To evaluate the acceptability and usability of the Hear Glue Ear mobileapplication to guide families and support speech and language development inchildren with otitis media with effusion (OME). To assess the validity ofthe app’s game-based hearing test to estimate changes in hearing levelsbetween audiology appointments.This evaluation examined 60 children aged 2–8 with and without OME, attendingCambridge Community Audiology clinics. Children’s performance in the app’shearing test was compared to their pure tone average (PTA) obtained inclinic. Children and caregivers completed questionnaires after their firstinteraction with the app, and after one week of using it at home. 18clinicians completed anonymous questionnaires after trialling the app.Results from the app’s hearing test show a significant correlation withclinic PTA values (Hear Glue Ear is acceptable to children, caregivers and clinicians as part ofOME management. The app’s hearing test provides a valid estimate offluctuating hearing levels. Hear Glue Ear is a free, accessible andfamily-centred intervention to provide trusted information and supportdevelopment, as NICE guidance recommends. This can impair thetransfer of sound to the ossicles, resulting in a conductive hearing loss.Children with cleft palate, skeletal dysplasia and other conditions such asDown’s Syndrome are at increased risk of developing OME.2 In most children, OME resolves spontaneously after an average of 6–10 weeks,3 but in some cases it may result in a chronic hearing loss which cansignificantly impact quality of life.4Otitis media with effusion (OME), also known as glue ear, is the leading cause ofchildhood hearing loss worldwide and affects an estimated 1 in 5 pre-schoolchildren in the UK at any one time.5 In particular lower frequency inputs that correlate to many speech soundsare often compromised, as illustrated in 6 In addition, the ages with highest prevalence of OME coincide with acritical window for speech and language development.7 As a result, auditory processing and learning can be adversely affected,and are often associated with social and behavioural difficulties which canpersist into teenage years.811 The diagnosis of OME isfrequently overlooked due to its non-specific presentation, resulting in manychildren being prematurely labelled with behavioural or educational issues. TheNational Institute for Health and Care Excellence (NICE) informs that hearingloss from OME can create social challenges for children and place strain on families.12 This is corroborated by feedback during PPV (Patient and Public Voice)research preceding this study, which revealed anecdotally that many parentsstruggle to manage their child’s behaviour while their hearing is reduced fromOME. Often this is due to challenges differentiating between whether their childneeds clear parental boundaries or is simply exhausted from struggling to listenand communicate at school.Chronic OME is associated with long term speech and language impairments, sinceknowledge about syntactic structure, which is critical for language learning,may be impaired by reduced exposure to acoustic-phonetic stimuli.12 If OME persists after this interval, the child may be referred to Ear,Nose and Throat (ENT) for consideration of hearing aids or ventilation tubes(grommets). The period of ‘watchful waiting’ for spontaneous remission combinedwith referral delays can result in children with persistent OME spending asignificant period of time with reduced hearing. In addition, OME has a highrecurrence rate, which results in many children being discharged from follow-upservices during a temporary period of resolution, only to deteriorate again.Review of the current literature highlights a need for additional supportbetween appointments, in order to minimise delays in speech and languagedevelopment. 60% of parents of children with mild or moderate hearing lossreport needing more support for their child.13 Actively supporting speech and language development following OMEdiagnoses could improve learning and behaviour in the long term. Behind-the-Ear(BTE) hearing aids during this period are unpopular due the fluctuating natureof the hearing loss and the challenge of selectively amplifying low frequency sounds.14,15 As an alternative or adjunct, NICE guidance highlights the need for“educational and behavioural strategies to minimise the impact of hearing loss”and tools to guide families on how to support their child from home.12 In particular, during the current COVID-19 pandemic, face-to-faceappointments and surgical interventions are restricted, so there is demand fromfamilies and clinicians to facilitate continuity of care by supportingchildren’s development and auditory processing remotely. The Hear Glue Ear appaddresses this need by providing focused activities to support children as wellas trusted information to guide families while their child’s hearing is reducedfrom OME.As summarised in 16 The role of mobile applications for healthcare is rapidly expanding, with regulation and review offered by the NHS App Libraryand ORCHA , inorder to offer doctors guidance and security with recommending apps. A number ofapplications exist for adult audiology , but there are currently very few high qualityapps addressing childhood hearing loss.17 This is surprising considering the increasing presence of technology inschools and evidence showing that children’s engagement with speech and languagesupport is increased when it is in electronic form.18 Barriers to the use of mobile applications for childhood hearing loss mayinclude accessibility and compliance in children and parental concerns aboutscreen time.19 There are concerns that increased screen time may adversely impact speechdevelopment in young children,20 although this association has been contradicted by a recent study.21 A key aim of this current study is to assess whether these concerns posesignificant barriers to the uptake of Hear Glue Ear, which is designed tosupport speech and language development as well as auditory processing andlistening skills.Health applications are an increasingly popular and cost-effective way forpatients to monitor, report and self-manage health conditions from home.3 The features of the app aim to meet NICE recommendations for supportingspeech and language development during management of OME3 and to address specific user needs, as summarised in 22 Hear Glue Ear is free to download from Apple and Android app stores, thusaddressing a demand for affordable support for children with hearing loss. Thiswas prioritised by the makers of the app since studies across several countrieshave shown that the impact of OME is greater in lower income families.23,24Hear Glue Ear was designed using a multidisciplinary and user-centred design(UCD) approach following recommendations from speech and language therapists,audiologists, ENT surgeons, paediatricians, parents and teachers of the deaf.The user interface is designed to be informative and accessible for children andcaregivers and is displayed in The Hear Glue Ear application contains a game-based hearing test, designed toprovide parents with an estimate of their child’s hearing level betweenaudiology appointments. It is known that hearing loss due to OME fluctuates, andas such it is often difficult for parents and teachers to identify when achild’s challenging behaviour is due to frustration and fatigue from strugglingto hear. This challenge may be so significant that some children with OME can beinitially referred for assessment of autism and learning disabilities. The HearGlue Ear app’s hearing test offers parents an estimate of their child’s hearinglevel between appointments, in order to gain confidence with managing theirbehaviour. It is not intended to be diagnostic of hearing loss or to replaceclinical audiology assessment.26 In paediatric patients, gamification within mHealth has been shown toincrease engagement and compliance with healthcare-related activities.27,28 The game-based hearing test within the Hear Glue Ear app presentschildren with a series of pure or warble tones at frequencies and volumescomparable to those used in standard pure tone audiometry tests . These arepresented within a game design, where the child is shown cartoon animals inwindows and must tap on an animal and wait for a sound. The test uses a yes/noparadigm where the child selects an option depending on whether or not theyheard the sound, which is recorded by the app. There is no positive or negativereinforcement following a child’s response to avoid biasing future responses.The frequency and volume of each sound is randomly distributed and variedbetween tests in order to avoid sequence learning, and there are always two muteanimals per game to discourage users from arbitrarily selecting ‘yes’ responses.The number of correct and incorrect answers per attempt is visible to parentsand clinicians  in the analytics section of the app asmatrix of volume against frequency, as shown in ‘Gamification’ is the process of applying game-design elements into a non-gamecontext in order to engage users and promote positive behavioural change.During the app set-up, the test environment is calibrated and the hearing test isallowed to proceed if the ambient noise is 35 dB or less, as shown in 29 This is thought to result from differences in the anatomy of theEustachian tube and increased susceptibility to middle ear infections. Childrenwith Down’s Syndrome frequently experience challenges with learning, speech andlanguage, and prolonged hearing impairment due to chronic OME can exacerbatethese developmental delays.30 Furthermore, there are significant discrepancies in access to hearingcare across the UK which can create variation in the speed of identification andthe level of speech and language support received.31 The Hear Glue Ear app could potentially provide remote speech, languageand listening support regardless of location for children with Down’s Syndrome,in order to minimise developmental delay.Numerous studies have shown that children with Down’s Syndrome have a higherprevalence of OME.Ethical approval was obtained from Wales REC 7 (IRAS ID: 262154).The study involved 60 children  aged 2–8 years oldand their accompanying caregiver(s), attending Cambridge Community PaediatricAudiology clinics. All children were attending the clinic with suspected orpreviously diagnosed OME. Following assessment, 25 children had an OME diagnosisand 35 did not. Additionally, 6 children had a diagnosis of Down’s Syndrome andno current OME diagnosis. 1 child had an unspecified learning disability otherthan Down’s Syndrome. The study also included 18 clinicians working inCambridgeshire Community Services (CCS), which consisted of paediatricians(n = 11), audiologists (n = 5), a GP and a paediatric psychologist.The experimental protocol is summarised in Children were allowed to navigate the app by themselves from the home screen andwere shown 2 audiobooks and 2 songs. They were also shown the ‘How’s MyHearing?’ hearing test and encouraged to complete it once. Children werepresented with an oral questionnaire on their impressions of the app, whilsttheir accompanying caregiver was given a written questionnaire to complete. Oneweek later, families who had consented were followed up via telephone or emailin order to assess level of uptake of the app and families’ experience of usingthe app at home. Not every family completed all of the stages within theprotocol due to limited time availability, children’s concentration andcompliance and families’ responsiveness to follow-up. Clinicians were shown theapp in person by a researcher or via an emailed link and were sent aquestionnaire which they completed and returned anonymously, in order tominimise bias.In order to assess the validity of the app’s game-based hearing test, the resultsobtained on the app were compared with the pure tone audiometry results obtainedby each child in clinic. The child was encouraged to complete the app’s hearingtest on their first interaction with the app and the percentage score of correctanswers was recorded by the researcher. The app’s hearing test presents soundsof 500, 1000, 2000 and 4000 Hz frequencies and between 20 and 70 dB pitch. Eachchild’s hearing level in the audiology clinic was assessed using pure toneaudiometry. This presents each child with sounds at 500, 1000, 2000 and 4000 Hzand the minimum audible volume in decibels is recorded. The pure tone average(PTA) is the mean of the minimum audible volumes in decibels heard at all 4frequency levels. Thus, the lower the PTA value, the ‘better’ a child’s hearinglevel. The PTA obtained in audiology clinic was compared to the percentage scoreobtained in the app’s hearing screen. Since all families chose to take part inthe study on the same day as their appointment, there was a maximum delay of30 minutes between audiometry assessment in clinic and completing the app’shearing test. Transient fluctuations in hearing level over such a short periodare unlikely to affect results and therefore need not be taken intoconsideration in statistical analysis.32 The child and caregiver preliminary questionnaires are included in onlineAppendix B.The children’s questionnaires consisted of 15 yes/no questions assessing theacceptability and accessibility of the app. The caregiver questionnaireconsisted of 18 written questions using a Likert scale (response agreement wasgraded from 1–5) to assess accessibility, acceptability and usability forfamilies. Free text spaces were available to express additional views on appimprovement. The first 13 questions of the caregiver and clinicianquestionnaires follow the standard SUS  for evaluationof an application, as used in previous app evaluations.The follow-up questionnaire consisted of 12 mixed yes/no and free-text questions.These assessed levels of uptake, engagement, use of specific features andfeedback on the appropriateness of the app for the home environment. The carerfollow up questionnaires are included in online Appendix B.The anonymous questionnaires given to clinicians consisted of 19 questions usinga Likert scale (1–5 grading) and free text space to assess the acceptability tohealthcare professionals of including the app within clinical pathways. Theclinician questionnaire is included in online Appendix B.33 The ‘app mean quality score’ is calculated as a mean average of theresponses to 19 questions in the categories ‘engagement’, ‘functionality’,‘aesthetics’, and ‘information quality’, and an ‘app subjective quality score’is calculated as a mean average of the responses to 4 questions assessingsubjective opinions of the app.10 adults (consisting of clinicians working in CCS and caregivers whose childrenwere attending paediatric audiology services) were shown the app and asked tocomplete a MARS questionnaire, which is included in online Appendix C. This is averified app-specific evaluation tool consisting of 23 questions using a Likertscale (graded 1–5).A Spearman rank correlation coefficient was used to calculate the relationshipbetween percentage scores obtained in the app’s hearing test and the pure toneaverage obtained in the audiology clinic for each child (where p < 0.05 issignificant). The results of each questionnaire were recorded as percentages ofoverall responses, with no further statistical analysis performed.34 The modal age of participants was 4 years old, which reflects the age ofhighest prevalence of OME.7 The inclusion of children with both glue ear (41.7% (n = 25)) and normalhearing (58.3% (n = 35)) diagnoses in the study group reflected the fluctuatingnature of hearing loss in glue ear, and enabled assessment of the app’susability during times of both impaired and normal hearing, both of which areseen in the watchful waiting period.60 children between the ages of 2 and 8 years old participated in the study. Nearequal gender distribution (51.7% (31/60) male; 48.3% (29/60) female) was ensuredto avoid skew, since mobile device use has been shown to vary with gender.There was a significant negative correlation between the percentage scoreobtained in the app’s game-based hearing test and the pure tone average (PTA)obtained in clinic for each child  and uses between appointments (73.9%) demonstrate families’enthusiasm for this type of support and suggest that parental concerns about screentime do not deter app uptake. These concerns may be partially assuaged by therecommendation of the app by a trusted clinician.Despite the growing popularity of mHealth, the number of high quality applicationsaddressing children’s speech and language development is relatively small.7 and confirms that the app is accessible for the most affected patient group.Children in this age group’s proficiency with using mobile apps may reflect theincreasing integration of mobile and tablet-based technologies in schools. In fact,a family whose 7 year old child had a Down’s Syndrome diagnosis commented that theapp was ‘too basic’, since she frequently used apps with more complex games andgraphics. This contrasted with the response of an 8 year old’s family who felt thatthe app was well-pitched for their child. These anecdotal findings were seenrepeatedly and suggest that within this age range, engagement with the app may beless dependent on chronological or development age, but rather on prior exposure tomobile applications. The majority of caregivers (82.6%) reported that their childseemed to be as engaged with the activities on the app as with other activities,which further demonstrates its appeal to children. This may be attributed to the‘gamification’ of the app’s interface and hearing test, which is well-known toincrease uptake and compliance with mHealth in children.27,28The high uptake of the app may also be explained by its practical design andaccessibility. In this study, 93.3% of children thought the app was easy to use and73.3% thought they could use it without an adult’s help, across the age range of2–8 years old, for which the app was designed. This range reflects the ages withmaximum prevalence of OME35 The high rate of uptake of the Hear Glue Ear app is therefore encouraging andmay be partially attributed to the app’s portability and ease of access: familiesreported using it most after school, before bed or during car journeys. The appprovides a focussed and reliable tool to guide caregivers to support their child,which may empower families to take a more active role in their child’s speech andlanguage development. It is known that increased parental involvement improvesspeech outcomes in children with hearing loss.25 Therefore a family-centred approach during the ‘watchful waiting’ period maysignificantly improve outcomes.Another perceived barrier to app use at home is families’ time availability. A 2015study by the National Literacy Trust showed that only 29% of caregivers read totheir child for more than 15 minutes per day, and this rate was influenced byfactors such as parental level of education.18The majority of clinicians (77.8%) agreed that they would like their patients to usethe Hear Glue Ear app regularly between appointments, demonstrating itsacceptability within the OME clinical pathway. However, only 55.6% felt that the appcould enable more accurate long term information about hearing abilities betweenappointments. In free-text spaces, clinicians explained their hesitance by concernsabout conveying the outcome of the app’s hearing test as a percentage score, sinceparents may interpret this as percentage hearing loss, which the app has not beenshown to reliably assess. To address this concern, the app display has been alteredto no longer display percentage scores to parents and a safety netting pop-upmessage clearly states that the test is not diagnostic or intended to replaceclinical audiology assessments (as shown in diagnose hearing loss). Any discrepancy which remains betweenthe app hearing test and clinic PTA results may be explained by normal variance inPTA values, since studies have shown a +/− 10 dB variance between PTA values takenwithin a short time of each other is normal and expected, due to testing variation.36,37 Furthermore, the app hearing test was performed a maximum of 30 minutesfollowing clinic assessment, which is insufficient time for fluctuations in actualhearing level to have influenced test results. The performance in the app’s test ofchildren with the poorest hearing levels results showed a significant correlation (39 A considerable advantage to the use of mobile health apps is the ability toupdate and improve in line with best practice, at minimal additional cost orinconvenience to end users. Following increasing appreciation of these benefits,technology in the field of paediatric audiology is rapidly evolving and exciting newinnovations have the potential to support and guide family-centred care withinmanagement pathways, as NICE advocates.12This study demonstrates that, despite the perceived barriers to the uptake of mHealthin the paediatric population, the Hear Glue Ear app is acceptable and accessible tochildren, their caregivers and clinicians working in the field, and its quality isverified by the MARS assessment. The app’s game-based hearing test is also validatedas a means of estimating trends in a child’s hearing level between appointments. Theaccessibility of the app depends on access to Wifi and ability to navigate its userinterface, and is unaffected by geographical location or ability to pay. Inscenarios where face-to-face appointments are limited, as in the current COVID-19pandemic, the app may provide ongoing support and continuity of care to familiesremotely. There is also the potential for use in low resource settings, where it mayprovide an estimate of changing hearing levels and support speech and languagedevelopment in communities with limited access to audiology services. Furthermore,there is the future possibility of using the app in conjunction with noveltechnologies such as bone-conducting headsets via Bluetooth, to further improveaccess and benefit for children with the most severe conductive hearing losses.


Histological lymphocytic reaction is regarded as an independent prognostic marker in colorectal cancer. Considering the lack of adequate statistical power, adjustment for selection bias and comprehensive tumour molecular data in most previous studies, we investigated the strengths of the prognostic associations of lymphocytic reaction in colorectal carcinoma by utilising an integrative database of two prospective cohort studies.KRAS, BRAF and PIK3CA mutations, and tumour neoantigen load.We examined Crohn’s-like reaction, intratumoural periglandular reaction, peritumoural reaction and tumour-infiltrating lymphocytes in 1465 colorectal carcinoma cases. Using covariate data of 4420 colorectal cancer cases in total, inverse probability-weighted Cox proportional hazard regression model was used to control for selection bias (due to tissue availability) and potential confounders, including stage, MSI status, LINE-1 methylation, PTGS2 and CTNNB1 expression, Ptrend < 0.002). Compared with cases with negative/low intratumoural periglandular reaction, multivariable-adjusted HRs were 0.55  in cases with intermediate reaction and 0.20  in cases with high reaction. These relationships were consistent in strata of MSI status or neoantigen loads (Pinteraction > 0.2).Higher levels of each lymphocytic reaction component were associated with better colorectal cancer-specific survival (The four lymphocytic reaction components are prognostic biomarkers in colorectal carcinoma. Therefore, a comprehensive study focusing on the prognostic role of lymphocytic reaction and its relationship with the aforementioned molecular features is needed.Host immune response in the tumour microenvironment plays a critical role in regulating cancer initiation and progression.22 (on the 4420 cases), which has not been used in the previous prognostic studies of immune response to tumour. In addition, we examined statistical interactions between lymphocytic reaction and MSI status or neoantigen load.In this study, we utilised two large US-nationwide prospective cohort studies with covariate data of 4420 colorectal cancer cases, and a molecular pathological epidemiology database of 1465 cases, to evaluate the relationships between lymphocytic reaction patterns and patient survival. We hypothesised that more intense host lymphocytic reaction to colorectal cancer might be associated with a favourable clinical outcome, after adjusting for other potential confounders including neoantigen load. To reduce potential bias due to the availability of tumour tissue, we utilised inverse probability-weighting (IPW) method23 Every 2 years, study participants have been sent follow-up questionnaires to collect information on lifestyle factors and medical history of physician-confirmed diseases including colorectal cancer. The National Death Index was used to ascertain deaths of study participants and identify unreported lethal colorectal cancer cases. Participating physicians reviewed medical records to confirm diagnosis of colorectal cancer, and to record tumour characteristics  classification), and causes of deaths for participants who were deceased. Formalin-fixed paraffin-embedded (FFPE) tissue blocks were collected from hospitals where participants diagnosed with colorectal cancer had undergone tumour resection. We included 1465 patients with available data on at least one of four histopathological lymphocytic reactions. We included both colon and rectal carcinomas based on the colorectal continuum model.24 Patients were followed until death or the end of follow-up , whichever came first. Informed consent was obtained from all study participants. This study was approved by the institutional review boards at Harvard T.H. Chan School of Public Health and Brigham and Women’s Hospital , and those of participating registries as required.We collected data on colorectal cancer cases within two prospective cohort studies in the United States, the Nurses’ Health Study  and the Health Professionals Follow-up Study .5 Tumour differentiation was categorised as well to moderate vs. poor . Four components of lymphocytic reactions  were examined  showed good concordance on grading of histopathological features, including lymphocytic reaction to tumour.5 For the analyses of lymphocytic reaction and patient survival in strata of tumour neoantigen load, each of the four lymphocytic reaction components was graded as negative/low (0) and intermediate/high . The overall lymphocytic reaction score (0–12) was calculated as the sum of scores for the above four reaction components, and was graded as low (0–2), intermediate (3–6) and high (7–12).FFPE blocks of tumour tissues were collected from hospitals throughout the United States, where colorectal cancer patients had undergone surgical resection. A single pathologist (S.O.), who was unaware of other data, reviewed haematoxylin- and eosin-stained tissue sections, and recorded histopathological findings, including tumour differentiation and lymphocytic reaction components, as previously described.ned Fig. . Crohn’s24 MSI-high status was defined as the presence of instability in ≥30% of the markers, and non-MSI-high as instability in <30% of the markers, as previously described.26 DNA methylation was measured in eight CIMP-specific promoters  and in LINE-1.28 CIMP-high was defined as ≥6 methylated promoters of eight promoters, and CIMP-low/negative as <6 methylated promoters. PCR and pyrosequencing were performed for KRAS , BRAF (codon 600) and PIK3CA (exons 9 and 20), as previously described.24 Neoantigen load, the number of proteins that likely give rise to immunogenic peptides in the tumour microenvironment, was predicted for 505 cases, by using a neoantigen prediction pipeline for somatic mutations based on whole-exome sequencing, and identifying peptides that bind to personal human leukocyte antigen (HLA) molecules with high affinity (<500 nM), as previously described.29 Using NetMHCpan ,30 we predicted the binding affinities of all possible 9- and 10-mer mutant peptides to the corresponding HLA alleles inferred by the POLYSOLVER algorithm.Genomic DNA was extracted from colorectal cancer tissue in whole-tissue sections of archival FFPE tissue blocks. MSI status was evaluated using ten microsatellite markers , as previously described.31 Immunohistochemical analyses for PTGS2 (cyclooxygenase-2), nuclear CTNNB1 (beta-catenin) and CD274  were performed using an anti-PTGS2 antibody , an anti-CTNNB1 antibody  and an anti-CD274 antibody , respectively, as previously described.32We constructed tissue microarrays of colorectal cancer cases with sufficient tissue materials, including up to four tumour cores from each case.P values were two-sided. We used a two-sided α level of 0.005 for our primary hypothesis testing.33 Our primary hypothesis testing was assessment of associations of four lymphocytic reaction components (negative/low vs. intermediate vs. high) with colorectal cancer-specific survival in the Cox proportional hazard regression model. All other analyses, including evaluation of individual hazard ratio (HR) estimates, assessment of stratum-specific risk estimates and of interaction with MSI status and neoantigen load, represented secondary analyses.All statistical analyses were performed using SAS software , and all To assess the association between ordinal categories of the level of lymphocytic reaction  and other categorical variables, the chi-square test was performed. To compare continuous variables (age and LINE-1), an analysis of variance assuming equal variances was performed.19 Multivariable IPW-adjusted Cox proportional hazard regression models were used to adjust for potential confounders. The multivariable IPW-adjusted Cox proportional hazard regression models initially included sex , age at diagnosis (continuous), year of diagnosis (continuous), family history of colorectal cancer in any first-degree relative (present vs. absent), tumour location , disease stage (I vs. II vs. III vs. IV), tumour differentiation (well/moderate vs. poor), MSI status (MSI-high vs. non-MSI-high), CIMP (low/negative vs. high), KRAS (mutant vs. wild type), BRAF (mutant vs. wild type), PIK3CA (mutant vs. wild type), LINE-1 methylation level (continuous), PTGS2 expression (positive vs. negative) and nuclear CTNNB1 expression (positive vs. negative). A backward elimination was conducted with a threshold P of 0.05 to select variables for the final models. Cases with missing data  and tumour location (0.4%)) were included in the majority category of a given categorical covariate to limit the degrees of freedom of the models. For the cases with missing data on LINE-1 methylation (13.0%), we assigned a separate indicator variable. For cases with missing information on MSI status (11.6%), CIMP status (14.6%), KRAS mutation (14.6%), BRAF mutation (10.7%), PIK3CA mutation (17.1%), PTGS2 (15.7%) and CTNNB1 (35.8%), we assigned a separate missing indicator variable. We confirmed that excluding the cases with missing information in any of the covariates did not substantially alter the results (data not shown). For the analyses using a subset of cases with available neoantigen load data, we included neoantigen load (continuous) to the multivariable IPW-adjusted Cox proportional hazard regression models in addition to the aforementioned potential confounders. The proportionality of hazards assumption in colorectal cancer survival was assessed by a time-varying covariate, which was an interaction term of survival time and the level of lymphocytic reaction (P > 0.27). We observed evidence on violation of this assumption in the hazards for four lymphocytic reaction components and the overall lymphocytic score in overall survival. However, the Schoenfeld residual plots supported the proportionality of hazards during most of the follow-up period up to 10 years (data not shown), and thus, we used Cox regression models limiting the follow-up period to 10 years. Cumulative survival probabilities were estimated using the IPW-adjusted Kaplan–Meier method, and a linear trend in survival probabilities across ordinal categories of the level of lymphocytic reaction was assessed using the weighted log-rank test for trend. For analyses of colorectal cancer-specific survival, participants were censored at the time of deaths from other causes.We utilised inverse probability-weighting (IPW) method using covariate data of 4420 colorectal cancer cases with or without tumour tissue, to adjust for selection bias due to tissue availability.27In secondary analyses, we assessed the statistical interaction between levels of four lymphocytic reaction components (negative/low vs. intermediate vs. high) and each of following features: MSI status (high vs. non-high), neoantigen load (high vs. low), year of diagnosis (1995 or before vs. 1996–2000 vs. 2001–2008) and tumour location , using the Wald test in the multivariable-adjusted Cox proportional hazard regression model for colorectal cancer mortality. We estimated HR for a unit increase of each lymphocytic reaction component in strata of MSI status, neoantigen load, year of diagnosis and tumour location using re-parameterisation of the interaction term in a single regression model.21 Using the multivariable logistic regression model for the entire dataset of colorectal cancer cases (regardless of available tissue), we estimated the probability of the availability of tumour tissue, as previously described.25 Each patient with complete data was weighted by the inverse probability. Weights greater than the 95th percentile were truncated and set to the value of the 95th percentile to reduce outlier effects.21 We confirmed that the results without weight truncation did not change substantially (data not shown). The Cox regression analyses without IPW yielded similar results to the IPW-adjusted model.In all survival Cox regression analyses, the IPW method was applied to reduce the potential bias due to the availability of tumour tissue.19 In 1465 cases, we examined lymphocytic reaction patterns: tumour-infiltrating lymphocytes , intratumoural periglandular reaction (1462 cases), peritumoural lymphocytic reaction (1456 cases) and Crohn’s-like lymphoid reaction (1195 cases) . During the median follow-up time of 12.3 years  for all censored patients, there were 885 all-cause deaths, including 432 colorectal cancer-specific deaths.We used covariate data of 4420 rectal and colon carcinoma cases in the two prospective cohort studies for the inverse probability- weighting (IPW) method to adjust for selection bias due to tissue availability.s) Table . All of Ptrend < 0.002) and better overall survival (Ptrend < 0.009) in multivariable Cox regression analyses. Compared with cases with negative/low intratumoural periglandular reaction, multivariable-adjusted HRs for colorectal cancer-specific mortality were 0.55 , 0.42–0.71) in cases with intermediate reaction, and 0.20  in cases with high reaction. The Cox regression analyses without IPW yielded similar results to the IPW-adjusted model   had weaker associations with survival. This supported the value of grading different lymphocytic reaction components to generate a composite lymphocytic reaction score. Few other studies have evaluated the prognostic significance of such composite score, but some have reported that the individual components of the lymphocytic reaction, including TIL and Crohn’s-like lymphoid reaction, are independently associated with lower colorectal cancer mortality after adjustment for MSI status.8 In this study, with an expanded sample size (1465 cases) and additional important potential confounders , we identified a significant association of each of the four lymphocytic reaction components with colorectal cancer-specific survival independent of the potential confounders. In addition, only a few studies, including ours, evaluated “true” TIL that exists on top of tumour epithelium,7 whereas most studies have not distinguished lymphocytes in tumour stromal regions  from the true TIL. Thus, this study supports the robust prognostic value of both the overall lymphocytic reaction score and its four components, suggesting that the comprehensive characterisation of the lymphocytic infiltrate in different areal regions provides valuable information about the host antitumour immune response. Finally, IPW was used to minimise the potential selection bias caused by biospecimen availability.22 The IPW method can utilise the information from all the incident 4420 colorectal cancer cases within the cohorts during the follow-up period in order to produce less-biased estimation of the prognostic association of lymphocytic reaction. The differences of the results between IPW-applied analysis and analysis without IPW were minor, which suggests that the selection bias may not be a major concern in this dataset, and supports the robustness of our current analyses.Lymphocytic reaction has been demonstrated to reflect local immune effector response in colorectal cancer, associated with patient survival.43 The assessment of host immunity against colorectal cancer in the tumour microenvironment is increasingly important in the translational research, and biomarkers representing tumour molecular characteristics and the immune microenvironment are likely to be more and more included in the future tumour pathology evaluation criteria.44 Thus, integrated analyses of the immune response and tumour molecular features are necessary for the development of new immune biomarkers. In this study, we have included important confounders, including MSI, CIMP, LINE-1 methylation, KRAS, BRAF and PIK3CA mutation, PTGS2 expression, nuclear CTNNB1 expression and neoantigen load. Neoantigens are the most interesting targets for immunotherapies since neoepitopes are not subject to central tolerance in the thymus.45 Peptides of neoantigens bound to HLA can be recognised by T cells, which initiate antitumour immune response. Our study further supports the finding that neoantigen load is positively associated with higher lymphocytic reactions in colorectal cancer patients. Importantly, the benefit associated with higher lymphocytic reaction was not significantly modified by the neoantigen load and other molecular features, confirming the independent role of lymphocytic reaction in colorectal cancer survival. To the best of our knowledge, there has been no previous study on lymphocytic reaction and patient survival, which has controlled as many molecular variables as we did in this study.Colorectal cancer represents a heterogeneous group of tumours that result from not only a progressive accumulation of somatic molecular alterations, but also various host–tumour interactions, including antitumour immunity.48 Innate immune response also plays a crucial role in the tumour immune microenvironment, and may interact with adaptive immune cells.40 Further identification of these immune cell types and immunoregulatory molecules, driving each component of the lymphocytic reaction, could contribute to better understanding of the tumour immune microenvironment. Finally, we had limited information of tumour pathological features in this study. Pathological features, such as lympho-vascular invasion, extramural vascular invasion, perineural invasion and tumour budding, represent potential unmeasured confounding factors of the current analyses.51We need to point out several limitations in our study. First, there is limited data on cancer treatments in our study cohort. However, it was unlikely that clinical treatment decisions were influenced by lymphocytic reaction, because these data were not available to treating physicians. In addition, given the advances in colorectal cancer treatment, as well as differences between treatment strategies of colon and rectal carcinoma, we conducted stratified analyses according to year of diagnosis and tumour location. Second, data on cancer recurrences were not collected. However, colorectal cancer-specific mortality is considered as a reasonable colorectal cancer-specific outcome, since these two cohorts had a long follow-up duration of censored cases. Third, our study was based on evaluation of immune cells by haematoxylin- and eosin-stained tissue sections. Accumulating evidence suggests that specific immune cell types are differentially involved in host immune response.52 This population-based colorectal cancer database enabled us to rigorously examine the interactive prognostic value of lymphocytic reaction and each of lymphocytic reaction components, controlling for potential confounders. The molecular pathological epidemiology method has been utilised to assess the combined influences of exposures and immunity in cancer. In addition, compared with our previous study, an increased number of cases allow us to control for a larger group of confounders, and we utilised the IPW method to reduce the potential bias by the availability of colorectal cancer tissue.The strengths of our study include utilising the two independent US prospective cohorts, which covered data on pathological findings and tumour molecular features.In conclusion, a higher overall lymphocytic reaction score, along with four lymphocytic reaction components, is strongly associated with better colorectal cancer-specific survival, independent of MSI status, neoantigen load and other tumour and patient characteristics. Our population-based data support the role of host immune response as an independent prognostic indicator in colorectal cancer.www.genenames.org. Gene symbols are italicised whereas symbols for gene products are not italicised.We use HUGO (Human Genome Organisation)-approved official symbols (or root symbols) for genes and gene products, including BRAF, CACNA1G, CD274, CDKN2A, CRABP1, CTNNB1, IGF2, KRAS, MLH1, NEUROG1, PDCD1, PIK3CA, PTGS2, RUNX3, and SOCS1; all of which are described at Supplemental tables and figure


Age-related hearing loss (ARHL) is associated with cognitive decline as well as structural and functional brain changes. However, the mechanisms underlying neurocognitive deficits in ARHL are poorly understood and it is unclear whether clinical treatment with hearing aids may modify neurocognitive outcomes. To address these topics, cortical visual evoked potentials (CVEPs), cognitive function, and speech perception abilities were measured in 28 adults with untreated, mild-moderate ARHL and 13 age-matched normal hearing (NH) controls. The group of adults with ARHL were then fit with bilateral hearing aids and re-evaluated after 6 months of amplification use. At baseline, the ARHL group exhibited more extensive recruitment of auditory, frontal, and pre-frontal cortices during a visual motion processing task, providing evidence of cross-modal re-organization and compensatory cortical neuroplasticity. Further, more extensive cross-modal recruitment of the right auditory cortex was associated with greater degree of hearing loss, poorer speech perception in noise, and worse cognitive function. Following clinical treatment with hearing aids, a reversal in cross-modal re-organization of auditory cortex by vision was observed in the ARHL group, coinciding with gains in speech perception and cognitive performance. Thus, beyond the known benefits of hearing aid use on communication, outcomes from this study provide evidence that clinical intervention with well-fit amplification may promote more typical cortical organization and functioning and provide cognitive benefit. Age-related hearing loss (ARHL), or presbycusis, affects more than 30% of adults over age 50 years and its prevalence roughly doubles with each decade of life, making it the third leading chronic health condition among aging adults . HearingBeyond the well-known negative effects of ARHL on communication, quality of life, physical functioning, and psychosocial status, ARHL has also been linked to cognitive decline. For example, large-scale epidemiological studies indicate a strong association between ARHL and risk for mild cognitive impairment and dementia, as well as accelerated decline in cognitive function over time . Though Cross-modal re-organization is a form of cortical compensation observed in deafness and lesser degrees of hearing loss, whereby the auditory cortex is recruited or “re-purposed” by intact visual and somatosensory modalities . For exaIn this study, we used high-density electroencephalography (EEG) to record visual evoked potentials (CVEPs) in response to visual stimuli in a group of adults with mild-moderate ARHL and in age-matched normal hearing (NH) controls to assess the relationship between visual cortical neuroplasticity, speech perception and cognitive function. We then fit the group of adults with ARHL with bilateral hearing aids to examine how increased audibility from amplification influenced cortical neuroplasticity, speech perception, and cognitive outcomes.This study was carried out in accordance with the recommendations of Belmont Report. The protocol was approved by the Institutional Review Board at the University of Colorado Boulder. All subjects provided written informed consent prior to participation in the study in accordance with the Declaration of Helsinki.SD = 4.68). Subjects were native speakers of English, with no reported neurological impairment and reported normal or corrected-to-normal visual acuity. Thirteen adults comprised the NH control group  and 28 adults comprised the ARHL experimental group . Independent samples t-tests were conducted to confirm that groups did not significantly differ in terms of age  or gender . It should be noted that it was difficult to recruit subjects in this age-range with normal hearing, likely due to high prevalence of ARHL. None of the ARHL subjects reported hearing aid use prior enrollment in the study.A total of 41 adults took part in this study  = 1.508, p = 0.140], noise exposure , or hypertension . No subjects reported history of diabetes or clinical depression. The two groups did not differ in terms of education level  or handedness . As expected with the presence of hearing loss, report of tinnitus was significantly higher in the ARHL group , with 68% ARHL subjects reporting some level of tinnitus. Interestingly, self-report of balance problems was significantly higher in the hearing loss group , with 25% of hearing loss subjects reporting balance disturbances and/or falls in the past year.There was no difference between groups on a variety of known demographic risk factors for hearing loss including smoking  and high frequency pure tone average (HFPTA) thresholds   between the right and left ears were not statistically different among subjects, averaged audiometric thresholds across the 2 ears were computed and used for subsequent analyses for each group. Average pure tone air conduction thresholds for each group and corresponding 95% confidence intervals are depicted in SD = 10.41) compared to the NH group  . Average HFPTA thresholds were approximately 33.5 dB HL poorer in the ARHL group  compared to the NH group  . On average, the ARHL group demonstrated a mild sloping to moderate hearing loss and the NH group demonstrated clinically normal hearing thresholds.Audiological inclusion criteria for the NH group were defined as pure tone audiometric behavioral thresholds for both ears ≤ 25 dB HL from 0.25 to 8.0 kHz, no presence of an air-bone gap (≥15 dB HL at 2 or more adjacent frequencies), and no sign of interaural asymmetry (≥15 dB HL at 2 or more frequencies). Audiological inclusion criteria for the ARHL group was defined as a high frequency pure tone average (HFPTA)  > 25 dB HL in both ears, no presence of an air-bone gap (≥15 dB HL at 2 or more adjacent frequencies), and no sign of interaural asymmetry (≥15 dB HL at two or more frequencies). Because pure tone average (PTA) thresholds  , N1 , and P2 CVEP latencies  in the right temporal ROI  were observed for the P1 (d = 1.66), N1 (d = 1.82), and P2 (d = 1.21) components.Plots of the grand average CVEP waveforms for the NH and ARHL groups across the occipital, right temporal, and left temporal ROIs are depicted in oral ROI . Large eAverage baseline CDRs for the NH and untreated ARHL groups are depicted for each CVEP component  in As can be observed in TM scores were significantly poorer in the hearing loss group compared to the NH group at baseline . Scores indicated a mild deficit in background noise (3–7 dB SNR) in the ARHL group  and normal performance (0–3 dB SNR) in the NH group   . Average = 2.31)  and are SD = 10.24) and there was no significant difference in performance between the NH and ARHL groups , indicating that adults with early-stage (mild-moderate) hearing loss do not derive greater relative benefit from visual (lip-reading) cues compared to age-matched NH control subjects. This finding is comparable to previously reported visual benefit using the same AzAv test materials in cochlear implant recipients, where average benefit from visual cues was 32–44%  benefit scores on the auditory-visual speech perception in noise scores for the NH and ARHL groups are shown in s 32–44%  in studis 32–44% . The rels 32–44% .SD = 2.80) compared to the NH group  and this difference was statistically significant . Executive function scores (BDS-2) were 3.06 points lower in the ARHL group  compared to the NH group, and this difference was also statistically significant . The ARHL group  performed 7.81 points poorer on the processing speed measure (SDMT) compared to the NH group at baseline, and this difference was significantly significant  . Percent recall scores on the visual working memory task (RST) were 6.92% poorer in the hearing loss group  compared to the NH group  . Percent recall scores on the auditory working memory task (WARRM) were 11.39% poorer in the ARHL group  compared to the NH group  . Together, these results suggest a negative impact on cognitive function even in mild hearing loss.Average results on cognitive measures and corresponding 95% confidence intervals are depicted in To evaluate the association between visual cortical cross-modal re-organization and behavioral outcomes, we correlated baseline CVEP latencies in ARHL group over the right temporal ROI to auditory performance (degree of hearing loss and auditory speech perception in noise), functional dependence on visual cues, and cognitive function.r = −0.672, p < 0.001), N1 , and P2  CVEP latencies in the right temporal ROI in the ARHL group at baseline, suggesting that more extensive cross-modal re-organization is apparent in greater degrees of hearing loss. This result is consistent with findings from Correlations between P1 CVEP and degree of hearing loss for the ARHL group are depicted in r = −743, p < 0.001), N1 , and P2  in the untreated ARHL group. This finding suggests that earlier CVEP latencies, considered an index of more extensive visual cross-modal re-organization of auditory cortex, are associated with poorer auditory speech perception performance. This finding is consistent with previous studies in deaf adults , N1 , P2 ]. While a significant association between visual cross-modal neuroplasticity and benefit from visual cues has been reported in deaf adults  cues in the ARHL group at baseline for any of the CVEP components , N1 , and P2  CVEP peak latencies with moderate to high effect sizes  . While average post-treatment amplitudes appear visually reduced, this difference was not statistically significant P1 , N1 , P2 . To our knowledge, no prior studies have evaluated clinical treatment with hearing aids on visual cross-modal plasticity in ARHL. Post hoc group comparisons between the NH group evaluated at baseline (n = 13) and the 6 months post-treatment outcomes in the ARHL group (n = 21) indicate no statistical difference in P1 , N1 , or P2  CVEP latencies over the right temporal ROI, suggesting that restored audibility from hearing aid use may promote more typical cortical visual processing patterns.Plots of the grand average CVEP waveforms for the ARHL group (n = 28) were compared to the group of ARHL adults who met minimum hearing aid usage criterion 6 months post-treatment with hearing aids (n = 21). While the ARHL exhibited occipital, temporal , and frontal and pre-frontal cortical activity  for the higher-order N1 and P2 CVEP components pre-treatment, there was a post-treatment reduction in auditory cortex recruitment for these components post-treatment, suggestive of a reversal in visual cross-modal re-organization by vision. In addition, post-treatment results indicate a reduction in frontal and pre-frontal cortex activation compared to baseline. Post-treatment CDR results in the hearing loss group are comparable those results observed in the NH group at baseline evaluation  = 4.643, p < 0.001]. While ARHL adults exhibited a mild auditory deficit (3–7 dB SNR) in background noise without hearing aids , treatment with hearing aids over yielded a 3.6 dB SNR improvement in performance , with performance comparable to NH adults (0–3 dB SNR).t(20) = −0.203, p = 0.841]. Average benefit from visual cues pre-treatment at the baseline evaluation (acutely aided condition) was 36.93% (SD = 9.69) and average benefit from visual cues at 6 months post-treatment follow-up (aided condition) was 37.66% (SD = 13.92). This finding suggests that hearing aid use does not modify auditory-visual integration in mild-moderate ARHL. However, given that adults with hearing loss did not have an advantage in lip-reading at the pre-treatment baseline compared to NH adults, it was not entirely unexpected that there would be no change in their results after treatment. No correlation was observed between average daily hearing aid use and change in auditory speech perception in noise performance (QuickSINTM)  or change in dependence on visual cues for auditory-visual speech perception in noise AzAv test: . Given high homogeneity of average daily hearing aid use among ARHL participants , this is not an unexpected finding.n = 15) of ARHL adults showed improved performance after 6 months of hearing aid use, 10% (n = 2) showed no change in performance, and 19% (n = 4) showed decreased performance. On the executive function measure (BDS-2), 90% of subjects (n = 19) showed improvement performance, 10% showed no change in performance (n = 2), and 0% (n = 0) showed decreased performance. On the processing speed measure (SDMT), 81% of subjects (n = 17) showed improved performance, 5% showed no change in performance (n = 1), and 14% (n = 3) showed decreased performance. On the visual working memory test (RST), 71% showed improved performance (n = 16), 0% showed no change in performance, and 24% showed decreased performance (n = 5). On the auditory working memory test (WARRM), 67% showed improved performance (n = 14), 0% (n = 0) showed no change in performance, and 33% showed decreased performance (n = 7). Pre-post cognitive test results on these cognitive assessments are depicted in t(20) = 2.878, p = 0.009]. Average improvement in executive function (BDS-2) after 6 months of hearing aid use was 3.09 points higher than pre-treatment scores, and this improvement was also significant . Significant improvements in processing speed (SDMT) by 4.52 points  and visual working memory (RST) by 5.30 percentage points  were also observed after 6 months of hearing aid use. We observed no significant improvement on the auditory working memory (WARRM) measure following treatment with hearing aids .Hearing aid use over the course of 6 months resulted in significant improvements in the domains of global cognitive function, executive function, processing speed, and visual working memory (but not auditory working memory). On the global cognitive function measure (MoCA), 71%  compared to situations where the acoustic speech signal is more optimal . For example, the correlations between unaided auditory speech perception in noise (QuickSINTM score) and performance on the global cognitive function task (MoCA)  and processing speed task (SDMT)  measured at baseline were stronger than the correlations between aided auditory speech perception in noise (QuickSINTM score) and performance on the global cognitive function task (MoCA)  and processing speed task (SDMT)  measured 6 months after hearing aid use. This finding is consistent with previous studies which show that acoustically degraded speech requires greater cognitive compensation (t(20) = 0.046, p = 0.843]; BDS-2: ; SDMT: ; RST: ; WARRM: ). Given high homogeneity of average daily hearing aid use among ARHL participants , this is not an unexpected finding.Further, ensation . No corrTM scores in the ARHL group. As shown, there was a significant negative correlation was observed between pre-treatment CVEP latencies in the right temporal ROI and auditory speech perception in noise outcomes for the P1 , N1 , and P2  components, suggesting that the cross-modal state of the auditory cortex pre-treatment is predicted of 6 months post-treatment auditory speech perception outcomes. No such association was observed between pre-treatment right temporal CVEP latencies and post-treatment dependence on visual (lip-reading cues) or post-treatment cognitive outcomes across the domains of global cognitive function, executive function, processing speed, auditory working memory, or visual working memory.TM , AzAv  or on majority of the cognitive tests  . Gender had no effect on performance outcomes on the QuickSINTM , AzAv , or majority of the cognitive tests  , though females performed slightly better than males on the global cognitive function test (MoCA)  and visual working memory test (RST)  post-treatment. Age was not correlated with performance outcomes on the QuickSINTM , AzAv , or majority of the cognitive tests   in the ARHL group, though older age was correlated with poorer executive function (BDS-2) .Tinnitus status in the ARHL group had no effect on performance outcomes on the QuickSINSD = 0.60) and average final ability with hearing aids 4.49 (SD = 0.44) (out of 5) on the COSI outcome questionnaire, indicating the ARHL group felt they were able to hear most of the time (>75%) with their hearing aids in the specific listening situations they identified as most important to them.Self-report of hearing aid benefit and satisfaction on the COSI, IOI-HA, and SADL is depicted in SD = 0.38), indicating significant benefit from hearing aid use (SD = 0.75), indicative of moderate self-reported hearing difficulty in background noise. Average global score across items 1–7 on the IOI-HA was 4.33 (SD = 0.38). Average scores across items 1–7 were 4.47 (= 0.60) for the use sub-score, 4.19 (SD = 0.99) for the benefit sub-score, 4.10 (SD = 0.54) for the residual activity limitation sub-score, 4.67 (SD = 0.67) for the satisfaction sub-score, 4.38 (SD = 1.07) for the participation sub-score, 4.57 (SD = 0.68) for the impact on others sub-score, and 3.95 (SD = 9.92) for the quality of life sub-score. These results were compared to normative data in adults with moderate-severe sensorineural hearing loss reporting “moderate” hearing problems on item 8 reported in On the IOI-HA outcome questionnaire, ARHL subjects were asked to provide a rating for 7-items assessing daily use of hearing aids, benefit, residual activity limitations, satisfaction, residual participation restrictions, impact on others, and quality of life at the 6 months follow-up visit. Ratings were provided on a 5-point scale, where a lower score indicates poorer outcome and a higher score indicates higher outcome for each item. Average global improvement rating was 4.33 ( aid use . An addiSD = 0.60). Average positive effect sub score was 5.15 (SD = 1.02), average service sub-score was 6.26 (SD = 0.83), average negative feature sub-score was 5.51 (SD = 1.32), and average self-image sub-score was 6.24 (SD = 0.75). Comparison of our results against normative data reported in On the SADL outcome measure, the ARHL group was asked to indicate their relative satisfaction with hearing aids across the areas of positive effect, service, negative features, and personal image . Higher Overall, the goal of this investigation was to explore the relationship between cortical visual cross-modal neuroplasticity and speech perception and cognitive outcomes in early-stage ARHL, and to assess treatment effects with well-fit hearing aids on these outcomes. Visual cross-modal re-organization was observed in the untreated ARHL group, as evidenced by earlier CVEP latencies over right auditory cortex and cortical source localization patterns indicating greater probable current densities in auditory cortex to visual motion stimuli. Visual cross-modal re-organization in the ARHL group was associated degree of hearing loss and poorer auditory speech perception outcomes, but not visual (lip-reading) benefit. More extensive cross-modal re-organization in the ARHL group at baseline was also associated with poorer cognitive performance in the domains of global cognitive function, executive function, processing speed, and auditory and visual working memory. As a group, clinical treatment with well-fit amplification reversed cross-modal recruitment of auditory cortex for visual processing in the ARHL group following 6 months of hearing aid use, coinciding with gains in auditory speech perception abilities and improvements in global cognitive function, executive function, processing speed, and visual working memory performance. Further, the cross-modal status of the right auditory cortex at baseline before hearing aid fitting was predictive of 6 months post-treatment auditory speech perception outcomes. To our knowledge, this is the first study to document reversal in visual cross-modal re-organization following clinical intervention with hearing aids, though reversal in cross-modal re-organization has been previously reported in an individual pediatric case of single-sided deafness following clinical intervention with a cochlear implant .A main finding from this study was more extensive cross-modal recruitment of auditory cortex by vision in the ARHL group prior to hearing aid fitting. We observed earlier P1, N1, and P2 latencies in the ARHL group relative to NH controls over the right temporal cortex at baseline, and CVEP current density source reconstruction patterns indicating greater cross-modal activity over the auditory cortex. This finding replicates results from a previous high-density EEG study in a smaller group of adults with mild-moderate ARHL using the same stimulus  as well Second, results from this study indicate that untreated mild-moderate ARHL is associated with deficits in auditory speech perception in noise and cognitive functioning. The untreated ARHL group exhibited significantly poorer cognitive performance across the domains of global cognitive function, executive function, processing speed, visual working memory, and auditory working memory compared to the NH group at baseline. Poorer global cognitive outcomes in ARHL have been reported in previous cross-sectional studies  and cohoThe pre-frontal and frontal cortex recruitment for visual processing that was observed in the ARHL group at baseline – which was absent in the NH group – is a new and unexpected finding. Previous studies have found that hearing impaired listeners may exhibit greater frontal cortex activity when processing incongruent audio-visual, auditory, and visual speech stimuli (McGurk Effect), which is presumed to reflect an increase in cognitive effort during auditory-visual integration tasks . RecruitNotably, as a group our ARHL subjects showed a reversal in visual cross-modal recruitment of auditory cortex within 6 months of hearing aid use. Moreover, this reversal in cross-modal re-organization coincided with recovery in auditory speech perception in noise performance. Performance on cognitive measures in the ARHL group also improved 6 months post-treatment in almost all cognitive sub-domains  except for auditory working memory, where test performance approximated performance of the NH control group at baseline. Thus, beyond the known benefits hearing aid use in improving speech perception and communication, our results provide preliminary evidence that hearing aid use may enhance cognitive function.r = 0.96) with re-test occurring 2 weeks apart (r = 0.75–0.92) over a range of 4-8 weeks or longer (r = 0.8) at 8 week and 6 months follow-up intervals (r = 0.7–0.9) when administered over the course of 2 weeks, 1 month, or 2 years intervals (r > 0.8) has also been reported for the auditory working memory measure (WARRM)  show high test-retest reliability  . HoweverIt is possible that reversal in visual cross-modal neuroplasticity in ARHL may provide an objective marker of treatment benefit. For example, less diffuse cross-modal re-organization has also been reported in deaf adults with good auditory speech perception outcomes following cochlear implantation, while deaf adults with poor auditory outcomes exhibit persistent cross-modal re-organization that persists even years after cochlear implantation .n = 40) fit with hearing aids and examined over the course of 6 months. However, different cognitive sub-domains were assessed in this study , study subjects were almost a decade older than the subjects in our study, and there was limited information provided with regards to the quality of the hearing aid fitting or the duration of hearing aid use during the study. The potential for hearing aid use to provide cognitive benefit may depend on a variety of factors  or take longer in some patients. Future studies should seek to understand this relationship. It is possible that audiological intervention may only provide neurocognitive benefit if treatment is delivered in a timely manner, before extensive functional and structural neural changes take place.To our knowledge, this is the first study to provide evidence that restored audibility with hearing aids may reverse compensatory changes in cortical resource allocation and promote typical more typical visual sensory processing patterns, coinciding with speech perception benefit and cognitive gains, though other studies indicate neurocognitive benefit from hearing aid treatment in adults with hearing loss. For example, findings from a recent study by Interestingly, the extent of visual cross-modal re-organization of auditory cortex pre-treatment  was predictive of auditory speech perception in noise outcomes 6 months post-treatment. This finding suggests that there may be an upper limit to reversing compensatory changes in cortical resource allocation. That is, recovery of auditory speech perception abilities after clinical intervention may be limited by the extent to which auditory cortex has become “re-purposed” by vision. For the small percentage of adults with ARHL who do seek treatment, treatment is typically sought out late, delayed 7–10 years after the hearing loss onset . Thus, aThis study was not a randomized controlled clinical trial and the NH subjects were not re-tested at 6 months follow-up. At baseline, the untreated ARHL group and NH control group demonstrated clear differences in CVEP latencies, cortical source activation patterns, and cognitive performance. After 6 months of hearing aid use, CVEP latencies significantly increased (right auditory ROI), cortical source activation patterns showed less extensive cross-modal re-organization, and cognitive performance improved in the ARHL group, with measures similar to the NH group at baseline. While preliminary findings from this study supports the idea that early and timely intervention with hearing aids  may provide the best chance of promoting typical cortical sensory functioning and good prognostic cognitive and behavioral outcomes, future randomized controlled trials can provide more robust evidence regarding cortical and cognitive benefit from hearing aid treatment.Longitudinal follow-up studies are also necessary to understand whether extended hearing aid use (beyond 6 months as reported in our study and previous studies) may modify long-term risk for cognitive decline, including dementia. Currently, there exists no universal screening of hearing loss in the United States and very few adults with clinically significant ARHL use hearing aids or cochlear implants. Research in this area may lead to better tools to diagnose auditory deprivation in its early stages and may also help target optimal timeframes for intervention.SD = 2.96). It is possible that poorly fit devices or low levels of compliance may reduce the efficacy of hearing aids in providing benefit. Future studies should seek to understand how the quality of hearing aid fitting and/or amount of daily hearing aid use may influence auditory, cortical, cognitive outcomes following intervention. Further, while this study focused on group-level differences in baseline auditory, cortical, and cognitive function in NH adults and adults with ARHL, future studies should seek to understand differences in individual characteristics and demographic variables that may affect cortical and cognitive outcomes following intervention for adults with ARHL. Such information may help inform best-practice guidelines and help guide clinical recommendations.It is important to note that ARHL subjects in our study were fit with well-fit hearing aids (± 5 dB of NAL-NL2 targets measured using probe-microphone measures). ARHL subjects in our study also complied with a high level of daily hearing aid use greater  may help maximize auditory function once hearing has been “restored.” If the extent of cross-modal re-organization is a limiting factor of post-treatment auditory outcomes, then aural rehabilitation or therapeutic techniques or treatments may help maximize treatment benefit for patients who may be struggling.The reversal in cross-modal recruitment of auditory cortex by vision and reduction in pre-frontal and frontal cortex activity in the ARHL group after 6 months of hearing aid use suggests that restored audibility from well-fit hearing aids may promote more typical sensory functioning comparable to activity observed in the NH group at baseline. Future studies will aim to examine the role of pre-frontal and frontal cortex activity during sensory processing tasks in ARHL, as it relates to behavioral outcomes of auditory function and cognition. Future studies should also aim to understand how restored audibility with amplification alters top-down attentional or cognitive modulatory control of sensory function.Finally, our results highlight the potential role of cognitive screening or evaluation in the clinical setting. Currently, only 25% of audiologists incorporate cognitive screening or other special tests into their clinical practice . If treaOne of the most remarkable capabilities of the human brain is its capacity for change. As a profession, the field of audiology is beginning to unearth the widespread effects of hearing loss on structural and functional changes in the brain. Ultimately, our clinical interventions  the neuroplastic ability for the brain to adapt to restored auditory input. With a more solid understanding of the mechanisms of neuroplasticity in ARHL, our profession may find new and innovative ways to leverage neuroplasticity in order to optimize treatment outcomes for our patients.All datasets analyzed for this study are included in the manuscript.The studies involving human participants were reviewed and approved by the Institutional Review Board, University of Colorado Boulder. The patients/participants provided their written informed consent to participate in this study.Both authors of this manuscript contributed to the design of the study, data analysis, interpretation, revision, and review of this manuscript.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.


An imbalance of angiogenesis contributes to many pathologies such as cancer, arthritis and retinopathy, hence molecules that can modulate angiogenesis are of considerable therapeutic importance. Despite many reports on the promising antiangiogenic properties of naturally occurring flavonoids, no flavonoids have progressed to the clinic for this application. This systematic review and meta-analysis therefore evaluates the antiangiogenic activities of a wide range of flavonoids and is presented in two sections. The first part of the study (Systematic overview) included 402 articles identified by searching articles published before May 2020 using ScienceDirect, PubMed and Web of Science databases. From this initial search, different classes of flavonoids with antiangiogenic activities, related pathologies and use of in vitro and/or in/ex vivo angiogenesis assays were identified. In the second part , 25 studies concerning the antiangiogenic evaluation of flavonoids using the in vivo chick chorioallantoic membrane (CAM) assay were included, following a targeted search on articles published prior to June 2020. Meta-analysis of 15 out of the 25 eligible studies showed concentration dependent antiangiogenic activity of six compared subclasses of flavonoids with isoflavones, flavonols and flavones being the most active (64 to 80% reduction of blood vessels at 100 µM). Furthermore, the key structural features required for the antiangiogenic activity of flavonoids were derived from the pooled data in a structure activity relationship (SAR) study. All in all, flavonoids are promising candidates for the development of antiangiogenic agents, however further investigations are needed to determine the key structural features responsible for their activity. Physiologically, angiogenesis is pivotal for tissue growth and regeneration  which isc agents ,8.Flavonoids are widely distributed in fruits, vegetables and nuts. They are one of the most important chemical classes of natural compounds showing various pharmacological profiles that include anticancer ,10,11, aThe antiangiogenic activity of flavonoids has been extensively studied over the last two decades. Several studies document the ability of flavonoids to inhibit the proliferation and migration of endothelial cells by interfering with key angiogenesis signaling cascades such as the mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Nevertheless, they can inhibit the expression of major proangiogenic factors such as VEGF and matrix metalloproteinases (MMPs) ,7,15.Researchers rely on different in vitro and in/ex vivo assays to quantitatively assess the effects of chemical compounds on angiogenesis ,17. EachDespite considerable research concerning the antiangiogenic activities of flavonoids, to date they have neither progressed to the market nor clinical trials for that purpose. Therefore, the aim of this review is to systematically assess the antiangiogenic activities of flavonoids to provide greater insight into their potential as therapeutic agents. This study is comprised of two parts: For The pool of studies included was classified with respect to: (a) flavonoid class , (b) flaThe majority of articles  focused on the implications of angiogenesis on cancer growth and metastasis. 7% of the articles studied antiangiogenic effects of flavonoids on other diseases such as diabetes, bone and eye diseases, whilst 11% focused on the antiangiogenic activity of flavonoids without application to a specific pathology a. A profThe second subset search, which is the basis of the meta-analysis forming The main study characteristics of the research articles included in 25 studies reporting the CAM assay for the in vivo evaluation of flavonoids were eligible for the meta-analysis. The number of blood vessels relative to the control was used as the outcome measure, the lower the ratio the higher the antiangiogenic activity. The studies were grouped into 3 sub-sets based on the controls used. In the first set (12 studies), the normal vasculature of the CAM was used as a control without any interventions that would induce angiogenesis. The second and third sets, however, tested the antiangiogenic activity of flavonoids on CAMs with abnormal angiogenesis using either proangiogenic factors for set 2 (4 studies) or cancer cell lines for set 3 (9 studies). 10 studies ,145,149 Set 1: Antiangiogenic effect of flavonoids under normal conditionsp-value < 0.00001), 0.50  and 0.26  for the low, mid and high concentrations, respectively. On the other hand, the anthocyanidines subgroup exhibited only a minor overall reduction of 18% at the highest concentration and a slightly proangiogenic effect  at 20 µM.To ensure consistency in our comparison, for the meta-analysis of set 1, the concentrations were grouped into three ranges i.e., low (10–20 µM), medium (40–50 µM) and high (100 µM). Flavonoids were sub grouped based on their chemical class as shown in In addition to the forest plot analysis that gives a general idea about the overall in vivo antiangiogenic activity of flavonoids and identifies trends of activity among the different subclasses, some structure activity relationship (SAR) conclusions were drawn from the pooled results .First, there was no correlation between the number of hydroxyl groups and antiangiogenic activity. However, the position of the hydroxyl groups appeared to be of importance as most of the highly active flavonoids had hydroxyl groups at positions 3, 5 and 7 and/or 4′ . The 7-OH group can be considered to be of greatest importance for activity since 7-OH flavone showed higher activity in the low and medium concentrations compared to the 5-OH analogue. Absence of the 3-OH group caused up to a 14% decrease in activity at the 50 and 100 µM concentrations, as demonstrated, for example, for 3-OH flavone vs. flavone, kaempferol vs. apigenin and 3,7-diOH flavone vs. 7-OH flavone. This was also true for quercetin vs. luteolin but with only a trivial drop of activity of 1 to 2%. However, this was not the case for 3,6-diOH flavone vs. 6-OH flavone where removal of the 3-OH group slightly increased the activity by 1 to 5% at the mid and high concentrations.Secondly, unsaturation of the C2 and C3 bond is a common feature of most of the highly active flavonoids and is important for activity. 7-OH flavone and 7-OH flavanone are two good examples that exemplify this, as demonstrated by a reduction of the number of vessels: 27%, 32% and 52% for 7-OH flavone and 10%, 22% and 39% for 7-OH flavanone at 10 µM, 50 µM, and 100 µM, respectively.3 flavone, reduction of number of vessels: 35% and 20% at 10 µM, 64% and 42% at 50 µM, 79% and 69% at 100 µM, respectively). On the other hand, conversion of the 7-OH group in 3,7-diOH flavone to a 7-OCH3 group in 3-OH-7-OCH3 flavone slightly improved the activity (reduction of number of vessels) from 18% to 20% at 10 µM and from 63% to 69% at 100 µM, respectively. Finally, glycosylation at positions 3 or 7 showed neither a pronounced nor a consistent effect on the antiangiogenic activity of flavonoids. While a decrease in activity was observed with quercetin vs. rutin, hesperitin vs. hesperidin and cyanidin vs. cyanidin-3-O-glucoside, an increase was observed in the cases of naringin vs. naringenin and delphinidin vs. delphinidin-3-O-glucoside.Third, there are examples of where the presence of a methoxy group at position 4′ increases activity . However, the presence of a methoxy group at C7 caused a decrease in the activity when compared to the unsubstituted analogue  and IL-6 induced angiogenesis in two reports ,147. TheSet 3: Antiangiogenic effect of flavonoids under tumor conditions.p-value < 0.00001). HIFα and VEGF were significantly downregulated, as evidenced by immunoblotting analysis of CAM OVCAR-3 tissues that were treated with apigenin or acacetin [Since angiogenesis plays a vital role in tumor growth and metastasis, several studies have focused on the antiangiogenic evaluation of promising cytotoxic agents. acacetin ,144. Theacacetin ,142. At acacetin . Expressacacetin  leading acacetin . Accordiacacetin .I2 > 80%) observed for all subgroups in the generated forest plots, except for the anthocyanidines subgroup at the mid and high concentrations analyses , was expected given that each class included different flavonoid molecules. In that context, a sensitivity analysis was conducted by a leave-one-out strategy to assess the robustness of the results and determine the contribution of each flavonoid to heterogeneity. Overall, the results showed good robustness and the overall summary estimates did not show significant changes upon the systematic removal of individual studies (I2 = 100%) to 0.74  and 0.38  upon removal of the Gacche 2015 (Silibinin) and Gacche 2015 (Taxifolin) flavonoids, respectively (I2) of the subgroups totals did not show significant change, with very few exceptions, upon implementation of the leave-one-out strategy  clinical: differences in participants, interventions or outcomes, (ii) methodological: differences in study design, risk of bias and (iii) statistical: variation in intervention effects or results . LookingThis review and meta-analysis were conducted according to Preferred Reporting Items for systematic reviews and Meta Analyses (PRISMA) guidelines .For With regards to the detailed meta-analysis for Studies were included in the n).Initially, articles’ titles and abstracts were screened based on relevance and inclusion/exclusion criteria. Full texts were checked in some cases when abstracts failed to provide a detailed description. Eligible articles were retrieved and data extracted into a specially designed form. The first set of extracted data for I2 measure where I2 ≥ 50% indicates substantial heterogeneity [Means of the number of blood vessels in a CAM relative to control were used as the outcome measure. Concentrations were reported in µM in all analyses except for analysis of wogonoside’s antiangiogenic effect on breast cancer cell lines in which ng/CAM was used. Values are represented as means ratio ± standard error of means (SEM). For studies reporting standard deviation (SD), the SEM was calculated by dividing SD by square root of the corresponding study sample size. Pool effect size was expressed as means ratio and 95% CI and was calculated using the inverse variance (IV) method. The random effects model was used because it accounts for between study variability. Heterogeneity was assessed using Higgins’ ogeneity . SensitiDespite the promising antiangiogenic activity of flavonoids presented in many literature studies, no flavonoids have reached clinical trials for this application. This systematic review and meta-analysis therefore aimed to provide further insight into this area by evaluating the in vivo antiangiogenic activity of flavonoids as determined by the widely reported, clinically relevant CAM assay. A comprehensive overview of the antiangiogenic activities of flavonoids with regards to the class of flavonoids, pathology and assays used was presented. Results have shown that the biggest fraction of studies focused on the flavone subclass, cancer related angiogenesis, and in vitro assays. Furthermore, an overall evaluation of the in vivo antiangiogenic activity of flavonoids was offered focusing on SAR and mechanistic considerations. Isoflavones, flavonols and flavones were found to be the most active classes of flavonoids where antiangiogenic activity was dose dependent. Several structural features were considered, from which it was concluded that the position of the hydroxyl substituents and the degree of unsaturation are key for high activity. Even though there were some limitations such as the miscellany of the studied flavonoids and the high heterogeneity, this study provided substantial information that will underpin further investigations by addressing current gaps in the literature regarding the antiangiogenic activity of flavonoids, and highlighting their future prospective as potentially clinically active antiangiogenic agents.