Twenty-nine patients received CRT plus VPA, and 53 patients received CRT alone. Clinical data were obtained via a database and chart review. Tests for differences in protein expression changes between radiation therapy (RT) with or without VPA were conducted for individual proteins using two-sided t-tests, considering p-values of <0.05 as significant. Adjustment for age, sex, and other clinical covariates and hierarchical clustering of significant differentially expressed proteins was carried out, and Gene Set Enrichment analyses were performed using the Hallmark gene sets. Univariate Cox proportional hazards models were used to test the individual protein expression changes for an association with survival. The lasso Cox regression method and 10-fold cross-validation were employed to test the combinations of expression changes of proteins that could predict survival. Predictiveness curves were plotted for significant proteins for VPA response  to show the survival probability vs. the protein expression percentiles. Results: A total of 124 proteins were identified pre- vs. post-CRT that were differentially expressed between the cohorts who received CRT plus VPA and those who received CRT alone. Clinical factors did not confound the results, and distinct proteomic clustering in the VPA-treated population was identified. Time-dependent ROC curves for OS and PFS for landmark times of 20 months and 6 months, respectively, revealed AUC of 0.531, 0.756, 0.774 for OS and 0.535, 0.723, 0.806 for PFS for protein expression, clinical factors, and the combination of protein expression and clinical factors, respectively, indicating that the proteome can provide additional survival risk discrimination to that already provided by the standard clinical factors with a greater impact on PFS. Several proteins of interest were identified. Alterations in GALNT14 (increased) and CCL17 (decreased)  were associated with an improvement in both OS and PFS. The pre-CRT protein expression revealed 480 proteins predictive for OS and 212 for PFS (p < 0.05), of which 112 overlapped between OS and PFS. However, FDR-adjusted p values were high, with OS  and PFS . The protein PLCD3 had the lowest p-value , and its elevation prior to CRT predicted superior OS and PFS with VPA administration. Cancer hallmark genesets associated with proteomic alteration observed with the administration of VPA aligned with known signal transduction pathways of this agent in malignancy and non-malignancy settings, and GBM signaling, and included epithelial–mesenchymal transition, hedgehog signaling, Il6/JAK/STAT3, coagulation, NOTCH, apical junction, xenobiotic metabolism, and complement signaling. Conclusions: Differential alteration in proteomic expression pre- vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA. Using pre- vs. post-data, prognostic proteins emerged in the analysis. Using pre-CRT data, potentially predictive proteins were identified. The protein signals and hallmark gene sets associated with the alteration in the proteome identified between patients who received VPA and those who did not, align with known biological mechanisms of action of VPA and may allow for the identification of novel biomarkers associated with outcomes that can help advance the study of VPA in future prospective trials.Background: Glioblastoma (GBM) is the most common brain tumor with an overall survival (OS) of less than 30% at two years. Valproic acid (VPA) demonstrated survival benefits documented in retrospective and prospective trials, when used in combination with chemo-radiotherapy (CRT). Purpose: The primary goal of this study was to examine if the differential alteration in proteomic expression pre vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA as compared to standard-of-care CRT. The second goal was to explore the associations between the proteomic alterations in response to VPA/RT/TMZ correlated to patient outcomes. The third goal was to use the proteomic profile to determine the mechanism of action of VPA in this setting. Materials and Methods: Serum obtained pre- and post-CRT was analyzed using an aptamer-based SOMAScan Glioblastoma (GBM) is the most common and the most aggressive brain tumor . The cur® assay) to measure the relative concentrations of 7596 protein targets (7289 human) for changes in expression using approximately 150 ul of serum [Twenty-nine patients who received concurrent valproic acid (VPA) were compared to 53 patients who received CRT alone. All patients had pathology-proven GBM (diagnosed 2005–2013) and were enrolled on NCI NIH IRB-approved protocols. The patients who received concurrent high-dose VPA were treated on the open-label, NCI NIH phase 2 study NCT00302159). To be included in this analysis in the VPA class, the patients needed to have received ≥1 week of VPA as per the original analysis  and have9. To be of serum ,36. Clinof serum , and outof serum  obtained® proteomic assay technology for changes in the expression of 7000+ protein analytes [® data were filtered to remove non-human and non-protein targets, resulting in 7289 aptamers targeting 6386 unique gene symbols. RFU values reported by SOMAScan® were log2-transformed.Serum samples were obtained before initiation of CRT ) and following completion of CRT ) with the time between pre- and post-sample acquisition averaging 49 days (range 27–83 days). Following the acquisition, samples were frozen at −80 for an average of 3442 days (range 800–5788 days) and then defrosted and screened using the aptamer-based SOMAScananalytes ,36. SOMAThere were 7596 proteins on the chip. We selected 7289 human proteins for the subsequent analyses. There were 82 patients with data before CRT (PRE) and post-completion of CRT (COT (Completion of Treatment)). Log base 2 (COT/PRE) represented the protein expression change between after-treatment and pre-treatment conditions.t-tests, considering p-values of <0.05 as significant. Adjustment for age, sex, and other clinical covariates was performed separately when appropriate. The Benjamini and Hochberg method was used to estimate the false discovery rate [Tests for differences in clinical characteristics and protein expression changes between CRT with or without VPA were conducted using two-sided ery rate . Hierarcery rate .p-value is below the specified threshold . GSEA with adjustments for age and sex were performed.Fifty Cancer Hallmark gene sets were downloaded from MSigDB. Gene Set Enrichment analyses were performed in BRB-ArrayTools . The Kolp-values < 0.05 as significant. First, we examined the association between the expression of individual proteins and survival in unadjusted and adjusted analyses. Using protein expression changes above and below 0 Kaplan–Meier survival curves were plotted using the R statistical package [In the previous iteration of proteomic sample analysis in this cohort, univariate and multivariate Cox analysis was carried out for OS . This it package .p-value that assesses whether expression data significantly enhances risk prediction compared to covariates. Cross-validated time-dependent ROC curves were employed for the model containing standard covariates and including both standard covariates and protein expression to evaluate whether the expression data provide more accurate predictions than those provided by standard clinical covariates in the case without separate test data [Two models were employed to examine whether alteration in the protein expression with the administration of VPA can provide additional survival risk discrimination to that already provided by the standard covariates and, if so, to evaluate how much risk discrimination power the protein expression can add to the clinical covariates. To test the combinations of expression changes of proteins that could predict survival, we used the Lasso Cox regression method ,43 impleest data .p-value < 0.005) to show the survival probability vs. the protein expression percentiles [Based on the pre-CRT protein measurement, predictiveness curves were plotted for each significant protein that is predictive for VPA response  . RPA wasp = 0.011) in Kaplan–Meier analysis (p = 0.015) in Kaplan–Meier analysis  and a robust differential survival probability vs. the protein expression percentiles , hedgehog signaling, Il6/JAK/STAT3, coagulation, NOTCH, apical junction, xenobiotic metabolism, and complement signaling . EMT hadThe ability to evaluate proteomic changes in a cohort of patients, such as the one in this study, represents a unique opportunity to analyze the impact of an additive component to the standard-of-care CRT by, in this case, examining the effect of VPA administered as part of a prospective protocol. We have previously shown that the patient population treated with concurrent VPA in this trial  had supeIn this study, we identified differential proteomic expression between patients who received CRT and those who received CRT plus VPA with 124 proteins differentially expressed, indicating that the effects of VPA provide a sufficiently enriched proteomic signal. We note that the sample size employed in this study ,50 compaWe found that in the OS and PFS models, the proteomic signal conferred additive risk discrimination power as compared to the proteome or the clinical data in isolation, and this was more pronounced for PFS in the context of VPA treatment. This is logical given that the signal alteration resulted from pre- vs. post-CRT proteome, which would more likely impact PFS since it represents a “closer” outcome endpoint to sample acquisition compared to OS, which occurs later. OS is also subject to multiple other factors, including further resection, additional systemic management, patient performance status, and comorbidities. It should also be noted that the ability to capture and interpret progression as an endpoint is limited and, thus, PFS remains flawed with clinical information limited by inconsistencies ; hence, presumably, PFS remains relatively insensitive. Based on this analysis, employing proteomic alteration with validation in larger cohorts may be possible to identify more robust signals and better predict progression.The GALNT14 protein was identified in both protein and protein-plus-covariate models, and was significant for both OS and PFS, with an increase following CRT and VPA treatment, measured as an improvement in OS and PFS and thus prognostic for both. GALNT14 is a member of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family comprised of enzymes that catalyze mucin-type O-glycosylation of proteins. Alterations in components of this family have been associated with several hallmarks of cancer, including migration, proliferation, and treatment resistance ,54. GALNp-value for both OS and PFS, in addition to VARS1, CYREN, KIR2DL4 all with p < 0.005 for both OS and PFS. PLCD3 had a higher value before CRT associated with the improved outcomes with the administration of VPA, and this bears further investigation, as do the additional predictive proteins identified in this study. Several predictive proteins identified have connections to known pathways in cancer, including PLCD3 (phospholipase C delta3), VARS1 , CYREN (Cell cycle regulator of NHEJ), KIR2DL4 (Killer cell immunoglobulin-like receptor 2DL4) and SKP1(S-phase kinase-associated protein 1), and have a direct link to GBM [PLCD3 emerged as one of the most statistically significant predictive proteins by k to GBM ,57 (Suppk to GBM ,59. Notak to GBM ,61. In ik to GBM . Additiok to GBM ,65,66,67The statistically significant proteins identified in this study and hallmark gene sets associated with the alteration in the proteome observed between patients who received CRT plus VPA and those who received CRT alone align with known biological mechanisms of action of VPA as an HDACi , includiThis study’s limitations include the small cohort and retrospective nature of the study, given the comparison with patients who did not receive VPA and were not treated on trial. The outcomes were superior to real-world data and thus may not be representative. The data set was missing MGMT in 37% of patients, and IDH status was unavailable. The data can not be validated with additional cohorts since no such cohorts are treated with high-dose VPA and available proteome, and large-scale proteomic data are lacking. Moreover, while adjusting for clinical covariates in the prognostic models, the potential interactions among these covariates were not incorporated. This omission could introduce bias if significant interactions exist and are associated with survival outcomes. Future directions include building prediction models wherein we will explore several techniques to mitigate class imbalance . There aDifferentially expressed proteins were identified in a large-scale proteomic panel carried out before and post-completion of CRT with the addition of high-dose VPA. GALNT14 and CCL17 were identified as potential prognostic markers based on their expression changes between pre- and post-treatment proteomic profiles. Additionally, PLCD3, SKP1, VARS1, CYREN, and KIR2DL4 were found to be potentially predictive biomarkers solely from the expression levels in the pre-treatment proteomic profile. Those findings were supported by existing data in cancer with several proteins specifically reported on in GBM with growing associations to treatment resistance or response. The predictive and prognostic ability following measurement in serum in GBM patients with the addition of VPA to the standard-of-care CRT is novel, as is the identification of several proteins that, collectively or individually, may be explored as biomarkers. Predictive markers may point to biological risk groups that may benefit from administering VPA to result in superior outcomes. Given these findings, VPA benefits from additional studies in prospective trials.


The construct of justice sensitivity has four perspectives that capture individual differences in the strength of reactions to injustice when becoming a victim of injustice (victim sensitivity), when witnessing injustice as an outsider (observer sensitivity), when passively benefitting from an injustice done to others (beneficiary sensitivity), or when committing an injustice (perpetrator sensitivity). Individual differences in these four justice sensitivity perspectives are highly relevant in moral research. With just eight items in total, the Justice Sensitivity Short Scales–8 (JSS-8) are a very efficient way to measure the four perspectives. JSS-8 was initially constructed in German  and later translated into English. In the present study, we empirically validated this English-language adaptation in a heterogeneous quota sample from the UK. The results show that the psychometric properties  of JSS-8 are good, and that they are comparable with those of the German-language source version. Because of the invariance of loadings, intercepts, and residual variances, researchers can compare manifest scale statistics  of JSS-8 across the UK and Germany. JSS-8 is thus particularly suitable for measuring justice sensitivity in various research areas with constraints on assessment time and questionnaire space. Questions of fairness and justice play a central role not only in people’s private lives but also at their workplaces and in society as a whole. Numerous disciplines, such as psychology, sociology, political science, and economics, investigate justice-related issues. The significance of justice in human lives is highlighted by several influential theories in the social sciences . The deTo enable justice-related research under severe time constraints , Baumert, Beierlein, et al.  and BeieFrequency of experienced injustice: Because highly justice-sensitive individuals focus more on justice-related information, they are likely to discover more situations of injustice than are justice-insensitive individuals. (b) Intensity of emotional reactions to injustice: Individuals with higher justice sensitivity are likely to experience more intense emotional reactions in situations they perceive as unjust. (c) Intrusiveness of thoughts about unjust events: Justice-sensitive individuals ruminate more about unjust treatment than do less justice-sensitive persons. (d) Motivation to restore justice: Highly justice-sensitive individuals have a stronger motivation to redress injustice and to support measures to restore justice .perspectives: victim sensitivity, observer sensitivity, beneficiary sensitivity, and perpetrator sensitivity. (a) Victim sensitivity refers to the sensitivity of becoming a victim of injustice. The dominant emotional reaction of victims of injustice is anger. Furthermore, victims tend to strive for retaliation against and punishment of the perpetrator of injustice. Accordingly, victim sensitivity describes individual differences in emotional reactions to perceived victimization and the motivation to redress such injustice. (b) Observer sensitivity is related to witnessing instances of injustice from an  uninvolved perspective. Observers of injustice may react with indignation and outrage in an unjust situation, and observer sensitivity captures the intensity of such reactions. (c) Beneficiary sensitivity refers to the perspective of someone who passively benefits from injustice. (d) Perpetrator sensitivity refers to the perspective of someone who perceives themselves as actively committing an injustice )In an extensive array of life contexts, such as personal relationships , work 22, 23, anFurthermore, justice sensitivity has been found to differ meaningfully among sociodemographic groups e.g., , 7, 27):, 27:7, 2Justice sensitivity not only differs notably between sociodemographic groups but also between countries: Wu and colleagues  looked aSchmitt and colleagues  found alJustice sensitivity perspectives are personality traits that are sufficiently different from other personality factors or facets e.g., , 13). Ba. Ba13]).N = 87; Baumert, unpublished data) that justice sensitivity perspectives are not correlated with working memory capacities or crystallized intelligence, but that they are correlated to a small to moderate positive extent with fluid intelligence. Furthermore, the justice sensitivity perspectives appear to be relatively independent of other justice-related dispositions, such as the belief in a just world and attitudes toward principles of distributive justice [Baumert and Schmitt  reported justice .Since its conceptualization as a personality trait, researchers have developed different psychometric scales to measure individual differences in justice sensitivity. In an early study, Schmitt et al.  proposedDrawing on this work, Schmitt et al.  construcAs the four scales proposed by Schmitt et al.  proved tSchmitt et al.  empiricaBaumert et al.  tested tNo studies to date have explored the psychometric properties of the English-language ultra-short version, JSS-8. In the present study, we aimed to fill this gap by validating the JSS-8 in a newly recruited, heterogenous quota sample and directly comparing its psychometric quality with that of the German-language source version, USS-8.We validated JSS-8 against personality and related constructs, political constructs, and sociodemographic variables such as the highest level of educational attainment and income. Based on the correlations reported in the “Theoretical background” section, we expected that higher victim sensitivity would be associated with lower Big Five Emotional Stability and Agreeableness, lower general self-efficacy, lower internal locus of control but higher external locus of control, lower interpersonal trust, lower optimism, and lower general life satisfaction. In line with other empirical findings, we further hypothesized that higher victim sensitivity would also be associated with stronger right-wing political preferences and right-wing political ideologies such as a preference for authoritarianism.By contrast, for the other-oriented justice sensitivity perspectives—namely, observer, beneficiary, and perpetrator sensitivity—we expected positive associations with Big Five Agreeableness. Further, we predicted negative correlations with preferences for right-wing political views and ideologies.In addition, we hypothesized that all the justice sensitivity perspectives would be positively associated with socially desirable responses. And finally, we expected that respondents’ self-reported physical health would be unrelated to the four justice sensitivity perspectives.In the present study, we could not validate the English- and German-language JSS-8 against other existing justice sensitivity scales or other well-researched related constructs . This was because our project also served to validate other short scales, and there was no option for specific validation scales besides the core scales of the project and some other relevant variables.https://ergebnisse.zensus2011.de) were the basis for selecting the two samples, representing the adult population of the respective countries in terms of age, sex, and educational attainment. That is, we kept the quotas equal across the samples from Germany and the UK, which helped to eliminate the potential effects of third variables on our results due to a different sample composition. We restricted the samples to native speakers only and financially rewarded all participants. The participants were informed about our research goal—to investigate the quality of several questionnaires. All respondents consented to participation in an online format . According to the local legislation and requirements of the institution, our study on human participants did not require review and approval by an ethics committee as we collected data without any reference to the participants’ identity. The data collection was completely anonymous. We adhered to ethical standards comparable to the 1964 Declaration of Helsinki. To evaluate the stability of justice sensitivity as measured by USS-8/JSS-8, we reassessed a subsample after approximately 3 to 4 weeks  in both countries.To examine the psychometric properties of JSS-8 and its comparability with the German-language source instrument, USS-8, we assessed the two versions in web-based CASI (computer-assisted self-administered interviewing) surveys conducted in parallel in January 2018 by the online access panel provider respondi AG. The survey to validate the English-language JSS-8 was conducted in the UK; the survey to assess the German-language source version was conducted in Germany (DE). Quotas from the latest German census  and NDE = 474 (retest: NDE = 117). We were in the lower range with 8% careless respondents compared to 5% and 15% careless respondents commonly reported in the literature : First,m e.g., , 35) was wasN = 5m . Third,m . In tote . Table does not apply at all) to 6 (applies completely).JSS-8 (like USS-8) consists of eight items measuring victim sensitivity, observer sensitivity, beneficiary sensitivity, and perpetrator sensitivity with two items per justice sensitivity perspective. We validated JSS-8 against personality and related constructs, political constructs, and sociodemographic variables. We compared the findings for the English-language JSS-8 with those for the German-language USS-8. To be able to analyze the predicted associations summarized in the “Aim of the present study” section, the following scales were also part of the comprehensive online surveys:disagree strongly) to 5 (agree strongly).The extra-short form of the Big Five Inventory–2  to 5 (completely agree).The General Self-Efficacy Short Scale–3 /Allgemedoes not apply at all) to 5 (applies completely).The Internal–External Locus of Control Short Scale–4 /Internado not agree at all) to 5 (completely agree).The Interpersonal Trust Short Scale /Kurzskanot at all optimistic/pessimistic) to 7 (very optimistic/pessimistic).The Optimism–Pessimism Short Scale–2 /Skala Onot at all satisfied) to 11 (completely satisfied).The General Life Satisfaction Short Scale /Kurzskaleft) to 10 (right).The political Left–Right Self-Placement scale (English- and German-language versions: ) consistdo not agree at all) to 5 (completely agree).The Authoritarianism Short Scale /Kurzskadoes not apply at all) to 5 (applies completely).The Social Desirability–Gamma Short Scale /Kurzskavery good) to 5 (very bad).Self-reported physical health was measured with a single-item question used in the European Social Survey . The 5-punemployed, 1, vs. employed, 2), income, educational attainment, age, and sex. We included descriptive statistics of all validation measures in the Additionally, we included the following sociodemographic variables: employment , using the packages lavaan , psych , and semWe estimated McDonald’s ω , CronbacWe calculated separate confirmatory factor analysis models per country to test factorial validity. To identify the model, we fixed the first loading of each factor to 1 and the first intercept of each factor to 0. We estimated each model with robust maximum likelihood estimation (MLR). We assessed the model fit of each tested confirmatory factor analysis model via heuristics for fit indices. A model fits the data well if the confirmatory fit index (CFI) is .95 or higher, the root-mean-square error of approximation (RMSEA) is .06 or lower, and the standardized root mean residual (SRMR) is .08 or lower . CFI valWe investigated the nomological network and the sociodemographic variables via correlations of manifest scale scores; thus, the correlations were lower-bound estimates of the true associations. We interpreted the correlation coefficients according to the guidelines proposed by Gignac and Szodorai , who intWe used multigroup confirmatory factor analysis models , 71 to aWe analyzed descriptive statistics and psychometric properties  of the English-language JSS-8 in the UK and compared them with those of the German-language source version, USS-8, in Germany. Additionally, we conducted measurement invariance tests across both countries to evaluate test fairness.rtt ≤ .68), albeit slightly lower than those in Germany (.61 ≤ rtt ≤ .75). The test–retest stability of the perpetrator sensitivity subscale in the UK was comparatively low  sufficiently explains the covariance among the items. As theoretically expected, the unidimensional model did not fit well in either country, as shown in First, we fit a unidimensional model to test the connectedness of Second, we tested a two-dimensional model, in which the items on victim sensitivity loaded on a factor separate from all other items, to investigate whether a victim sensitivity and an other-oriented factor  sufficiently explained the covariance between items. The factors were allowed to covary. The two-dimensional model did not fit well in either country see .Thus, third, we tested the theoretically assumed four-dimensional confirmatory factor model. The factors were allowed to covary. However, we ran into a problem: The residual variance of Item 6  was negative in the UK. When we restricted the residual variance of Item 6 in the UK to be larger than zero, we obtained an acceptable model fit in both countries see .The negative residual showed the instability of a model, which was just identified via covariances between latent variables. We tested an alternative model with further restrictions: Factor loadings were restricted to be the same within one latent variable . Such models are known as essentially tau-equivalent models . The esDue to the instability of the congeneric model and the still acceptable fit of the essentially tau-equivalent model, we accepted the latter one. r = .37–.60/.44–.61) correlated more strongly with each other than with victim sensitivity (UK/DE: r = .13–.32/.04–.23). The only exception was the relatively high correlation between victim and observer sensitivity (UK and DE: r = .47).Notably, the correlations of the subscales in the essentially tau-equivalent model were consistent with theoretical expectations see : Observer ≥ .19).Next, we examined the nomological network and the sociodemographic correlates of JSS-8 (compared with USS-8). In both countries, we found that higher victim sensitivity was associated with lower Agreeableness, lower Emotional Stability, lower dispositional optimism, lower interpersonal trust, lower life satisfaction, and higher external locus of control in both countries. Higher victim sensitivity was also associated with more authoritarian views in the UK. Notably, correlations of validity constructs with other-oriented justice sensitivity perspectives were often small and diverged between the two countries. We found medium to strong correlations of validity constructs with other-oriented justice sensitivity perspectives in only one of the two countries each: Lower Emotional Stability and higher external locus of control were associated with higher observer sensitivity in the UK, lower Conscientiousness and higher external locus of control was associated with higher beneficiary sensitivity in the UK, and lower conventionalism  and higher Agreeableness (in Germany) was associated with higher perpetrator sensitivity.We observed that nearly all perspectives of JSS-8 (and USS-8) were affected either by exaggerating positive qualities (observer sensitivity in Germany) or by minimizing negative qualities .Correlations with sociodemographic variables and health were mainly negligible or small. We observed only medium to strong correlations of selected justice sensitivity perspectives and age: Individuals with higher values on victim sensitivity and beneficiary sensitivity were typically younger.To quantify the cross-national similarity of correlations of the justice sensitivity perspectives with the nomological network and sociodemographic variables, we estimated Pearson correlations of all correlations across countries see . These mr = |.11| to |.63| in the UK and from r = |.08| to |.76| in Germany, which shows that the perspectives have different information content and are not simply copies of each other.Further, we could show that the correlation patterns with external variables are quite differentiated for all four perspectives of justice sensitivity measured by JSS-8 (and USS-8): We estimated Pearson correlations of all correlations across perspectives but within countries. Thus, within each country, we used all correlations of each justice sensitivity perspective and correlated them with those of another justice sensitivity perspective  in the UK, which points to the instability of the measurement model (which is solely identified through the factor covariances). Thus, we constrained the negative residual variance to be larger than zero. The resulting configural model fit was acceptable in absolute terms see . The fitSRMR see . The scaSRMR see . When thSRMR see . Thus, tTo facilitate objective application, JSS-8 (like USS-8) includes fixed instructions , and the order of the items is fixed (ranging from Item 1 to 8), as is the number of labeled response options . We recommend not using a total scale score but rather building unit-weighted  mean scores for each of the four, theoretically and empirically distinct, justice sensitivities. Thus, the instrument is accompanied by strict rules on sum score derivation to facilitate objective evaluation. Additionally, to facilitate objective interpretation, we provide reference values  in The ultra-short Justice Sensitivity Short Scales–8 (JSS-8) measure four aspects of justice sensitivity—victim, observer, beneficiary, and perpetrator sensitivity—with two items each. JSS-8 is the English-language adaptation of the original German-language instrument, the Ungerechtigkeitssensibilität-Skalen–8 . The puWhen looking at the descriptive statistics of JSS-8/USS-8 across countries, we found that means of victim, observer, beneficiary, and perpetrator sensitivity were only minimally higher in the UK than in Germany. It is no surprise that descriptive statistics are so similar across countries, considering the similarity of both countries based on some of Hofstede’s cultural dimensions . In partWe were able to confirm the postulated four-factor structure of justice sensitivity for JSS-8 and USS-8. Correlations across subscales varied substantially; therefore, we do not recommend using a total scale score across all four justice sensitivity perspectives. Instead, unit-weighted mean scores should be computed separately for each perspective. Individual answers should be aggregated to the subscale level only if there are no missing values for any of the two items. In the case of missing data, appropriate methods for handling missing data, such as multiple imputations or full information maximum likelihood estimation, should be applied.Moreover, cross-national measurement invariance testing revealed uniqueness invariance  for JSS-8/USS-8. Uniqueness invariance implies that researchers can compare manifest scale scores  and correlations of JSS-8/USS-8 across Germany and the UK without introducing biases .We found adequate to good reliability  for all JSS-8/USS-8 subscales in our samples. Internal consistencies were sufficiently high for research purposes , 80⸺thisWhereas other researchers have found that all perspectives of justice sensitivity are relatively stable traits , 29, 30,r = |.00| to |.18|, similar to what has been found in other studies investigating correlations between justice sensitivity and a broad range of other constructs [As we considered a broad range of constructs from the nomological network, several correlations between justice sensitivity perspectives  and variables of the nomological network were relatively low . Still,nstructs , 13, lownstructs , 13, lownstructs , 6, lowenstructs , 6, highnstructs —all relanstructs , 6, 26).nstructs , 13. We We observed that nearly all perspectives of JSS-8 (and USS-8) were affected by social desirability⸺a typical phenomenon with self-reporting data . The useIn the present study, we did not validate the English- and German-language JSS-8 against other existing justice sensitivity scales or other well-researched related constructs . However, Beierlein et al.  providedIt is important to note that none of the items from JSS-8/USS-8 is reverse-keyed. Thus, all items are positively pooled in relation to the underlying factor. We cannot exclude that response styles such as acquiescent responding inflate results . This is not a problem specific to JSS-8/USS-8 but relevant for all scales without reverse-keyed items .Further, respondents should rate the items of JSS-8/USS-8 on a 6-point Likert scale. Thus, they have no possibility to choose a neutral midpoint response. Excluding a neutral midpoint response is typically recommended when responses are likely to be influenced by social desirability —such as For practical reasons, we selected only the UK as the English-speaking country for our study: The online access panel provider respondi AG can recruit English-speaking participants only from the UK. However, we do not expect a different conclusion on the psychometric quality of JSS-8 in other English-speaking countries.In conclusion, the ultra-short instrument JSS-8 measured the four perspectives of justice sensitivity well across the UK and Germany. JSS-8 can now be used for research purposes in settings with extreme constraints in time or questionnaire length and in cross-national studies.S1 Appendix(PDF)Click here for additional data file.S2 AppendixJustice Sensitivity Short Scales–8 (JSS-8).(PDF)Click here for additional data file.S3 AppendixUngerechtigkeitssensibilität-Skalen–8 (USS-8).(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(PDF)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file.


Studies that assessed the efficacy of pre-operative immune checkpoint blockade (ICB) in locally advanced urothelial cancer of the bladder showed encouraging pathological complete response rates, suggesting that a bladder-sparing approach may be a viable option in a subset of patients. Chemoradiation is an alternative for radical cystectomy with similar oncological outcomes, but is still mainly used in selected patients with organ-confined tumors or patients ineligible to undergo radical cystectomy. We propose to sequentially administer ICB and chemoradiation to patients with  muscle-invasive bladder cancer.The INDIBLADE trial is an investigator-initiated, single-arm, multicenter phase 2 trial. Fifty patients with cT2-4aN0-2M0 urothelial bladder cancer will be treated with ipilimumab 3 mg/kg on day 1, ipilimumab 3 mg/kg plus nivolumab 1 mg/kg on day 22, and nivolumab 3 mg/kg on day 43 followed by chemoradiation. The primary endpoint is the bladder-intact event-free survival (BI-EFS). Events include: local or distant recurrence, salvage cystectomy, death and switch to platinum-based chemotherapy. We will also evaluate the potential of multiparametric magnetic resonance imaging of the bladder to identify non-responders, and we will assess the clearance of circulating tumor DNA as a biomarker for ICB treatment response.This is the first trial in which the efficacy of induction combination ICB followed by chemoradiation is being evaluated to provide bladder-preservation in patients with  urothelial bladder cancer.The INDIBLADE trial was registered on clinicaltrials.gov on January 21, 2022 (NCT05200988). Although mostly non-invasive at diagnosis, approximately 25% of the patients has a more aggressive bladder tumor, which involves the muscle-layer surrounding the bladder . The staRadical cystectomy is associated with an increased risk of both morbidity and mortality. This is reflected by a complication rate of 58.5% and a mortality rate of 4.7% within 90 days after radical cystectomy . NeverthTo improve outcomes in patients with urothelial bladder cancer, immune checkpoint blockade (ICB) has been studied intensively in the last decade. Pembrolizumab, an antibody targeting programmed cell death protein 1 (PD-1), resulted in superior OS in comparison to second-line chemotherapy  in metastatic urothelial cancer . MaintenCombination ICB has been studied in earlier stages of urothelial cancer as well. The rationale for this strategy is based on encouraging results of combination ICB in other resectable malignancies –25. In tBased on the promising anti-tumor effects with respect to pCR and clinical outcome upon combination ICB observed in the NABUCCO trial –28, we aAs we hypothesize that combination ICB followed by chemoradiation could be an effective bladder-sparing approach in patients with  urothelial cancer of the bladder, we initiated the INDIBLADE trial. We aim to enroll 50 patients for induction treatment with ipilimumab plus nivolumab followed by consolidative chemoradiation to assess bladder-intact event-free survival (BI-EFS).The Pure-01 study showed that multiparametric magnetic resonance imaging (mpMRI) of the bladder was predictive for pCR following neo-adjuvant pembrolizumab . In the To enable personalized medicine, liquid biopsies could be used as biomarkers to evaluate response to treatment and potentially de-intensify therapy. As the absence of plasma ctDNA after neo-adjuvant ICB was associated with treatment response in NABUCCO , we aim 22.1The INDIBLADE trial is an investigator-initiated, multicenter, single-arm, open label phase 2 trial investigating the efficacy of induction therapy with ICB followed by consolidative chemoradiation to spare the bladder in patients with  muscle-invasive urothelial bladder cancer. Participating sited are the Netherlands Cancer Institute , University Medical Center Utrecht , and Erasmus Medical Center .2.2Adult patients with cT2-4aN0-2M0 urothelial bladder cancer who are considered fit for chemoradiation are eligible for enrollment. We recognize that patients with nodal disease have a high risk for recurrence and may not traditionally be viewed as patients where cystectomy and pelvic lymph node dissection can be omitted. However, these patients are mainly at risk for distant recurrence. We believe that efficacy of systemic induction therapy with ICB, as was observed in node positive patients in NABUCCO , 27, cou2.32.3.1Eligible patients will undergo study treatment which consists of ipilimumab 3 mg/kg on day 1, ipilimumab 3 mg/kg plus nivolumab 1 mg/kg on day 22, and nivolumab 3 mg/kg on day 43 Figure 12.3.2Both the European and the American Urology Association recommend either cisplatin or mitomycin C (MMC) combined with 5-Fluorouracil (5-FU) as radio sensitizing chemotherapeutic agent, as these two regimens have been studied most extensively , 36. To 2.3.3Radiation treatment will be administered during four to six weeks  using intensity modulated radiation therapy. Fifty-five gray or a bioequivalent dose covering ≥95% of the total bladder as clinical target volume and visible tumor for focal boosting should be applied. Solitary tumors can be irradiated with a partial boost after lipiodol injections, bladder fiducials [e.g. BioXmark ] or MRI 2.42.4.1The primary objective of the INDIBLADE trial is to establish efficacy of induction therapy with ipilimumab plus nivolumab followed by chemoradiation by determining BI-EFS, as this is a clinically meaningful endpoint for the population included in this trial. Events are defined as muscle-invasive recurrence in the bladder or in the ureter - distal of the crossing with the common iliac artery -, nodal or distant recurrence, cystectomy, death by any cause and/or switch to platinum-based chemotherapy. If possible, histological or cytological confirmation of disease recurrence is preferred. BI-EFS will be determined starting from initiation of the study drug with the use of cystoscopy and CT-imaging of the chest and abdomen. We expect the patient population in INDIBLADE to consist of approximately 40% cT2N0 tumors, 40% cT3-T4aN0 tumors, and 20% patients with node positive disease, which represents a population with a poor prognosis. This is underlined by results of a large retrospective study in which patients with mainly cT2 tumors were evaluated after either cystectomy or chemoradiation, demonstrating a median OS of 3 years and 2.7 years respectively . In addi2.4.22.4.2.1Secondary objectives to evaluate efficacy include OS and PFS. OS will be measured from patient enrollment until death. If information about survival is lacking, OS will be censored on the last date the patient was known to be alive. PFS will be measured from initiation of the study drug until one of the following events: muscle-invasive recurrence in the bladder or in the ureter - distal of the crossing with the common iliac artery, nodal or distant recurrence, switch to platinum-based chemotherapy or death by any cause. Performing a cystectomy will not be considered as a PFS event as PFS is meant to determine efficacy of induction therapy with ICB, regardless of loco-regional treatment. We will report on OS and PFS after two and five years.2.4.2.2As treatment-related adverse events (AEs) potentially influence  related) outcome, we will evaluate safety at various time points within this trial: during and after induction therapy with ICB and during chemoradiation. AEs of all grades irrespective if related to treatment will be provided as measured according to CTCAE 5.0 until 100 days after study drug initiation. Once this period of 100 days after study drug initiation has expired, only treatment-related AEs will be registered.2.4.2.3The evaluation of neo-adjuvant treatment response in  bladder cancer, using conventional imaging, remains challenging. This is partially due to the changes after TUR-B, including an inflammatory response. MpMRI is hypothesized to more accurately distinguish TUR-B effects from cancer (recurrence) , 45. Ana2.4.2.4Current assays to measure plasma ctDNA use highly sensitive sequencing methods to detect in a set of mutations in plasma that is present in a patient’s tumor. Clearance of ctDNA, as measured by these assays, has shown striking predictive power for clinical outcome after treatment with neo-adjuvant or adjuvant atezolizumab in resectable bladder cancer , 47. Bas2.4.2.5Data regarding HR-QoL upon treatment for MIBC are scarce, in particular due to short follow-up periods and the lack of sufficient prospective studies. Nevertheless, results of two prospective trials demonstrate that radical cystectomy can result in long-term effects on HR-QoL, with sexual dysfunction being a main domain compromised by surgery , 49. Bla2.5To achieve successful patient recruitment and adherence to the study protocol, patient participation is of vital importance. We installed a patient advisory committee at an early stage of the development of this trial to ensure that both the trial conduct and outcome measurements are comparable to patients’ treatment goals. Also throughout patient enrollment in this trial, we will continue to involve the opinion of representatives of the patients’ association by scheduling yearly meetings to optimize patient participation.2.6The INDIBLADE trial obtained authorization by the medical ethical committee of the Netherlands Cancer Institute (NKI) in Amsterdam and by the Central Committee on Research Involving Human Subjects. Additionally, the medical ethical committee of all participating sites reviewed the study protocol before initiation of the study site. In case of a substantial amendment to the protocol, a review will be performed by the medical ethical committee of the NKI before integrating the amended protocol. According to the Declaration of Helsinki, all participants give written informed consent before enrollment in the study. The INDIBLADE trial is registered at clinicaltrials.gov (NCT05200988). At the NKI, patient enrollment started in February 2022. By May 2023, 31 patients have been recruited.3Here, we provide a description of the clinical protocol of the INDIBLADE trial. To our knowledge, this is the first clinical trial in which ICB is administrated as induction therapy followed by consolidating chemoradiation to patients with  muscle-invasive bladder cancer to evaluate BI-EFS. There are several ongoing trials assessing bladder-preserving strategies using ICB and chemoradiation, but in contrast to the current trial, these trials simultaneously administer ICB and chemoradiation. In a phase 2 trial evaluating bladder-intact disease-free survival at two years, patients with cT2-T4a MIBC are treated with pembrolizumab followed by TMT with gemcitabine as radio sensitizing agent together with pembrolizumab . In anotA biological argument for sequential administration of ICB and chemoradiation includes the immunosuppressive effects of radiation on the TME, which could diminish the immune response evoked by ICB . HoweverConversely, sequential administration of chemotherapy and ICB has shown positive results. In a phase 3 trial evaluating maintenance avelumab in patients with metastatic urothelial cancer who did not progress on first line chemotherapy, avelumab prolonged OS, compared to best supportive care . The phaIn the current trial, we initially treat patients with ICB as induction treatment before continuing with chemoradiation. As was observed in the NABUCCO trial, we expect tumor volume to shrink upon ICB, thereby enabling omission of cystectomy, including in patients with high-risk disease. In addition, we hypothesize that as the primary tumor has not been removed, the presence of multiple neo-antigens elicits a broader immune response , 57. OncIn conclusion, we here provide the design and rationale for the INDIBLADE trial, which is the first clinical trial in patients with cT2-4aN0-2 bladder cancer evaluating BI-EFS upon induction therapy with combination ICB and subsequent consolidative chemoradiation. Results of this trial will not only provide information about the efficacy of ICB and chemoradiation as a bladder-preservation strategy, but will also give insight into the potential of ctDNA assessment and mpMRI of the bladder to identify patients who qualify for a bladder-sparing approach and those who may need alternative systemic therapy. Furthermore, HR-QoL assessment of our bladder-sparing approach is a vital component of this trial.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 humans were approved by Medical Ethical Committee of the Netherlands Cancer Institute and the Central Committee on Research Involving Human Subjects. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.MH, ES, BR, BS, RM, DR, JB, MF, SB, TB and J. Nooteboom  contributed to the design of the INDIBLADE study. CS and MH wrote the current manuscript. All co-authors reviewed and approved the manuscript before submission.


Both groups underwent training for 30 min five times a week for 8 weeks. The gait ability of each was assessed using the Dynamic Gait Index (DGI), Timed-Up-and-Go (TUG) test, 10-meter walk test, and Figure-of-8 walk test (F8WT). (3) Results: The curved-path gait training group showed significant differences in the DGI, TUG test, 10-m walk test, and F8WT pre- versus post- intervention (p < 0.05). The general gait training group showed no significant difference in F8WT pre- versus post-intervention (p > 0.05). Additionally, there was a statistically significant intergroup difference in gait ability (p < 0.05). (4) Conclusions: Curved-path gait training resulted in greater improvement in gait ability than general gait training. Therefore, curved-path gait training can be a meaningful intervention for improving the gait ability of patients with stroke.(1) Introduction: This study aimed to investigate the effects of curved-path stride gait training on the gait ability of patients with stroke. (2) Materials and Methods: Thirty patients with stroke were randomly assigned to curved-path stride gait training ( Gait, an interaction between the body and central nervous system, is a complex technique involving intentional movements that quickly adapt to changing circumstances ,2,3. GaiPatients with stroke show gait disorders resulting from damage to the motor and sensory pathways ,9. TheseCommon gait training attempts to achieve a normal range of motion in the joints and inhibit abnormal movements. In patients with stroke, gait training involves teaching weight loading and using sensory stimuli to move the load to the affected lower extremities . SeveralMoreover, to successfully assume a curved gait, the head and eyes must first move along the curved path ,7. In otHowever, patients with stroke have limited information about their body position owing to poor joint stability and proprioceptive senses on the affected side, which reduces efficacy and efficiency during movement and reduces stride and gait speed during walking . These pAs shown above, a curved-path gait enhances the proprioception of patients with stroke, giving them a more efficient gait ability. Regarding the effect of curved-path walking, Kim et al. (2012)  reportedThe study participants were recruited from the Gyeonggi K Hospital. A total of 34 middle-aged patients who were diagnosed with hemiplegia caused by middle cerebral artery stroke more than 6 months prior and agreed to participate in the study were included. Informed consent was obtained from all participants. The training period was 8 weeks long. The inclusion criteria were as follows: first stroke, ability to walk at least 6 m independently without assistance , Mini-Men = 15) and general gait training groups (n = 15). Both groups received gait training for 30 min five times a week for 8 weeks , main exercise (20 min), and cool-down (5 min). During this program, participants maintained a forward gaze, focusing on the front. If the participant adapted to the program without losing their balance and showed improved walking speed compared to the initial speed, the walking speed was gradually increased, and the assistance was reduced.The 10-meter walk test, which is commonly used to assess the gait ability of patients with neurological impairments ,27,28, iThe Dynamic Gait Index (DGI) is a tool that was developed to assess an individual’s stability and risk of falling during walking ,31,32,33The Figure-of-8 walk test (F8WT) was used to assess curved walking ability ,37,38,39The Timed-Up-and-Go (TUG) test is used to assess the mobility, balance, and motor ability of stroke patients ,41. In td = 1.34 was used to calculate the sample size based on the result of the F8WT (time and steps) in a prior pilot test [d = 1.34, significance level of 0.05, and power of 0.90 [This study used a single-blind randomized controlled trial design. G power 3.1.9.2 software   was usedlot test . Twenty- of 0.90 . An addit-test was performed to analyze the differences between the dependent variables according to the measurement period (before and after the experiment), while an independent t-test was used to compare the effects of gait training on gait ability between the two groups.The statistical analysis was performed using the Statistical Package for the Social Sciences software . All measured values are presented as mean and standard deviation. The Levene’s homogeneity of variance test was conducted to test the general characteristics and homogeneity of the participants before the experiment. A paired p < 0.05.The effect size was calculated to explain the arbitration effect more objectively. The significance level was set at p > 0.05) in general characteristics  for DGI, F8WT speed, and F8WT steps.The dynamic gait ability results with curved-path versus general gait training in patients with stroke are shown in This study aimed to determine the effect of a curved-path gait training program on walking in patients with stroke. Significant improvements were observed in both curved-path gait training and general gait training pre- and post-intervention after the 8-week intervention; however, the curved-path gait training showed a significant improvement in gait ability compared to the general gait training group. Both training methods improved weight load, weight shifting, and speed changes on the affected side. It is important to note the significantly greater increase in walking speed observed after the intervention, particularly in the curved-path gait training group. This improvement in walking speed indicates enhanced muscle activation on the affected side, improved weight shifting and bearing, increased stance time on the affected side, and ultimately results in a more balanced walking pattern compared to general gait training. Eng et al. (2008)  also repOn the TUG test, the curved-path gait training group showed statistically significant improvements compared with the general gait training group. Consistent with the results of this study, Kim et al. (2012)  noted thThe 10-meter walk test results for both groups showed statistically significant improvements in gait speed after training. The curved-path gait training group showed statistically significant improvements compared to the general walking training group. In a similar study, Lee et al. (2003)  reportedOn the F8WT, the curved-path gait training group demonstrated improved gait speed and stride compared with the general gait training group. Similar to the results of this study, Kim et al. (2012)  reportedChen et al. (2014)  reportedOur study showed that the curved-path gait training improved overall gait ability compared to general gait training. The decreased number of steps observed in the F8WT after curve-path gait training may be related to an increase in step length and a decrease in step width. These changes in step length and width resulted in improved gait speed compared to the general gait training. The increased gait speed indicates an improvement in the activation of knee extensor muscles on the affected side, ultimately resulting in a longer stance time on the affected side and an increased swing phase time on the unaffected side. The prolonged swing time helps to compensate for the difference in stride length between both legs, leading to a more symmetrical movement between both sides. In other words, it is considered positive that the curved-path walking training has resulted in an increase in walking speed compared to general walking training, as it creates more symmetrical movements through spatial and temporal changes such as increased weight acceptance on the affected side, increased step length and swing time, and decreased step width.This study had some limitations. First, it included only participants with chronic middle cerebral artery stroke. It would be necessary to verify the effectiveness of various stroke cases as well, as the remaining function and prognosis of patients may differ depending on the damaged blood vessels . Second, one-sided focus training on the affected side was not performed during gait training. Third, we failed to measure the relationship between vestibular sense and proprioception or between muscle activity and improved walking ability. Fourth, we compared only curved-path gait training and general gait groups; no control group was included. Although there may be ethical issues with including a group of patients with stroke who receive no intervention, it could serve as an important control group. Fifth, the study did not consider upper limb movements, such as the trunk and shoulder, in relation to the gait ability of patients with stroke.This study investigated the effects of curved-path gait training on gait ability in stroke patients with hemiplegia. Curved-path gait training resulted in significantly improved gait ability compared with general gait training. This improvement ultimately results in a more balanced walking pattern compared to general gait training. These results suggest that curved-path gait training can be a meaningful intervention for improving the asymmetrical gait ability of stroke patients with hemiplegia. However, the neurological effects of this type of training have not been clearly defined. Further research is needed to investigate the effects of curved-path gait training on other neurological patients with gait impairment and other related neurological conditions.


Cytological evaluation of lymph nodes (LN) during staging of canine Mast Cell Tumors (MCTs) is a hot topic. While histology is currently the gold standard of LN evaluation, cytology would have the advantage of in vivo investigation that allows for the planning of a therapeutical approach. However, at present, LN cytological examination and interpretation needs to be standardized. In the present work, the significance of the nodal mast cell number in dogs with and without MCT has been investigated, comparing different counting methods and different sample preparation techniques. Our results suggest that, while counting methods and sample preparation technique do not influence MC count, the nodal MC number can discriminate between metastatic and non-metastatic LNs, but fails to distinguish between metastatic and possibly metastatic LNs.Cytological evaluation of lymph nodes (LN) in canine cutaneous mast cell tumors (MCT) has a key role in MCT staging. However, cytological discrimination between metastatic and reactive LNs is debated and diagnostic criteria inconsistent. The aim of this study was to retrospectively quantify nodal mast cells (MCs) in non-oncological (NOD) and MCT-bearing dogs (MCTBD), using different sample preparation techniques, to evaluate the significance of the MCT number. Cytological specimens from NOD-LNs (10 fine-needle aspirates—FNAs) and MCTBD-LNs  were evaluated. MCTBD-LNs were grouped in: non-metastatic, possibly-metastatic, and metastatic based on current literature criteria. MCs were counted in 4, 8, and 20 high-power-fields, and over 500, 1000, and 2000 total cells. MCs were significantly more numerous in MCTBD-LNs than in NOD-LNs and in “metastatic” samples than in “non-metastatic”. There was no significant difference between “metastatic” and “possibly metastatic” samples. Sample preparation techniques did not influence these results. A negative correlation between MCs number and sample cellularity was observed. Results were confirmed regardless of the counting method applied. MCs counting per se cannot distinguish possibly metastatic and metastatic cytological samples. Sample preparation technique and the counting method applied seem to have no influence on cytological quantification of nodal MCs in MCTBDs. Mast cell tumor (MCT) is the most common cutaneous neoplasm in dogs ,2,3,4,5.Lymph nodes evaluation for determining metastatic status is, therefore, highly recommended ,16,17,18Unfortunately, cytological interpretation of LN in canine MCT is still controversial ,21. The At present, no clear consensus exists regarding the quantity of MCs that can be present in normal or reactive canine LNs in dogs ,30,32,33To the best of our knowledge, only four studies so far included the quantification of MCs in LNs from MCT-bearing dogs ,21,23,30More standardized and feasible criteria for LN cytological assessment that would correspond as much as possible to the histological classification by Weishaar et al. are, therefore, still needed ,17,21,28Moreover, the growing attention toward LN mapping techniques in veterinary medicine has pushed forward the need for standard criteria in interpreting lymph nodes metastatic status ,44,45,46The purpose of the present study was, therefore, to perform a preliminary investigation to verify whether the quantification of nodal MCs is a useful feature per se to evaluate the presence or absence of MCT nodal metastasis in canine cytological samples, regardless of the sampling method applied to obtain the specimen. The number of nodal MCs in MCT-bearing dogs (MCTBDs) and dogs affected by non-neoplastic conditions (NODs) was compared. Different sample preparation techniques  were also compared to verify if the sampling method significantly impacts on the number of nodal MCs in samples from MCT-bearing dogs.The database of the Diagnostic Pathology Service of the Department of Veterinary Medicine and Animal Science (DIVAS—Università degli Studi di Milano) was retrospectively searched for canine cytological nodal samples collected between 1 January 2016 and 31 December 2018. Cytological samples obtained in vivo during daily clinical activities had been submitted by the University Veterinary Teaching Hospital and by external private veterinary clinics. Additionally, cytological samples were prepared by the authors ex vivo from surgically extirpated LNs sent for histopathological evaluation to assess regional or, more rarely, systemic/multicentric lymph-adenomegaly conditions, or for neoplasm staging. Most samples prepared via scraping, smearing and/or touch imprinting were obtained from clinically non-palpable SLNs surgically extirpated from MCTBDs .All cytological slides were air-dried and manually stained with May-Grünwald-Giemsa .Each cytological sample was considered as a single independent “case” even if belonging to a different LN from the same dog or belonging to the same LN sampled with different techniques. According to the cytological diagnosis reported in the database as well as to the information contained in the medical record of each dog, cases were classified as: NODs  or MCTBDs (cases from MCT-bearing dogs).Cases were excluded from the study if belonging to dogs lacking detailed clinical history, or to dogs affected concurrently by MCT and other neoplasms.Only cases obtained by fine-needle aspiration (FNA) were included for NODS, while cases from MCTBDs were further classified based on the sample preparation technique: MCT-F (FNA), MCT-S (scraping smearing), or MCT-T (touch imprinting).Each case was considered eligible for the study if at least 1 cytological slide was available for review, and if at least 20 adequately cellular high-power fields (HPF) were available. Each microscopic HPF evaluated with routine light microscopy  was considered as “adequately cellular” when at least 50% of its surface was represented by well-preserved cells arranged in a single layer without overlapping.Among eligible cases, the 10 most recent ones for the NODs category and for each subcategory of MCTBDs  were arbitrarily selected, for a final caseload of 40 cases .Details regarding each LN included in the study  and signalment data of dogs were reported in For each case obtained from MCTBDs, the metastatic status was cytologically evaluated applying the criteria proposed by Krick et al.  modifiedFor each investigated case, microphotographs of 20 hotspots, adequately cellular HPFs (each one corresponding to an area of 434.97 × 327.51 microns) were taken with a digital camera Olympus DP70  supported by the software Olympus DP-Soft . “Hot-spot” was defined as those characterized by the best balance between cellularity and cell preservation.Each microphotograph was then randomized and blinded for category, subcategory, and signalment information. Microphotographs were then uploaded in the Fiji software 1.8.0_172  and manuFor each counting method applied to each category and subcategory (from now on indicated as “groups”), both the median (with minimum-maximum range) and the mean (with standard deviation) absolute and percentage number of MCs were calculated to facilitate comparison of our results with those reported in previous studies.Data distribution for each counting method applied to each group mentioned above was investigated with the Shapiro-Wilk normality test. Considering that the majority of datasets did not pass the normality test, non-parametric tests  were applied to investigate the difference in the median number of MCs for each counting method among the following groups: NODs versus MCTBDs; MCT-NM versus MCT-PM versus MCT-M; MCT-F versus MCT-S versus MCT-T. Additionally, the error coefficient (obtained dividing the standard error of the mean by the mean) for each counting method applied to MCTBDs was calculated.The difference in the mean cellularity among the different groups was also investigated. Mean cellularity of each case was defined as the mean cellularity observed in the first 5 microphotographs entirely counted during counting “over cells”. Although all the datasets reporting mean cellularity data passed the Shapiro-Wilk normality test, non-parametric tests  were applied to maintain consistency with the other statistical analyses. Finally, to verify whether cellularity influenced the number of nodal MCs, the Spearman correlation coefficient was calculated.www.graphpad.com). Statistical significance was set at p-value ≤ 0.05.Statistical analyses were performed with GraphPad Prism software  electronical records of canine LN-cytological specimens collected between 2016 and 2018 were revised . Twenty-Cytological samples were obtained from 25 dogs ; 17 patiAll details concerning dogs signalment, LN localization, and pathological process (NOD and MCT) are provided in The median range of nodal MC absolute number was 0–0.5 and 3.90–28 for NODs and MCTBDs, respectively; the mean range was 0–1.7 and 79.22–428.3 for NODs and MCTBDs, respectively (details for each counting method are reported in p < 0.0001), regardless of the counting method applied .Although not statistically significant, mean cellularity was higher in cases obtained from NODs (median: 286.7) than from MCTBDs (median: 222.2). Spearman correlation analysis revealed a significant inverse correlation between MC number and cellularity in samples obtained from MCTBDs , but not for samples obtained from NODs  than MCT-NM cases, but not compared to MCT-PM cases , which instead was not observed in MCT-NM  and MCT-PM cases .Despite not being significantly different, mean cellularity showed a decreasing trend, being slightly higher in MCT-NM cases (median: 251.8) compared to MCT-PM ones (median: 238.6), which in turn were characterized by increased cellularity compared to MCT-M cases .Mean cellularity showed an increasing trend, being lower in MCT-F (median: 147.2) compared to MCT-S (median: 245.8) and slightly lower in MCT-S compared to MCT-T , and between MC number and cellularity in MCT-F exclusively for counting over 500 cells , although the Spearman coefficient was also constantly negative for the other counting methods . On the contrary, Spearman correlation analysis did not reveal any significant correlation between MC number and cellularity in MCT-T, regardless of the counting method applied for MC quantification .Spearman correlation analysis revealed a significant inverse correlation between MC number and cellularity in MCT-S with all counting methods  except for counting of MCs in 8 HPFs , and dogs with MCT ,30,32,33To the best of our knowledge, only four studies included the quantification of MCs in cytological LN samples from MCT-bearing dogs ,21,23,30Specifically, Mutz et al.  comparedMarconato et al.  evaluateSulce et al.  applied Finally, Sabattini et al.  applied To the best of our knowledge, this is the first study quantifying MCs in cytological nodal specimens from MCTBDs sampled with different techniques. The differences in the number of MCs among the different sample preparation techniques were not statistically significant, suggesting that the sample preparation technique does not influence the cytological evaluation of LN metastatic status in MCT-bearing dogs. This result opens the road to future investigations on the intraoperative use of cytology for the real-time evaluation of surgically extirpated SLNs. Future studies on a larger cohort of cases, possibly applying all three different methods to the same lymph node, are advisable to confirm our data.Additionally, it is noteworthy that the cytological diagnoses obtained from the same lymph node by scraping smear and by touch imprint were not consistent in three cases . SpecifiA decreasing trend in the number of MCs from FNAs to touch imprints has been observed and might be explained by potential correlations between the size of sampled LNs and their metastatic status. Specifically, at least 8/10 of MCT-F were reported as megalic , among wWhen the mean cellularity was investigated between the groups evaluated in the current study, the only significant differences were observed between MCT-F and MCT-T. The lower cellularity of scraping smears compared to touch imprints could be related to the greater number of broken cells resulting from the traumatic action on the neoplastic cells during smear preparation, although the literature reports that the scraping smears should provide an increased cellularity compared to FNA ,50. AddiThe statistically significant differences observed among the groups were maintained irrespectively of the counting method applied for MC quantification. Counting methods “over cells” are still considered more reliable because they are based on a proportionality ratio that is not theoretically affected by the extremely variable cellularity that can characterize cytological samples ,21. NoneThe lack of systematical comparison of the cytological evaluation of the LN metastatic status with the corresponding histological diagnosis and of repeated evaluation by multiple investigators represent the major limitations of the current work.The retrospective nature of the study and the experimental design determined a few further limitations. For instance, the different number of cases assigned to each of the subcategories according to the cytological diagnosis may have influenced the statistical analysis.In conclusion, although MCs counting might be an objective method to evaluate nodal metastasis in MCTBDs, this technique seems not to be useful per se to distinguish among non-metastatic, possibly metastatic, and metastatic cytological samples. Nonetheless, according to the observation that the system by Krick et al.  is mainl


The aim of this study is to evaluate the utility of binocular chromatic pupillometry in detecting impaired pupillary light response (PLR) in patients with primary open-angle glaucoma (POAG) and to assess the feasibility of using binocular chromatic pupillometer in opportunistic POAG diagnosis in community-based or telemedicine-based services.In this prospective, cross-sectional study, 74 patients with POAG and 23 healthy controls were enrolled. All participants underwent comprehensive ophthalmologic examinations including optical coherence tomography (OCT) and standard automated perimetry (SAP). The PLR tests included sequential tests of full-field chromatic stimuli weighted by rods, intrinsically photosensitive retinal ganglion cells (ipRGCs), and cones (Experiment 1), as well as alternating chromatic light flash-induced relative afferent pupillary defect (RAPD) test (Experiment 2). In Experiment 1, the constricting amplitude, velocity, and time to maximum constriction/dilation were calculated in three cell type-weighted responses, and the post-illumination response of ipRGC-weighted response was evaluated. In Experiment 2, infrared pupillary asymmetry (IPA) amplitude and anisocoria duration induced by intermittent blue or red light flashes were calculated.In Experiment 1, the PLR of POAG patients was significantly reduced in all conditions, reflecting the defect in photoreception through rods, cones, and ipRGCs. The variable with the highest area under the receiver operating characteristic curve (AUC) was time to max dilation under ipRGC-weighted stimulus, followed by the constriction amplitude under cone-weighted stimulus and the constriction amplitude response to ipRGC-weighted stimuli. The impaired PLR features were associated with greater visual field loss, thinner retinal nerve fiber layer (RNFL) thickness, and cupping of the optic disk. In Experiment 2, IPA and anisocoria duration induced by intermittent blue or red light flashes were significantly greater in participants with POAG than in controls. IPA and anisocoria duration had good diagnostic value, correlating with the inter-eye asymmetry of visual field loss.We demonstrate that binocular chromatic pupillometry could potentially serve as an objective clinical tool for opportunistic glaucoma diagnosis in community-based or telemedicine-based services. Binocular chromatic pupillometry allows an accurate, objective, and rapid assessment of retinal structural impairment and functional loss in glaucomatous eyes of different severity levels. Glaucoma is the most frequent cause of irreversible blindness worldwide and is characterized by a decline in retinal ganglion cells (RGCs) and visual function damage  relies on the integrity of the connection between the outer  and inner (ipRGC projection) retinal photoreception , is an informative clinical sign indicating unilaterally or asymmetrically affected eyes  with binocular occulted head mount displays (HMDs) and ramping-up lighting stimuli designed with sequential tests, to lower the environmental requirements of the examination. We applied the full-field stimuli to evaluate the global response of rod-, cone-, and ipRGC-weighted PLR sequentially and the intermittent chromatic flashes to evaluate the inter-eye asymmetry of PLR. We extracted the PLR features to correlate these functions with different severities of glaucoma. Furthermore, we evaluated the performance of our binocular chromatic pupillometer as a potential diagnostic tool for evaluating POAG. To our knowledge, this is the first investigation that employs alternating chromatic stimuli for RAPD testing in pupillometry studies of POAG patients.Seventy-four patients with POAG (26 women and 48 men) and 23 visually healthy controls (12 women and 11 men) were included in this clinic-based, cross-sectional study conducted between May 2021 and October 2022. Based on previous studies . The research followed the tenets of the Declaration of Helsinki, and the study was approved by the human subjects review committee of the Eye and ENT Hospital of Fudan University. Written informed consent was obtained from each participant. All participants underwent comprehensive ophthalmologic examinations including best-corrected visual acuity, intraocular pressure measurement, slit-lamp stereo biomicroscopy, color vision , and direct ophthalmoscopy, OCT, and SAP. Patients with clinically confirmed POAG were identified by a fellowship-trained specialist as having glaucomatous optic neuropathy  and a compatible visual field defect  the presence of two or more adjacent points with p < 1% probability of normal range and one or more points with p < 2% probability  and the G standard white/white TOP program. All SAP tests were required to meet reliability criteria. SAP tests were defined as abnormal when they met one of the following conditions: (1) the presence of three or more adjacent points in the superior or inferior field with Optical coherence tomography was performed with the RTVue-100 spectral domain optical coherence tomography , and the standard glaucoma protocol was applied, including a three-dimensional optic disk scan for the definition of the disk margin, an optic nerve head (ONH) scan, and a standard ganglion cell complex (GCC) scan.The setup consisted of an HTC VIVE Pro Eye, with an integrated eye tracker . The visual stimuli were generated in Unity 2018, transmitted to the HMD utilizing SteamVR 2.0, and run on a laptop computer . Participants were fitted with HMDs that used near-infrared (940 nm) illuminating diodes and infrared cameras to track pupil data (sample rate 90 Hz). The pupil diameter of the eye was obtained through SRanipal SDK supported by Tobii XR and was calculated by multiplying the diameter of the pupil on the acquired image and by a scaling distance factor between the eye and the sensor. The stimuli were presented to one eye, and the pupillary data from both eyes were recorded. Both eyes were tested in turn. The stimuli consisted of short-wavelength (“blue” dominant wavelength of 465 nm) and long-wavelength (“red” dominant wavelength of 642 nm) pulses of light according to different experiments. Stimulus wavelength and luminance were verified using a spectroradiometer .2 was presented. For the melanopsin-dependent PLR test, a blue flash with a high luminance of 100 cd/m2 was shown. For the cone-weighted PLR tests, the tested eye was first presented with a 6-cd/m2 blue field for 2 min to suppress rod activity, followed by a red flash (10 cd/m2).Experiment 1: PLR was induced by full-field chromatic light stimuli designed to activate the rod-, cone-, and melanopsin-weighted PLRs  or red (100 cd/m2) flash to both eyes (1 s*12 times). The direct and consensual response was recorded . Data were analyzed offline using custom scripts programmed in MATLAB , which automated interpolated eye movement and filtered short-time (<500-ms) eye blinks.In Experiment 1, the following components were extracted:Baseline pupil diameter : mean pupillary diameter during the initial 10 s before light onset.*100%.Rod-weighted PLR: maximum constriction amplitude induced by rod-weighted PLR  = BPD-maximum constriction pupil diameter/BPDTime to maximal constriction induced by rod-weighted PLR : the interval between the beginning of the rod-weighted stimulus and peak constriction.Time to dilation induced by rod-weighted PLR : the interval between the beginning of the rod-weighted stimulus and the recovery of the pupil diameter to 90% BPD.Constriction velocity of rod-weighted PLR : velocity of pupillary constriction induced by rod-weighted stimulus.Dilation velocity of rod-weighted PLR : velocity of pupillary dilation to 90% BPD following rod-weighted stimulus.at6safterflashoffset/BPD*100%.ipRGC-weighted PLR: In addition to the parameters  measured in the rod-weighted PLR test, the post-illumination pupillary response (PIPR) was also calculated. Sustained response was defined as the pupil diameter at 6 s after melanopsin-weighted stimulus flash offset. PIPR: BPD-PD Cone-weighted PLR: The constriction amplitude, latency, velocity of constriction, and dilation were calculated as above mentioned.In Experiment 2, the infrared pupillary asymmetry (IPA) curve was recorded during alternating stimuli  = |pupil diameter (OD)– pupil diameter (OS)|. The duration of an IPA >1 mm was calculated (intermittent blue/red flash induced).post-hoc analysis (LSD) or Dunn's method was further performed to identify significant group differences. Comparisons between the two groups were evaluated using the one-way ANOVA or the Wilcoxon rank-sum test (J). The relationship between the cumulative effect of pupillometric features and glaucoma severity was assessed using logistic regression. Comparison between the AUCs of pupillometric features, OCT, or SAP was performed using DeLong's method with Bonferroni correction. The threshold for significance for all statistical tests was set at 0.05. Statistical procedures were performed using R Version.4.2.2: A Language and Environment for Statistical Computing .Ophthalmologic examination outcomes and pupillometric features are represented as the means ± standard deviation or median (interquartile range). Comparisons between controls and the different glaucoma severity groups were evaluated using a one-way analysis of variance (ANOVA) or Kruskal–Wallis one-way analysis of variance on ranks test, and a sum test . The corThis study included 194 eyes from 97 participants, including 148 eyes from 74 patients with POAG, and 46 eyes from 23 healthy controls. The age in the control group was 42.00 ± 11.08 years, and the age in the glaucoma group was 42.46 ±13.81 years. POAG patients were further divided into mild–moderate and severe glaucoma groups. The description of the clinical characteristics and the pupillometric parameters under full-field stimuli of all the groups are provided in pAmplituderod = 0.001; pAmplitudeipRGC < 0.001; pAmplitudecone < 0.001). The velocity of pupil constriction was slower in POAG patients (p < 0.001 in three comparisons). In rod-weighted PLR and ipRGC-weighted PLR, dilation after transient constriction was prolonged in POAG patients , and no obvious delayed dilation was observed in cone-weighted PLR (p = 0.143). However, the velocity of dilation was significantly slower in patients with POAG who responded to the three stimuli . The post-illumination response of ipRGC-weighted PLR was different between healthy controls and patients with different severities of POAG . The constriction amplitude responded to ipRGC-weighted stimuli  and the constriction amplitude of cone stimuli  also showed strong correlation with the clinical parameters.The correlation between PLR features under full-field stimuli and current parameters to assess the severity of POAG is presented in pBlue − IPA < 0.001, pRed − IPA = 0.001). The duration of the anisocoria induced by intermittent blue or red light flashes was significantly longer in patients with POAG than in healthy controls . Among 74 POAG patients, 39 (52.7%) presented RAPD during intermittent blue light flashes, and 20 (27.0%) presented RAPD during intermittent red light flashes, which were more frequent than among healthy controls . IPA and anisocoria duration had good diagnostic values . The cumulative effect of two paired parameters showed a higher AUC for diagnosing patients with POAG . The deficit in the RAPD tests was correlated with the inter-eye asymmetry of retinal structure and MD score .In Experiment 2, the IPA induced by intermittent blue or red light flashes was significantly larger in patients with POAG than in healthy controls , followed by constriction amplitude responding to cone-weighted stimuli (amplitude cone) and constriction amplitude responding to ipRGC-weighted stimuli (amplitude ipRGC) and PIPR. We also highlight a close relationship between time to pupillary redilation to bright blue light with changes in retinal structure  and visual function (MD score). In RAPD tests, the inter-eye PLR asymmetry in patients with POAG was quantified by the IPA value and anisocoria duration, which correlated with the inter-eye asymmetry of retinal structure and MD score. These findings suggest that the impairment of pupillary responses could be detected using binocular pupillometry, and the PLR tests could potentially be used to estimate the severity of POAG and the inter-eye asymmetry in glaucoma severity.2 (wavelength 465 nm) was presented. In cone-weighted PLR tests, a blue field of 6 cd/m2 (wavelength 465 nm) was used to suppress rod activity for 2 min, followed by a red flash . The constriction amplitude, latency, and velocity of constriction and dilation were calculated in the rod/cone-weighted PLR tests. In the ipRGC-weighted PLR tests, a blue flash with a high luminance of 100 cd/m2 (wavelength 465 nm) was presented on the head-mounted screen. The 6-s PIPR and the abovementioned pupillometric parameters were calculated and evaluated as diagnostic indices for glaucoma.A PLR test using bright white light flashes is commonly used in clinical practice to roughly assess retinal sensitivity. However, a white light flash covers a wide spectrum of wavelengths and does not selectively favor the activation of excitatory photoreceptors  or simulated (2 milliwatts/cm2) afferent pupillary defects. Stimulus cycles were set for alternating eye stimulations of 200 ms in duration every 1.2 s. Cohen et al.  in 76 eyes from 38 patients with primary open-angle glaucoma. Duque-Chica et al. (2/s) of blue and red lights in patients with early-stage POAG, the alteration of PLR correlates with structural thinning of the RNFL, but not with visual field scores. Adhikari et al.  but not the RNFL asymmetry. In our study, we used binocular pupillometry and sequenced chromatic stimuli to better quantify the RAPD value. In Experiment 2, we found that the amplitude and duration of the anisocoria induced by intermittent chromatic light flashes were significantly larger in patients with POAG than in healthy controls. The inter-eye difference in the MD value was inversely correlated with the IPA value and anisocoria duration. The RAPD induced by intermittent blue flashes was more frequently observed in both controls and POAG patients. This result could be explained by the fact that the PLR to bright blue light was a combination of cone and ipRGCs, and the more post-illumination response was attributed to these cells. Consistent with previous studies, 52.7% of POAG patients exhibited RAPD, and 13.0% of healthy controls exhibited RAPD (blue flash induced) in our study (Chang et al., Glaucoma damage is often more severe in one eye (Tatsumi et al., d et al.  made useOne limitation of our study is that all the participants were dark-adapted shortly before stimuli; hence, rhodopsin was not entirely regenerated in rods to optimally capture light. The PLR to each stimulus could not be entirely isolated to a single photoreceptor type, and the response could only be considered as having one specific type with major participation (Kardon et al., In this study, we provide proof of concept for the use of binocular chromatic pupillometry for identifying POAG with a strong correlation to retinal structural measurements and visual functional impairment. This could be especially useful for patients who are unable to use the chinrest or for children. Binocular chromatic pupillometry could accurately and rapidly detect glaucomatous damage. The low-cost and patient-friendly program could be used to screen for multiple retinal and optic neuropathies in population-based screening.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 the human subjects review committee of the Eye and ENT Hospital of Fudan University. The patients/participants provided their written informed consent to participate in this study.XSu and YQ: conceptualization. YQ, HD, ZZ, YS, and JH: methodology. YQ, ZZ, and RL: data analysis. YQ and XSu: writing—original draft preparation. TL, TZ, JW, GZ, XSo, and YZ: writing—review and editing. XSu and GZ: funding acquisition. All authors contributed to the article and approved the submitted version.