separately within cement space parameters.A total of 1200 images were obtained for all tested samples (20 sections X 60samples), including ten occlusal veneer /subgroup and four measuring value /slicewith 4800 measuring values. A summary of mean gap values, standard deviations,minimum and maximum gap values) in micrometer (µm) for both materials (Vita Enamicand Vita Suprinity) at the four tested sites  for the three tested cement spaces  are shown in .Regarding the effect of material factor, the two-way ANOVA test indicated nostatistically significant effect for the four tested locations  . Regarding the effect of the cement spaces factor, the two-way ANOVA test indicatedthat there was no statistically significant effect for both occlusal and axialsites)  while there was a statisticallysignificant effect for both marginal and absolute marginal discrepancy . One-way ANOVA test for multiple comparison statistics at marginal and absolutemarginal discrepancy revealed that from 30 to 40 µm cement spaces, there were nostatistically significant differences in the gap distances for both tested materials . From 30 to 50 µm and from 40 to 50 µm there werestatistically significant differences in the gap distances for both tested materials. The clinical significance of this study is that the outcomes may help in the clinicalassessment of the least cement space setting providing optimal marginal adaptationfor Hybrid all-ceramic, and zirconia-reinforced lithium silicate glass-ceramicocclusal veneers.Based on the results of this study; cement space parameters only affected themarginal adaptation of occlusal veneer restorations, while, the type of material didnot affect, so the null hypothesis would be partially rejected.,The material’s relationship with the luting agents plays an important role in theclinical success of occlusal veneer restorations ,A previous study has postulated that the ideal value for marginal adaptation shouldbe 100 to 200 µm to be clinically acceptable In this study, an extremely high resolution of microCT (9.77 μm) was used. The highresolution increases the contrast of the image layers, which subsequently improvesthe gap measurements. The microscopic evaluation inherently involves projectionerror due to the frontal view contrary to the sectional view used in microCT.Furthermore, the standard selection of measurement points around the multiplereplicas is challenging In the present study, the same reproduced die was used for each veneer, andstandardized positions were used for scanning. To ensure the standardization of thecomputed tomography images for all slices, each die with its corresponding occlusalveneer was placed in a prefabricated special mold with thin stainless-steel wire(0.1mm) parallel to the longitudinal axis of the mesial side as a fixed startingreference point.,Manual adjustment of restorations after visual inspection could greatly decrease themarginal gap and AMD, but this technique was not applied in this study contrary toother studies Polymer-infiltrated ceramic exhibited marginal and internal gap values lower thanthat for zirconia-reinforced lithium silicate ceramic without statisticallysignificant differences, this may be attributed to reduced brittleness ofpolymer-infiltrated ceramic providing thin and smooth margin which enhances themilling procedure during manufacturing process unlike Zirconia reinforced lithiumsilicate, which retains their crystal structure and rupture during the millingprocess ,The mean internal gap values of all tested groups did not imitate the designed cementspaces in the design software, this result is in accordance with that of the otherstudies ,,In agreement with other previous studies ,The absolute marginal discrepancy is the distance between the preparation margin andrestoration margin , therefore, it must be greater than or in anideal state equal to the marginal gap Absolute marginal discrepancy values of this study were lower than those evaluated byYildirim et al using vita Enamic crowns Based on the microCT measurements, the comparison of the different virtual cementspace settings for each material showed a statistically significant differenceconcerning the accuracy of fit. These results indicate that an increase in thecement space setting parameters improves the marginal fit. This result is inagreement with Shim et al ,In this study, axial mean gap values were lower than those at the occlusal surface.This is similar to other studies The major disadvantage of microCT evaluation is the formation of radiation artifactscaused by the differences of radiation absorption coefficients between differentmaterials during the evaluation of the gap distances


Pathway inference methods are important for annotating the genome, for providing insights into the mechanisms of biochemical processes and allow the discovery of signalling members and potential new drug targets. Here, we tested the hypothesis that genes with similar impact on cell viability across multiple cell lines belong to a common pathway, thus providing a conceptual basis for a pathway inference method based on correlated anti-proliferative gene properties.To test this concept, we used recently available large-scale RNAi screens to develop a method, termed functional pathway inference analysis (FPIA), to systemically identify correlated gene dependencies.AKT1, MTOR and PDPK1 were among the most correlated with those for PIK3CA (encoding PI3Kα), as returned by FPIA, whereas negative regulators of PI3K/AKT/MTOR signalling, such as PTEN were anti-correlated. Following FPIA, MTOR, PIK3CA and PIK3CB produced significantly greater correlations for genes in the PI3K-Akt pathway versus other pathways. Application of FPIA to two additional pathways (p53 and MAPK) returned expected associations (e.g. MDM2 and TP53BP1 for p53 and MAPK1 and BRAF for MEK1). Over-representation analysis of FPIA-returned genes enriched the respective pathway, and FPIA restricted to specific tumour lineages uncovered cell type-specific networks. Overall, our study demonstrates the ability of FPIA to identify members of pro-survival biochemical pathways in cancer cells.To assess FPIA, we initially focused on PI3K/AKT/MTOR signalling, a prototypic oncogenic pathway for which we have a good sense of ground truth. Dependencies for cordial’ freely available from https://github.com/CutillasLab/cordial.FPIA is implemented in a new R package named ‘Bioinformatics online. Signal transduction pathways decode extracellular signals into appropriate biological responses, thereby regulating most cell processes. Components of signalling pathways form complex networks of biochemical reactions and are frequently dysregulated in diseases such as cancer, neurodegeneration and diabetes. Therefore, discovering new signalling pathway members is important to understand the mechanisms of cellular biological processes and to uncover new anti-cancer drug targets. Pathway inference methods used for this purpose are based on literature mining techniques  and catalytic  subunits. When activated by upstream signals, such as those emanating from receptor tyrosine kinases (examples of which include EGFR and the insulin receptor), PI3K phosphorylates phosphatidylinositol 4,5 bisphosphate (PIP2) to produce phosphatidylinositol 3,4,5 trisphosphate (PIP3). This lipid second messenger nucleates protein complexes leading to the activation of an intracellular protein kinase cascade involving PDK1 (gene PDPK1), AKT1 and MTOR. The lipid phosphatase PTEN dephosphorylates PIP3, thus opposing and terminating PI3K signalling. The PI3K system therefore offers a system for which we have a good sense of ground truth and thus allows assessing the ability of FPIA to produce biologically meaningful results. FPIA was then further tested for its ability to return meaningful associations to p53 and MEK1.To test this concept, we developed a method named functional pathway inference analysis (FPIA), which uses gene dependency data (cell viability data as a function of genome-wide gene targeting) to reveal genes whose inhibition produces similar cell survival phenotypes across cell lines. This is distinct from previously published methods where the aim was to identify genetic or other perturbations correlated to gene dependencies  in RStudio (v1.4.1717).FPIA utilized as input cancer dependency datasets from the Cancer Dependency Map (DepMap) project—a collaboration of the Broad Institute  and the Wellcome Sanger Institute . Data produced by the DepMap project are made publicly available under a Creative Commons licence and are accessible through the portal  were subjected to the Benjamini–Hochberg multiple testing adjustment method with stats::p.adjust to control the false discovery rate calculating q-values; ‘cordial’ functions have the capacity to set the adjustment method (parameter method) which is passed to stats::p.adjust  to facilitate downstream analysis. The ‘cordial’ package was designed to be flexible so as not to be dependent on the input data and as such it can be used for correlation analysis of datasets beyond the molecular/genetic screens studied in this research.FPIA implemented Pearson correlation analysis to assess the similarity in dependency data via three principal functions of the ‘adjust . The genfuture package to provide the parallel backend, and vectorized mapping functions from the furrr package that process input in parallel . Prior to function usage, a multisession plan (plan) was created that generates multiple R sessions which resolve futures asynchronously  in separate R sessions equal to the number of physical processor cores; for convenience, this process has been incorporated into the ‘cordial’ function start_parallel. The input supplied was split, distributed to the workers and returned to the main R session to be combined once all chunks have been processed. This method of spawning multiple background R sessions ensures compatibility across all operating systems since the alternative of forking processes is not supported by Windows.Correlation analysis was performed in a vectorized and parallelized manner with the dataset) containing values which may also include associated metadata, or alternatively an optional dataset can additionally be supplied that may hold the metadata instead (parameter metadata). The data structure for both inputs is a data.table as this allowed for the subsequent efficient filtering of rows and subsetting of columns. Filtering (parameter filter_rows) was programmed by taking the input and a named list, whereby names specify column names to filter on, and performing a cross-join. If the dataset required subsetting to limit the correlations to be made  then a vector of column names can be provided (parameter select_cols). The resultant correlations of cor_map are symmetrical, specifically pairwise correlations are produced between all combinations of the columns specified (parameter select_cols). Unlike cor_map, cor_targets takes an additional argument of a single target column name (parameter target). The function cor_target_map calls cor_targets internally which is mapped in parallel with future_map to the argument of a vector of column names. In contrast with cor_map, cor_target_map creates non-symmetrical correlations, that is for each specified target column (parameter target), pairwise correlations are produced with the columns specified (parameter select_cols).The function arguments consisted of an input dataset (parameter stats) were used to determine the statistical significance of results and check assumptions were met. These included: Shapiro–Wilk test for normality (shapiro.test), F-test for homogeneity in variances (var.test) and Welch two-sample unpaired t-test (t.test). Fisher z-transformation (psych::fisherz) of the Pearson correlation coefficient r was used prior to testing of statistical significance for the population correlation coefficient ρ to overcome the skewed distribution observed as the sample correlation coefficient r nears ±1.Statistical methods , which minimized within-cluster variance $weight) was assigned as absolute r-value; the edge colour attribute (E(graph)$color) was assigned to the direction of the correlation (positive/negative); the edge width parameter (edge.width) was assigned inside the call to the plotting function plot.igraph as −log10. The vertex (node) colour attribute (V(graph)$color) was assigned to being a target gene. The vertex size parameter (vertex.size) was assigned in plot.igraph as weighted vertex degree with strength. Network groups were calculated in the plot.igraph parameter (mark.groups) as connected components (groups(components(graph))). The layout of the network graph ) was the Fruchterman and Reingold force-directed layout where larger weight values  produced shorter edges (degree (the number of adjacent edges of a vertex) and betweenness (the number of shortest paths through a vertex)  .cor_target_map was studied by Term Enrichment Analysis of genes correlated to the target gene in Reactome ; the false discovery rate was controlled for by calculating q-values with the Benjamini–Hochberg method  to target genes, of which 300 were in hsa04151 from the 342 hsa04151 genes present in the RNAi dataset, producing a pathway coverage of 87.7% (INPPL1 (91) followed by INPP5E (75), and this was transposed for significant correlations of genes corresponding to other pathways (PIK3R1 (71) and PDPK1 (69). INPPL1 and INPP5E are involved in PI-associated signalling and thus can only indirectly influence AKT signalling by manipulating the pool of PI lipid substrates, whilst PIK3R1 encodes a regulatory subunit of PI3K  followed by t-test of hsa04151 genes versus genes of all other pathways. This analysis revealed six target genes where the means of the significant correlations that belong to the pathway were significantly different to those of other pathways. Of these, MTOR, PIK3CA and PIK3CB exhibited means that were significantly greater for correlations to genes in hsa04151 than other pathways (FPIA found 13 725 genes in the dataset (81.6%) that were significantly correlated  which was homogenous for all key target genes , there is support to suggest that this may be a true observation as both have similar mutation patterns in cancer  for analysis. Although this represents a restriction, it is of the input dataset rather than the method and therefore will improve as more cancer models are studied. The three tumour lineages that cumulatively covered the greatest number of significantly correlated (q < 0.05) genes in hsa04151 were lung, breast and skin (PIK3R4 (42), whereas in the breast it was PDPK1 (21), whilst in skin it was INPP4B (21). The three topmost target genes ranked by number of significantly correlated genes were PDPK1, MTOR and PIK3R4, which contrasted from the gene-specific analysis in hsa04151 across all cell lines  within individual tumour lineages. Of the 31 lineages represented in the dataset, 12 had a sufficient number of cell lines without missing values  and MEK1 . Genes that positively or negatively regulate p53, including MDM2, MDM4, TP53BP1 and CHEK2, were the most significantly associated genes with TP53 (MAP2K1 include upstream regulators of this kinase (BRAF and STK38) as well as downstream effectors  , and by unbiased over-representation analysis in which the respectively associated pathways were enriched in the new unbiasedly identified network. Consequently, the FPIA method, and the associated cor_target_map function of the ‘cordial’ package, provides insights into signalling pathways and may be used to uncover novel interactions in a global, genome-wide manner. A potential limitation of the method is that it relies on functional analysis of genes that impact cell viability and therefore it may only be applied to the discovery of members of pathways that regulate cell survival and the proliferative process. Nevertheless, since dysregulated cell proliferation is a key hallmark of cancer, and the aim of most anti-cancer drugs is to inhibit cell viability/proliferation, FPIA has the potential to advance our understanding of biochemical networks involved in cancer and to inform new therapeutic targets and approaches.This work tested the hypothesis that genes with similar impact on cell viability across multiple cell lines belong to a common pathway, thus implying that the correlation of anti-cancer genetic dependencies may be used for biological pathway inference and for the annotation of genes with functional information. To test this idea, we developed a method termed FPIA to systemically determine correlated gene dependencies from large-scale gene silencing screens. Our gene-specific analysis revealed significant gene correlations within canonical signalling cascades with the potential to identify novel interactions. This was evident from inspection of PI3K, p53 and MEK1 correlated genes which returned the canonical pathway (btac769_Supplementary_DataClick here for additional data file.


We investigated safety and preliminary efficacy of anodal transcranial direct current stimulation (atDCS) paired with swallowing exercises in improving post-stroke dysphagia from an acute unilateral hemispheric infarction (UHI). We conducted a double-blind, early phase-2 randomized controlled trial, in subjects (n = 42) with moderate-severe dysphagia [Penetration and Aspiration Scale (PAS) score ≥ 4], from an acute-subacute UHI. Subjects were randomized to Low-Dose, High-Dose atDCS or Sham stimulation for 5 consecutive days. Primary safety outcomes were incidence of seizures, neurological, motor, or swallowing function deterioration. Primary efficacy outcome was a change in PAS scores at day-5 of intervention. Main secondary outcome was dietary improvement at 1-month, assessed by Functional Oral Intake (FOIS) score. No differences in pre-defined safety outcomes or adjusted mean changes in PAS, FOIS scores, between groups, were observed. Post-hoc analysis demonstrated that 22 /24 subjects in the combined atDCS group had a clinically meaningful dietary improvement (FOIS score ≥ 5) compared to 8 /14 in Sham . atDCS application in the acute-subacute stroke phase is safe but did not decrease risk of aspiration in this early phase trial. The observed dietary improvement is promising and merits further investigation.Dysphagia is a serious stroke complication but lacks effective therapy It is independently associated with increased risk of pneumonia and malnutrition, early stroke mortality and institutionalization6. Treatments that help restore normal swallowing can therefore improve stroke outcomes. Unfortunately, such therapies are currently lacking and management usually revolves around providing nutritional support with nasogastric or percutaneous endoscopic gastrostomy tubes until swallowing recovers spontaneously, if at all7; patients fed via these means remain vulnerable to serious medical complications and have been observed to have high mortality rates10.Dysphagia is a very common complication of a stroke and carries major implications for stroke survivors11. This improved understanding of swallowing behavior and its regulation aligns well with the observation that patients with isolated unilateral hemispheric strokes that spare the brainstem structures also have a high incidence of persistent dysphagia12.The prevailing view of swallowing and its control has evolved to incorporate the great diversity of swallowing behavior in health that can range from reflex swallowing to volitional eating or a combination of volitional and reflexive swallowing such as brisk drinking. Both cerebral hemispheres, the subcortical regions and the central pattern generators in the medullary pontine regions intricately mediate normal swallowing15. The presence of inherent redundancies in swallowing control, where projections from both swallowing cortices converge on a common pool of brainstem structures , can enable independent unilateral hemispheric regulation of bilateral “swallowing-centers”16. These unique features make the undamaged swallowing cortex an attractive target for cortical modulation techniques to augment the naturally occurring neuroplastic changes within this region and to enhance its role as a mediator of recovery18.Recent insights on the neurophysiology of swallowing control in health and the neuroplastic processes that drive swallowing recovery after stroke also provide a basis to develop and test targeted therapies for post-stroke dysphagia. Separate investigations show that while unilateral hemisphere infarction can produce dysphagia by damaging the swallowing motor cortex or its projections, the contralateral healthy swallowing cortex possesses the capacity to reorganize sufficiently to enable restoration of normal swallowing19. A non-invasive brain stimulation technique, like transcranial direct current stimulation (tDCS) may be especially well suited to improve swallowing in this stroke population17. It has been demonstrated that recovery of swallowing functions in patients with hemispheric lesions occurs via compensatory reorganization of the undamaged cerebral hemisphere and shows a predictable pattern of expansion of the pharyngeal representation in an anterolateral direction, irrespective of lesion site or laterality15. This reorganization is preceded by an increase in excitability of the swallowing cortex in the undamaged hemisphere suggesting that the excitability changes drive cortical reorganization15. We therefore hypothesize that with hemispheric lesions, where the brainstem and peripheral structures are intact but the upper echelons of the swallowing apparatus are dysfunctional, a cortical stimulation technique can be effective by engaging the intact swallowing motor cortex and helping to restore swallowing functions.As hemispheric strokes are the commonest stroke sub-type in the population, they likely contribute disproportionately to the overall dysphagia burden21. Building upon these experiences, we systematically examined the safety and feasibility, and performed a preliminary assessment of efficacy of atDCS paired with swallowing exercises in improving swallowing functions after an acute unilateral hemispheric infarction in a randomized controlled clinical trial (RCT).We and others have previously reported on the effect of anodal tDCS (atDCS) for dysphagia recovery after a stroke in pilot studieshttp://www.clinicaltrials.gov. Unique identifier: NCT01919112) on 8/08/ 2013.The study was reviewed and approved by the Institutional Review Board at Beth Israel Deaconess Medical Center. We confirm that all research procedures was performed in accordance with relevant regulations and in accordance with the Declaration of Helsinki. A written informed consent was obtained for all trial participants or their legal guardians. A Data Safety Monitoring Board with assistance from a Medical Safety Monitor reviewed the progress of the trial. This clinical trial was registered on Clinicaltrials.gov  in Boston, MA. We enrolled patients with an acute-subacute unilateral hemispheric infarction, day 2-day 6 since the qualifying stroke, between 21–90 years of age, with moderate to severe dysphagia, defined as a Penetration And Aspiration Scale23 (PAS) score ≥ 4 on a standardized videofluoroscopic study (VFSS). The main exclusion criteria were pre-stroke swallowing difficulties, severe stroke (NIHSS score ≥ 25), or presence of any other contraindication for tDCS22.FEASt is an early phase-2 double-blind, single-center RCT conducted from September 2013–September 2019. The rationale, design and protocol of the trial have been published previouslySubjects were randomized to 1 of 3 intervention arms–a Low-Dose tDCS, a High-Dose tDCS, or a Sham group, using computer based randomization stratified according to the baseline PAS scores (4–6 versus 7–8). Randomization codes were generated electronically by an independent study Data Coordination Center (DCC). Three individuals, not involved in any other study procedure received confidential electronic notification about the randomization codes and programmed the tDCS device accordingly. They also interrogated the device after each session to verify its fidelity to the stimulation allocation. All de-identified VFSS were sent electronically to the Boston University Medical Center laboratory for detailed analysis and results were fed directly to the DCC. These processes ensured complete concealment of intervention allocation and blinding of all investigators and subjects.24 and underwent a swallow evaluation by a Speech-Language pathologist (SLP) if suspected of having dysphagia. Those with moderate to severe dysphagia (PAS ≥ 4) on a standardized VFSS evaluation were enrolled if they fulfilled all study criteria. The VFSS protocol employed 3 consistencies  and 5 swallows at trial enrollment (baseline) and after the 10th or last stimulation session (post-stimulation). The arithmetic mean of the PAS score was computed for each subject (composite PAS score) at these time points. A Functional Oral Intake Scale (FOIS) score25 was computed by the study SLP at baseline, after 5th and final session; in addition, at 30-days after the index stroke, an investigator, blinded to the intervention allocation, collected a FOIS score over the phone or in-person using a standardized questionnaire with the subject or a legally authorized representative familiar with subject’s current dietary status. An investigator certified in the performance of the NIH Stroke Scale (NIHSS)26 assigned scores for each participant at enrollment, after every second stimulation sessions and after the last session. All subjects underwent a brain MRI with diffusion weighted imaging (DWI) prior to enrollment, which were used to create a binary map of the acute stroke lesion and construct a novel radiological variable, the corticobulbar tract-lesion load (CBT-LL) (below).All consecutive acute ischemic stroke (AIS) patients admitted to BIDMC were screened for trial participation. All patients received a standardized dysphagia screening using a 3-oz water swallow test22 from baseline to post-stimulation.. PAS is specifically designed to measure the depth of airway invasion from a swallow bolus (degrees of penetration and aspiration) and the compensatory response to expel this bolus from the airways 23. We had selected PAS scores as a primary measure of efficacy since aspiration has been reported to be a major contributor to pneumonia and dietary restriction after stroke27. We supplemented our assessment of intervention efficacy with our secondary outcomes which included objective measures of dietary improvement assessed by a change in FOIS scores 30-days post-stimulation25 and changes in swallowing physiology parameters on VFSS from baseline to post-stimulation 30. The primary safety outcomes were incidence of seizures, deterioration in global neurological functions , motor functions (≥ 2 points increase in the motor sub-item of the NIHSS score on the same limb), swallowing functions (increase in PAS score by ≥ 2 points compared to baseline), and stroke specific mortality during the period of active stimulation.The primary efficacy outcome was a change in PAS scores based on VFSS31. Overlaying the manually drawn lesion maps derived from the acute DWI sequences of trial participants onto this canonical probabilistic CBT allowed us to calculate a CBT-lesion-load variable. This overcomes previous shortcomings in describing lesion volume and lesion location by creating a combined lesion load variable that combines lesion volume and relevant lesion location that overlay a relevant system such as the CBT-lesion load. For clinical interpretability, the CBT-LL variable was dichotomized for each subject based on their lesion-load measures , into 2 groups  to assess for effect modification of this variable on the experimental intervention.The CBT-LL variable was derived by creating a canonical tract of the corticobulbar tract in spatially standardized space using the swallowing related cortical fMRI activation and the posterior pons as seed regions. The fMRI experiment consisted of repeated swallowing trials contrasted with whole hand opening and closing tasks, done at the same frequency as the swallowing tasks. The fMRI sequences were acquired with a gradient-echo T2*-weighted MR pulse sequence using our own modification of a sparse temporal sampling method with clustered volume acquisition to overcome imaging artifacts caused by swallowing motion. The voxel clusters of significant cortical activation were used as a seed region  with the second seed region in the posterior pons. The DTI scans were high resolution studies obtained in 12 healthy elderly controls. Image acquisition, analysis, construction of corticobulbar tracts and computation of CBT-lesion load were performed as previously described by Zhu et al.32. This cut-off corresponds to a dietary intake of a single consistency or worse and demonstrated to correlate with significant reduction in protein, energy and fluid intake, and suggested as an indication for gastrostomy tube placement after stroke35.We performed a post-hoc analysis using a FOIS score threshold ≥ 5 as clinically meaningful improvement in dietary intake based on a recent noteworthy publication that utilized this threshold to develop an instrument for prognosticating dysphagia recovery21. The electrode positioning was re-confirmed in a subset of trial participants using anatomical brain MRI scans. The atDCS electric field (V/m) distribution was modeled using a freely available software package called SimNIBS36  or sham was delivered via a battery-driven, constant current stimulator (NeuroConn-DC Stimulator Plus) using saline soaked electrodes . We targeted the healthy swallowing motor cortex using the 10–20 EEG electrode placement system, placing the anode mid-distance between C3/T3 [left] or C4/T4 [right] over the unaffected hemisphere and the reference electrode over the contralateral supraorbital region. We had previously verified the location of the stimulating electrode using a combination of functional brain MRI (fMRI) and anatomical brain MRI scansThe High-Dose tDCS group received 2 mA atDCS twice daily for a total of 20 min ; Low-Dose group received 2 mA alternating with sham stimulation daily for a total of 20 min  and Sham group received sham stimulation twice daily.38. All study SLPs and investigators were trained by an expert (SL) on eliciting these maneuvers. Occurrence of an effortful swallow was verified by a laryngeal microphone attached to the patient’s throat. We aimed to obtain 40 effortful swallows during each session, which were recorded and entered into the study website.All stimulation sessions were conducted concurrently with standardized effortful swallowing exercisesWe planned to randomize 99 subjects- 33 in each arm, estimated to detect a difference of 1.0 and 1.15 standard deviations (SD) between groups in the mean primary outcome measure with a type I error rate of 2.5% and power 80% and 90%, respectively.The primary efficacy analysis was performed in an intent-to-treat approach. Our outcome variable was a change in the arithmetic mean PAS scores , before and after intervention and compared across groups. We used a linear model using PROC GLM in SAS to analyze the mean change in PAS scores with the intervention after adjusting for baseline NIHSS scores and age, which were included as covariates based on clinical considerations after screening for other confounders. Similar analyses were performed on the dietary outcome (mean change in FOIS scores at 30-days) and other secondary analysis on changes in swallowing physiology . The incidence of safety outcomes were captured as a whole and compared separately across groups. Intraclass correlation (ICC) for PAS scores was performed on a subset of VFSS  between 2 reviewers (JP and SL). Regression models using interaction terms were used to assess for heterogeneity of intervention effects across the CBT-LL.39; 2) the total number of experimental sessions (10 or < 10) for every subject was used as a covariate in an adjusted analysis for our primary outcome; 3) Fisher-exact test was conducted to compare the proportion of subjects with a FOIS score ≥ 5 in the combined atDCS group versus sham. Treatment comparison p-values were considered statistically significant at the two-sided 0.05 level of significance; interaction p-values were considered statistically significant at the 0.15 level of significance given the relatively low power for tests of interaction to detect a true interaction. SAS Version 9.4 was used to carry out all analyses.In addition, we conducted the following post-hoc analysis: 1) PRESS score, a recently validated prognostic tool for spontaneous swallowing recovery after an AIS, was computed for each participant and incorporated in an adjusted analysis for the efficacy outcomesPresented in part at International Stroke Conference, February 20, 2020; Los Angeles, CA and American Neurological Association, October 8, 2020.p = 0.78); male , female  (p = 0.54); 218 (76.2%) white and 48 (16.8%) (p = 0.86) The enrolled cohort had moderately severe strokes at study entry . There were 3 deaths (one per arm) and 1 subject was lost to the 30-day follow-up. Overall 18 (43%) subjects completed all 10 sessions and 41 (98%) completed at least 5 stimulation sessions. Forty effortful swallows were completed in 291/ 382 (76%) stimulation sessions. No subject withdrew consent or stopped stimulation due to discomfort; 38 (90%) completed a 30-day follow-up. The mean times in hours (SD) from stroke onset to initiation of experimental intervention was 93.5 (38.8), 101.6 (32.5), 84.1 (34.7), in sham, low-dose and high-dose tDCS groups, respectively (p = 0.45).There were no major protocol violations.A total of 328 subjects were screened for the trial and 42 were enrolled  . In the post-hoc analysis adjusting for baseline PRESS score, the mean changes in PAS scores were − 0.97 (0.34) in sham, − 0.83 (0.36) in Low-Dose and − 0.31 (0.36) in High-Dose; pairwise differences (p > 0.40). The mean change in PAS scores between subjects completing 10 sessions (n = 18) versus those who did not (n = 24), were − 0.99 (SD 1.44) and − 0.49 (SD 1.41), respectively; after adjustment for age, baseline NIHSS and PAS scores, the least square estimate of the difference between group mean was − 0.57 (SE ± 1.41), p = 0.22. The ICC of the raw PAS scores on randomly selected 303 swallow evaluations between the 2 evaluators was 0.67.The relevant parameters for our primary efficacy outcomes are summarized in Table p > 0.15). A post-hoc analysis of FOIS scores at 30-days showed that 22 /24 subjects in the combined tDCS group had a FOIS score ≥ 5 compared to 8 /14 in sham .The relevant parameters for our dietary outcome are outlined in Table p = 0.116) and FOIS (p = 0.017) outcomes , PDT  and HLPE (semi-independent movements that close off the airway and shorten the pharynx)24. A theoretical criticism of this scale has been that patients may advance their diet despite persistent swallowing impairment and professional recommendations. We believe this is less likely to have influenced our result. All subjects were randomized and had been clinically stable at their respective dietary levels, the assessors were blinded and computed the scores based on a structured interview. Higher doses of atDCS were associated with higher FOIS scores but group differences were not statistically significant though this comparison may have been underpowered. A post-hoc analysis for minimal clinically important difference (MCID) in dietary intake using a Fisher-exact test , revealed significantly better intake at 30-days in the combined atDCS compared to sham group43.The overall objective of dysphagia therapies is to safely restore a normal diet. Unlike, PAS, which primarily assesses pharyngeal and laryngeal events during swallowing, changes in diet, as measured by FOIS scores can provide a more holistic assessment of swallowing functions. FOIS is a functional outcome that provides a validated measure of dietary level, is sensitive to changes in swallowing functions and has been tested and validated in stroke population9. We expected that other factors might influence the response to our intervention44. We used the CBT-LL variable  to help create a combined variable of lesion size and relevant lesion location . This combined variable overcomes limitations of previous lesion descriptions that relied mostly on lesion volume or the relative components of gray- and white matter in a lesion. We used CBT-LL to better understand the variability in response and found a significant heterogeneity of intervention effect across this variable. These findings are hypothesis generating but suggest that the effects of atDCS are primarily intracortical and its effects on swallowing behavior can be influenced by the extent of damage to the corticobulbar tracts with more damage to one CBT making the contribution and modulation of activity in the other intact hemisphere more important. We anticipate that novel radiological variables such as the CBT-LL may help stratify patients in future studies and assist in predicting responses to experimental interventions in future RCTs.Dysphagic stroke patients usually have more severe deficits and overall fare poorly compared to their non-dysphagic counterparts45. Future trials will require a better selection of swallowing parameters as well as further optimization in their acquisition and analysis. It will also need validated metrics to identify minimal clinically important differences (MCID) in meaningful change in swallowing functions. There was suggestion of a dose effect reflected in some of the swallowing measures but not in others. This needs further corroboration and exploration of other doses that may be more effective.A significant limitation was accruement of lower than anticipated sample size during the funding period that may have affected the power to detect our efficacy endpoints. Analyses of VFSS based outcomes showed significant variability and our main outcome PAS had suboptimal ICC, which may have undermined our ability to detect a real change. While PAS scores assignments based on VFSS captures the subject’s ability to swallow a limited number of prepared boluses without aspirating it does not translate to true functional swallowing ability in a natural environment. FOIS scores, on the other hand, identifies the patient’s current diet level but does not assess dysphagia related quality of life. Our post-hoc analysis for investigating intervention effects using dichotomized FOIS scores however are susceptible to type I error ; they should therefore be considered hypothesis generating and require further confirmation. Including other outcome measures, such as the Swallowing Quality-of Life could have been a useful adjunct for capturing the subjective perceptions of wellbeing in our trial cohort39. A shorter course of in-hospital stimulation, if effective, also carries pragmatic implications, based on observations from FEASt where most subjects were able to complete 5 sessions but adherence declined for more sessions. Meta-analyses from separate studies on motor recovery have suggested a dose–effect relationship although only studies up to 2 mA were included this metanalysis46. More recent investigations have revealed that higher dose tDCS (4 mA) is feasible, well tolerated, safe, and leads to stronger activity changes48. Furthermore, there is growing recognition that improvements in modeling of tDCS induced electrical fields in the human head and utilization of neurophysiological techniques to account for variability in skull conductance can help optimize stimulation parameters for individual subjects49.The results of FEASt demonstrate that application of atDCS to the unaffected hemisphere paired with swallowing exercises in acute stroke patients with post-stroke dysphagia is safe and feasible though it did not decrease the risk of aspiration events in this early phase-2 RCT. It improved dietary intake, which needs further validation in a larger trial. The science behind non-invasive brain stimulation has continued to advance since the inception of this trial. A recent single-center trial demonstrated that 1 mA of daily tDCS significantly improved swallowing function in AIS patients after 4 sessionsIt is necessary for future trials to consider modifications in stimulation parameters including alternate atDCS doses that incorporate accurate models of electric potential distributions and neurophysiological data, and use of clinically meaningful swallowing outcomes. The heterogeneity of intervention effects across CBT-LL observed in this trial merits further investigation and can be used to stratify subject enrollment and analysis. Our trial experience highlights challenges but also the promise of this technique in improving stroke related dysphagia, which should be investigated further in a confirmatory RCT. A major attraction of this technique is its ease of use, portability, safety profile, and low-cost. It is feasible to use it in multiple settings including at patient’s home using remote monitoring or device settings that allow safe administration. Therefore, if proven effective, this method can have a high penetrance not only in academic, tertiary care settings but can also be scaled up for other outpatient settings.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.