The intergenerational transmission of language/reading skills has been demonstrated by evidence reporting that parental literacy abilities contribute to the prediction of their offspring’s language and reading skills. According to the “Intergenerational Multiple Deficit Model,” literacy abilities of both parents are viewed as indicators of offspring’s liability for literacy difficulties, since parents provide offspring with genetic and environmental endowment. Recently, studies focusing on the heritability of musical traits reached similar conclusions. The “Musical Abilities, Pleiotropy, Language, and Environment (MAPLE)” framework proposed that language/reading and musical traits share a common genetic architecture, and such shared components have an influence on the heritable neural underpinnings of basic-level skills underlying musical and language traits. Here, we investigate the intergenerational transmission of parental musical and language-related (reading) abilities on their offspring’s neural response to a basic auditory stimulation  and later phonological awareness skills, including in this complex association pattern the mediating effect of home environment. One-hundred and seventy-six families were involved in this study. Through self-report questionnaires we assessed parental reading abilities and musicality, as well as home literacy and musical environment. Offspring were involved in a longitudinal study: auditory processing was measured at 6  months of age by means of a Rapid Auditory Processing electrophysiological paradigm, and phonological awareness was assessed behaviorally at 5  years of age. Results reveal significant correlations between parents’ reading skills and musical traits. Intergenerational associations were investigated through mediation analyses using structural equation modeling. For reading traits, the results revealed that paternal reading was indirectly associated with children’s phonological awareness skills via their electrophysiological MisMatch Response at 6  months, while maternal reading was directly associated with children’s phonological awareness. For musical traits, we found again that paternal musicality, rather than maternal characteristics, was associated with children’s phonological phenotypes: in this case, the association was mediated by musical environment. These results provide some insight about the intergenerational pathways linking parental reading and musical traits, neural underpinnings of infants’ auditory processing and later phonological awareness skills. Besides shedding light on possible intergenerational transmission mechanisms, this study may open up new perspectives for early intervention based on environmental enrichment. SpecifiPrevious studies have additionally shown that such low-level auditory processing skills are highly heritable , with heAre reading skills and musical aptitudes associated? Associations between reading skills and melodic vs. rhythmic musical aptitudes were investigated in the sample of adults included in the study as parents.Are parental reading skills/musical aptitudes associated with their offspring’s auditory processing and phonological awareness? In an intergenerational longitudinal design, we explored the associations between parental reading skills/musical aptitudes and their children’s skills. More specifically, the neural correlates of low-level auditory processing (P1 and MMR) were included as a possible intermediate phenotype common to the language and music system, and phonological awareness skills at 5 years of age as a reading-related trait.Do parental reading skills and their musical aptitudes influence the environment  provided to children? How does this environment influence children’s auditory processing and phonological awareness? In the intergenerational longitudinal design, we further explored how parental reading skills/musical aptitudes influence the environment provided to children and what role such an environment plays in children’s development.Focusing on this electrophysiological paradigm, in the present study, we analyzed data from a broad longitudinal Italian sample  in orderSince both reading and musical aptitudes are heritable traits e.g.,  we folloBased on this literature, we specifically expected (1) reading skills and musical aptitudes to be associated ; based o2.2.1.Infants of 6 months of age and their parents were recruited to take part in a longitudinal study  which foM = 6.6 months, SD = 0.5) through a Rapid Auditory Processing task (see paragraph 2.4) and concurrent cognitive development was assessed using the Bayley Scales of Infant and Toddler Development  infants’ EEG/ERP data at 6 months, (2) children’s phonological awareness data at 5 years, (3) parents’ reading and musical assessment, (4) information about literacy and musical environment. Not every measure was available for all families, e.g., due to scheduling problems for parents’ reading assessment, rejection of EEG/ERP data, or because children had not yet reached the 5-year-old session; furthermore, the questionnaire assessing musical skills and environment was added to the research protocol only for later participants. Therefore, families were included in the sample when at least two out of these measures were available. Other inclusion criteria were (1) both parents were Italian native speakers, (2) infants’ gestational age was ≥35 weeks, (3) infants’ Bayley Cognitive Scale Score at 6 months of age was ≥7.n = 2) or an older sibling (n = 40) had a certified diagnosis of Developmental Language Disorder (n = 21), Developmental Dyslexia (n = 11) or both (n = 10). The final sample consisted of 176 families in which the mother, the father and one child  took part in the study. In 42 of these families, a parent  to validate self-reported measures, thus preliminarily exploring the relationship between task-based reading ability and self-reported measures.2.2.2.The Home Literacy Environment Questionnaire HLEQ;  was coll2.3.2.3.1.To estimate their musical sense and skills, parents were asked to fill out the Brief Music Experience Questionnaire BMEQ; , a 53-it2.3.2.ad-hoc questionnaire concerning musical environment, measured in how much they engaged in joint musical activities with their children during their first year of life. For example, each parent had to estimate how much time (quantified in average minutes per day) they spent listening to music with their child, singing to their child, and lulling their child rhythmically. A total score comprehensive of all musical activities for each parent was used for the analysis . This measure was available for 71 mothers and 51 fathers.Mothers and fathers separately completed a short 2.4.2.4.1.Auditory processing was assessed by means of an electrophysiological non-speech multi-feature paradigm tapping the ability to process rapidly changing and complex auditory stimuli and eliciting responses for two different auditory attributes . The paradigm and stimuli were identical to those used in previous research .Since the paradigm was originally implemented in order to specifically address early Rapid Auditory Processing, pairs of complex tones with a rapid inter-stimulus interval (ISI) of 70 ms were presented. The first tone in the pair always had a fundamental frequency of 100 Hz with 15 harmonics (6 dB roll-off per octave) and 70 ms  duration. For standard tone-pairs (STD) the same tone was repeated twice . Two deviant tone-pairs differing with respect to the second tone were presented: for the frequency deviant (DEVF), the second tone had a fundamental frequency of 300 Hz and 70 ms duration; for the duration deviant (DEVD), the second tone had a duration of 200 ms and a fundamental frequency of 100 Hz.The stimuli were presented in a passive oddball paradigm where 1,200 stimuli  were pseudo-randomized, so that at least three standard tone-pairs were presented before each deviant pair. The intertrial interval  randomly varied from 700 to 900 ms. All stimuli were presented at an intensity of 75 dB via speakers located on either side of and equidistant (95 cm) from the subject  with vertex as the online reference. Sampling rate was 250 Hz with 0.1–100 Hz online bandpass filter.M = 125.7, SD = 23.1, min = 60, max = 184; DEVF condition, M = 69.1, SD = 9.9, min = 60, max = 104; DEVD condition, M = 68.5, SD = 10.3, min = 60, max = 102).After recording, EEG data were exported to a MATLAB  compatible format and processed using EEGLAB , and ERP2.4.3.P1: peak amplitude was extracted in the time window 100–300 ms.MMR: we first computed the different waveforms: DEVF-STD  and DEVD-STD . Mean amplitude was then extracted in the following time windows: 350–550 ms for MMRF and 420–620 ms for MMRD. Mean amplitude instead of peak amplitude was extracted for the MMR because this component is typically wide and without clearly identifiable peaks.To examine the role of auditory processing, we focused on the first positive deflection occurring at about 150 ms from stimulus onset (P1) reflecting low-level stimulus detection and registration, and on the following large positive response corresponding to the mismatch response (MMR), reflecting a neural change detection process. Time windows and electrode sites to be submitted to statistical analyses were selected based on mass univariate analyses applied to a subset of ERP data . This prThese measures were available for 132 infants.2.5.M = 4.95 years; SD = 0.26) using the test “Competenze Metafonologiche”  = 0.281, p = 0.001; Rhyme detection and Pseudo-word minimal pairs, r(101) = 0.235, p = 0.007; Syllabic synthesis and Pseudo-word minimal pairs, r(101) = 0.191, p = 0.028). Since no specific difference between tasks was expected in relation to the other measures in the study, a composite score made of the mean accuracy score was calculated and entered in the analysis. This measure was available for 103 children.Phonological awareness was measured between 4.5 and 5.5 years of age . Descriptive statistics are reported in N = 344, 172 mothers and 172 fathers). Since the self-report measure yielded significant correlations with both Reading-accuracy  and Reading-speed, , only this self-report measure is reported in the main analysis, in order to maximize sample size.Indirect assessment of parental reading skills was performed through the self-report ADCL questionnaire, available on a larger sample  = 38.954, p < 0.001, and no effect of Stimulus Type, F = 0.769, p = 0.465 nor the two-way interaction, F = 1.204, p = 0.302. Following these preliminary results, two composite variables were created for the P1 peak, collapsing stimulus types and keeping separate responses in the two hemispheres .The ANOVA on the P1 peak included Stimulus type (STD vs. DEVF vs. DEVD) and Hemisphere (left vs. right) as within-subject factors. It revealed a main effect of Hemisphere, F = 15.762, p < 0.001, and a significant interaction Stimulus Type x Hemisphere, F = 4.484, p = 0.036. Following these preliminary results, four separate variables were included in the following analysis for the MMR amplitude .The ANOVA on mean amplitude of the MMR was computed on the difference waveforms, and included Stimulus Type (MMRF vs. MMRD) and Hemisphere (left vs. right) as within-subject factors. It revealed a main effect of Stimulus Type, Despite these differences between hemisphere and stimulus type, all the electrophysiological variables were highly correlated among each other .2.6.3.Since our second and third experimental questions pertain to the influence of parental reading/musical skills and home environment  on children development, we preliminarily investigated the possible intervening effect of the socio-demographic variables reported and described in 2.6.4.Descriptive statistics and Pearson’s bivariate correlation among the selected variables were performed using SPSS, Version 28.0 . These correlational analyses were exploratory and finalized to the generation of more specific data-driven hypotheses to be tested in the following mediation models. Due to the exploratory and preliminary nature of these correlational analyses, we did not adjust significance levels for multiple testing, in order to avoid type-II errors and not to miss potentially relevant associations to be tested in the further models .Mediation models were tested by using Structural Equation Modeling (SEM) as implemented in the Mplus 8.1 software package . We esti3.Descriptive statistics of all the measures included in the analyses are reported in 3.1.N = 152, including 85 mothers and 67 fathers with both measures).Associations between reading skills and musical aptitudes were investigated in the overall sample of parents with both measures (r(150) = −0.223, p = 0.006, and with reactive musical behavior, r(150) = −0.189, p = 0.020. Adults with lower scores in the ADCL questionnaire (corresponding to better self-reported reading skills) were characterized by higher scores in the two subscales of musical aptitude.Scores in the ADCL questionnaire were negatively associated with both innovative musical aptitude, 3.2.N = 129) and paternal reading skills (N = 129).Associations between parental measures and offspring electrophysiological measures of auditory processing were measured separately for maternal (As shown in r(100) = −0.254; p = 0.011, and r(101) = −0.248; p = 0.013, respectively.Interestingly, both paternal and maternal reading skills were associated with the offspring’s phonological awareness at 5 years of age, r(56) = −0.297, p = 0.023], and an association between paternal innovative musical aptitude and children’s phonological awareness   = −0.090, p = 0.294, and r(139) = −0.033, p = 0.697, respectively], whereas both maternal and paternal musical aptitudes were related to the musical environment provided by each parent , with the exception of paternal reactive musical behavior, r(48) = 0.217, p = 0.135 that was not related to paternal music environment.Correlations were analyzed separately to explore the relationship between (1) parental reading skills and literacy environment, and (2) parental musical aptitudes  = 0.230, p = 0.027; paternal musical environment, r(39) = 0.398, p = 0.010, with the exception of maternal musical environment, r(51) = −0.120, p = 0.390). However, against expectations, the musical environment provided in the first year of life did not influence auditory processing skills at 6 months of age  = 52.752, p < 0.001; RMSEA = 0.000, CI (90%) = 0.000–0.000; CFI = 1.00; SRMR = 0.000] and explained 27.9% of the variance in children’s phonological awareness.Having shown that individual differences in children’s phonological awareness were associated with maternal and paternal reading skills and with specific measures of early auditory processing skills in infants (amplitude of the MMR in the right hemisphere), we used SEM to test the mediation model depicted in β = 0.397, p < 0.001). Children with higher discrimination skills (as reflected in the amplitude of the MMR on the right hemisphere) have higher phonological awareness scores at age 5 years. A significant effect was found from paternal reading skills to both measures of auditory discrimination : better paternal reading skills predict higher amplitude of the MMR.Standardized estimates of path coefficients are shown in β = −0.075; SE = 0.037; 95% CI : paternal reading was associated with more positive MMRF at age 6 months which, in turn, influenced phonological awareness skills at age 5 years. Conversely, maternal reading was directly associated with children’s phonological awareness  = 18.693, p = 0.002; RMSEA = 0.000, CI (90%) = 0.000–0.000; CFI = 1.00; SRMR = 0.000] and explained 21.8% of the variance in children’s phonological awareness.Having shown that individual differences in children’s phonological awareness were associated with parental musical aptitudes and with the musical environment provided by fathers to infants in the first year, we used SEM to test the mediation model depicted in β = 0.321, p = 0.002). Children whose fathers provided richer musical environment during the first year of life had higher phonological awareness scores at age 5 years. A significant effect was found from paternal musical aptitudes  to paternal musical environment provided to the children : fathers with higher musical aptitudes provided richer musical environment to their children.Standardized estimates of path coefficients are shown in β = 0.113; SE = 0.058; 95% CI .More interestingly, using 5,000 bootstrap CI 95%, we found a significant indirect effect from paternal musical aptitude to children’s phonological awareness scores via the musical environment . Weak but significant associations were found between the two domains (both self-reported). We could have expected the rhythmic properties of musical ability to be specifically related to phonological awareness and reading skills, based on a previous study conducted on developmental population  characterized by the first stimulus in every pair being always identical, we could speculate that the auditory detection of this first stimulus might be subjected to the phenomenon of repetition suppression, i.e., the decrease of neural activity that can be observed when a certain stimulus is presented repeatedly . The association from this electrophysiological response at 6 months of age and phonological awareness 4.5 years later was expected based on the original theories of Rapid Auditory Processing  predictiN = 58). Since we hypothesized that music and reading-related traits shared underlying heritable mechanisms, we expected parental musical aptitudes to be related to both infants’ auditory processing and later phonological awareness. Our findings showed that only paternal musical aptitude, concerning the melodic aspects of musical skills, was associated with children’s phonological awareness outcomes. However, infants’ neural signatures of low-level auditory processing did not appear to be influenced by either paternal or maternal musical skills and aptitudes. This result contrasts with previous findings reporting that infants with musically trained parents had better neural entrainment to beat and meter compared to infants with musically untrained parents  and musical traits and environment (N = 58). Since such differences prevented us from building a solid comprehensive model that could include both aspects, we showed how parental reading and musical traits separately influence children’s phonological awareness through different mediators. Larger and more similar sample sizes are needed in order to investigate how the association between the two traits impacts the intergenerational pathway, i.e., whether it is possible to encompass both parental traits and the environment in a single overall model, and shed more light on how they interact in influencing children’s language competence. A second limitation of the study is the use of self-report measures of parental skills instead of directly assessing them as done with children. Regarding reading skills, however, the ADCL questionnaire is considered as a reliable measure of screening for (suspected) dyslexia in adults  skills and added some pieces of evidence on the potential mechanisms underlying parent-specific pathways. Specifically, maternal reading skills seem to directly predict children’s phonological awareness, whereas paternal reading skills seem to exert an indirect effect, mediated by infants’ neural underpinning of auditory discrimination skills. Interestingly, the home literacy environment had a significant effect on children’s phonological awareness skills but was not directly associated with either maternal or paternal reading skills. This suggests that the effects of parental reading skills on children’s phonological awareness are more likely to be explained by a predominantly genetic transmission rather than an environmental pathway. As for musical traits, we found again that paternal musical aptitudes and skills, rather than maternal characteristics, were associated with children’s phonological phenotypes: in this case, the association was mediated by the musical environment provided at home. Besides shedding light on possible intergenerational transmission mechanisms concerning reading and musical traits, this study may open up new perspectives for early intervention based on environmental enrichment: our findings suggest that home literacy and musical environment may be suitable targets for early intervention.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical Committee of the Scientific Institute IRCCS Eugenio Medea. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.CC, MM, and ML designed the study. CC and CD ran the experiment and collected the data and drafted the manuscript. CC, CD, and VR analyzed the data. CC and ML interpreted the results. All authors edited and revised the manuscript.This work was supported by the Italian Ministry of Health (Ricerca Corrente).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of this study was to investigate the factors predictive of clinical outcome in advanced hepatocellular carcinoma patients receiving ramucirumab treatment.We conducted a retrospective study using a multi‐institutional electronic medical records database in Taiwan. We included advanced HCC patients newly receiving ramucirumab as second‐line or beyond systemic therapy between January 2016 and February 2022. The clinical outcomes were median progression‐free survival (PFS) based on the modified Response Evaluation Criteria in Solid Tumors (mRECIST), overall survival (OS) and adverse events. We applied Kaplan–Meier methods to estimate median PFS and OS. Uni‐variable and multi‐variable Cox regression models were applied to identify the prognostic factors.We included 39 ramucirumab naive users with a median age of 65.5 (IQR: 57.0–71.0) years and treatment time of 5.0 (3.0–7.0) cycles, of whom 82.1% were male and 84.6% were Barcelona Clinic Liver Cancer (BCLC) stage C. After median follow‐up time of 6.0 months, 33.3% of patients' AFP level had decreased more than 20% within 12 weeks. The median PFS and OS were 4.1 months and non‐reach, respectively. Moreover, tumor burden beyond the up‐to‐11 criteria  and a decrease in estimated glomerular filtration rate of more than 10% within 12 weeks  were significantly related to PFS in the multi‐variable analysis. No patient discontinued ramucirumab during the treatment on account of side effects.Ramucirumab was an effective treatment option with good AFP response for advanced HCC patients in real‐world experience. Tumor burden beyond the up‐to‐11 criteria and a decrease in estimated glomerular filtration rate were independent predictive factors for progression‐free survival. The CGRD comprises seven hospitals and covers 10% of in‐hospital care and 14% of cancer patients in Taiwan.2.2We recruited all patients with BCLC stage B or C HCC, newly receiving ramucirumab between January 1, 2016, and February 28, 2022. The first date of ramucirumab use was defined as the index date. Included patients were followed from the index date until loss to follow‐up, death, or May 31, 2022, whichever came first. To avoid short‐term use, we excluded patients who received only one dose of ramucirumab. To evaluate real‐world effectiveness of ramucirumab as a second‐line or beyond treatment, we also excluded patients without baseline imaging records or those combining with other chemotherapy .2.3Ramucirumab was administered every 2 weeks with a dosage of 8 mg/kg. All patients received intravenous agents in hospital where nurses recorded any adverse events (AEs) and physicians arranged renal function, liver function, and tumor marker tests at least every 4 weeks, in order to assess the grade of AEs, based on the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI‐CTCAE) version 5.0.2.4Baseline information regarding prognostic factors including sex, age, weight, Eastern Cooperative Oncology Group (ECOG) performance status, BCLC stage, tumor size, tumor numbers, up‐to‐7 or 11 criteria, presence of macrovascular invasion (MVI) and/or extrahepatic spread (EHS), presence of ascites, Child–Pugh class, albumin‐bilirubin (ALBI) grade and common biochemical examination were recorded at the index date or within the 3 months closest to the index date. Patient HCC etiology, comorbidities, and co‐medication including HBV, HCV, alcohol consumption, Charlson comorbidity index (CCI), and chronic diseases were recorded from 1 year prior to the index date.2.5We described patients' characteristics and analyzed tumor responses and AEs using median and interquartile range (IQR) for continuous variables, and absolute and relative frequencies for categorical variables. We applied the Kaplan–Meier method to estimate median progression‐free survival (PFS) and OS among ramucirumab users based on different prognostic factors. We also applied univariable and multivariable Cox proportional hazard models to estimate hazard ratios (HR) for the different prognostic factors. In the multivariable model, significant variables in the univariable analyses were selected using stepwise regression analysis. We performed all analyses using SAS Enterprise Guide, version 8.3 (SAS Institute).33.1Within the study period, 94 patients newly commenced ramucirumab treatment. After excluding short‐term uses and chemotherapy combinations, 39 patients remained for further assessment of effectiveness and safety. The details of study population selection are shown in Figure 3.2p < 0.05). Otherwise, patients with AFP decreased by more than 20% had better PFS , and those with eGFR decreased by more than 10% also had better PFS .During the median 6.0 months follow‐up, 3 (7.7%) and 15 (38.5%) patients had partial response (PR) and stable disease (SD), respectively. Among all patients, the disease progression rate was 41.0%. Of the 39 patients, 13 (33.3%) showed a decline in AFP of more than 20%. Furthermore, the median PFS and OS were 4.1 months and non‐reach, respectively . As regards eGFR dynamic change, the eGFR significantly decreased after ramucirumab treatment .As regards the safety evaluation, treatment‐related ascites and eGFR decrease occurred in 20 51.3%) and 31 (79.5%) patients, respectively. AEs for any reason were reported in 37 patients (94.9%), whereby the most common AE was edema . These AEs were of grade 2 in four patients (10.2%) and none of the patients reported events of grade 3–5. Table % and 31 3.3In the univariable Cox regression analysis, we found that out of the up‐to‐11 criteria, better tumor response, decrease of AFP and eGFR were significantly associated with better PFS. For the multivariable analysis, we excluded the variable of tumor response in the model due to the surrogate endpoint of PFS. Finally, up‐to‐11 criteria  and eGFR decrease of 10%  were identified as independent prognostic factors for PFS  is one of the critical molecular targets in cancer treatment and has been used in several successful pivotal combination regimens for not only HCC but also other cancers.Higher AFP level correlated with higher expression of the VEGFR2 receptor.Until now, ramucirumab has been the only systemic treatment with biomarker “AFP”. AFP level correlates with tumor burden in HCC patients.Owing to the simplicity of targeting the receptors, ramucirumab has a good side effect profile. In the REACH‐2 trial, the incidence of AEs was low with good maintenance of liver function during treatment.There were some limitations to the current study. First, it was a retrospective and non‐randomized study. Selection bias was inevitable. Second, the cohort size was small and included patients with systemic treatment beyond second line. Third, follow‐up duration was not long enough to evaluate the OS.In conclusion, ramucirumab was an effective treatment option with good AFP response for advanced HCC patients in real‐world experience. Tumor burden beyond up‐to‐11 criteria and the decrease in estimated glomerular filtration rate were independent predictive factors for progression‐free survival. Despite the safety profile being tolerable, liver function fluctuations and renal function during treatment require ongoing monitoring.Po‐Ting Lin: Conceptualization ; data curation ; writing – original draft ; writing – review and editing . Min‐Hua Hung: Data curation . Shih‐Chieh Shao: Conceptualization ; formal analysis ; methodology ; writing – original draft ; writing – review and editing . Hui‐Yu Chen: Project administration ; resources . Yuk‐Ying Chan: Project administration ; resources . Kai‐Cheng Chang: Conceptualization ; data curation ; formal analysis ; methodology ; software ; writing – original draft ; writing – review and editing . Shi‐Ming Lin: Methodology ; project administration ; writing – review and editing . Huang‐Tz Ou: Supervision ; writing – review and editing .This study was supported by grants from Chang Gung Medical Research Fund (CMRPG3M039).None reported.The datasets generated and/or analyzed during the current study are not publicly available for reasons of data privacy.Informed consent requirement was waived due to the retrospective study design.Not applicable.Not applicable.Figure S1.Click here for additional data file.


To explore the mediational effect of activities of daily living (ADL) and kinesiophobia on the cardiac function and health status of patients with chronic heart failure (CHF).From October 2021 to January 2022, a total of 244 CHF patients treated in the Department of Cardiology of general hospitals were recruited by the convenience sampling method. They were investigated with the Tampa Scale for Kinesiophobia Heart (TSK‐SV Heart), the Barthel index for assessing ADL, and the EuroQol five‐dimensional questionnaire (EQ‐5D) for assessing the health status.r = −.390 and −0.410, respectively, both p < .01). Besides, the ADL of CHF patients was positively correlated with the health status . The cardiac function of CHF patients was negatively correlated with the ADL , but positively correlated with kinesiophobia . The mediation proportion of ADL plus kinesiophobia between the cardiac function and health status of CHF patients was 43.48%. Both ADL and kinesiophobia partially mediated the effect of cardiac function on health status in CHF patients, but their mediational effects showed no significant difference (p = .777).The cardiac function and kinesiophobia of CHF patients were both negatively correlated with their health status  programs based on the cardiac function, ADL and kinesiophobia of CHF patients may contribute to reduce the medical burden and improve the well‐being of affected people. 1.Only 2.5% of chronic heart failure(CHF) patients were engaged in Cardiac rehabilitation(CR) programs and 77.9% of them participated in daily activities/exercise, the top three of which were walking (73.8%), housework (27.9%) and jogging (21.3%). Kinesiophobia was detected in 170/244 (69.5%) CHF patients who had limited or low‐intensity daily activities/exercises. Both activity of Daily Living (ADL) and kinesiophobia partially mediated the effect of cardiac function on health status in CHF patients, the mediational effects are equal. Kinesiophobia should be relieved and ADL enhanced to improve the health status and prolong the life expectancy of CHF patients. Exclusion criteria: (1) Subjects with unconsciousness or cognitive impairment; (2) Subjects with malignant tumors, severe liver or renal failure. This study was approved by the Ethic Committee of the First Affiliated Hospital with Nanjing Medical University (2019‐SR‐474).A consecutive sample of 244 HF patients were recruited from the Department of Cardiology of the First Affiliated Hospital with Nanjing Medical University from October 2021 to January 2022 by convenience sampling method. Inclusion criteria: (1) CHF diagnosed based on the 2.2Pretrained investigators were responsible for explaining the aim and clinical significance of this study using the unified instructions. The subjects were asked to fill in questionnaires by themselves after obtaining the written informed consent. For those who were unable to write or read by themselves, investigators read the items one by one, and recorded their answers. All completed questionnaires were collected within 48 h before discharge, and checked on the spot. All missed or incorrectly filled items were revised by reinquiring subjects.2.2.1The self‐designed questionnaire for 14 baseline characteristics of subjects included the sex, age, marriage, educational level, occupation, current work situation, current per capita monthly household income (yuan), medical insurance, smoking history, drinking history, comorbidities at admission, course of HF, cardiac function classification, and primary disease.2.2.2The self‐designed questionnaire for exercise status and type included the involvement of CR programs, daily exercises/activities, the frequency, time and type of exercises/activities, and fitness centers near your home.2.2.3The 10‐item Barthel index was calculated for assessing ADL, with a total score of 100 points. A higher Barthel index indicated better ADL.2.2.4α for the TSK‐SV Heart was .859.The Chinese version of TSK‐SV Heart developed by Lei et al.2.2.5α for the EQ‐5D was 0.761. The EQ‐5D score was transformed using the time trade‐off model.The 6‐item EQ‐5D was used to assess health status in five dimensions, including the mobility, self‐care, usual activities, pain/discomfort and anxiety/depression.2.3α = .05. p < .05 was considered as statistically significant.Statistical analysis was performed using SPSS 25.0. Enumeration data were expressed as the frequency and constituent ratio, and measurement data as mean ± standard deviation. The correlation between two variables was identified by the Spearman's rank correlation. Analysis of variance single factor analysis of variance was used. The structural equation modeling and path analysis were performed using IMB SPSS Amos 26.0. The mediation analysis with two mediators was performed by the bootstrap test at 33.1A total of 244 eligible CHF patients with a mean age of 64.45 ± 11.076 (30–93) years and a mean number of 3.05 ± 1.796  comorbidities were recruited, involving 138 (56.6%) men and 106 (43.4%) women. Their baseline characteristics were listed in Table 3.2Only 6 2.5%) of CHF patients used to or were now participating in CR programs. A total of 190 (77.9%) CHF patients could perform ADL. The frequency, time and type of daily activities/exercises are listed in Table .5% of CH3.3Kinesiophobia was detected in 170 (69.5%) of CHF patients who had a minimum TSK‐SV Heart score of 37 points. There were 202 (82.2%) with a minimum Barthel index of 60‐points, suggesting their good ADL (Table 3.4p < .01), but positively correlated with the kinesiophobia (p < .01). In addition, ADL of CHF patients was positively correlated with the health status (p < .01), but negatively correlated with the kinesiophobia (p < .01). A negative correlation was identified between kinesiophobia and health status in CHF patients (p < .01)  Table .3.5χ2/df, goodness‐of‐fit index (GFI), adjusted GFI (AGFI), comparative fit index, Tucker‐Lewis index and the root mean square error or approximation of the model were 1.614, 0.954, 0.924, 0.951, 0.933, and 0.053, suggesting the good fitness of the structural equation model. Furthermore, the significance of mediational effects was tested by bootstrapping in IMB SPSS Amos 26.0 with the sample size of 5000. It is shown that the 95% confidence interval (CI) of bootstrap was 0.036−0.532, in which 0 was not included, suggesting the statistical significance of mediational effects of kinesiophobia and ADL between cardiac function and health status of CHF patients. In the mediation analysis, the total effect c and the direct effect c΄ were 0.023 (p < .01) and 0.013 (p = .001), respectively. The indirect effect (ab) was calculated as ab = a1 × b1 + a2 × b2 = 0.006 + 0.004 = 0.01. Therefore, the mediation proportion of ADL plus kinesiophobia in the correlation between cardiac function and health status, which was calculated as the ratio of the indirect effect to the total effect, was 43.48%. The difference between the mediational effects of two mediators (ADL and kinesiophobia) was 0.001 , suggesting that both ADL and kinesiophobia partially mediated the effect of cardiac function on health status of CHF patients, but their mediational effects had no significant difference , housework (27.9%) and jogging (21.3%). CHF patients were less involved in other low‐risk activities/exercises, like dancing (7.0%) and Tai Chi (1.6%). Despite strong evidence supporting the preventive advantages of CR, previous research has indicated that CR is underutilized by clinicians and patients.Kinesiophobia was detected in 170/244 (69.5%) CHF patients, suggesting that kinesiophobia was highly prevalent in CHF patients, who had limited or low‐intensity daily activities/exercises. Previous studies reported higher Kinesiophobia scores (40.66 ± 5.56‐points) in patients with acute cardiovascular disease and elderly people with CHF than those in our findings, which may be attributed to the acute phase of disease, stronger fear on pain and negative emotions in the elderly.4.3Pairwise correlations were identified among the cardiac function, health status, ADL and kinesiophobia in CHF patients. CHF patients with the severer classification of cardiac function possessed worse ADL, health status and kinesiophobia. Cardiac function could mainly reflect the clinical manifestations, health status and prognosis of HF patients. Decreased exercise capacity is a main symptom of HF, manifested as fatigue, dyspnea, precordial pain, and intolerance to high‐intensity exercise.p < .001).n = 16) and NYHA Ⅳ  class were included in this study. Current studies mainly highlight the correlation between kinesiophobia and activity status in patients with cardiovascular diseases, and influencing factors. However, there is currently a limited number of interventions available to address kinesiophobia. Sahin et al.The presence of kinesiophobia poses a significant risk to the life and health of patients in numerous other diseases, associated with disability, depression, pain and even disuse.4.4Our findings revealed that the cardiac function not only directly influenced the health status of CHF patients, but also indirectly influenced it through kinesiophobia and ADL. The cardiac function of HF patients is closely linked with the pulmonary, renal, endocrine and metabolic functions, often accompanied by pain, depression, gastrointestinal discomfort, fatigue and other complications.Kinesiophobia and ADL mediated the influence of cardiac function on the health status of CHF patients, and their indirect effects accounted for 43.48% of the total effect. Notably, no significant difference in the mediational effect was detected between kinesiophobia and ADL, both being able to predict the health status. Somatic symptoms can be aggravated by negative emotions. As a negative psychological state, kinesiophobia leads to negative cognitive and emotional changes in CHF patients, and decreased ADL is an independent risk factor for acute HF onset and death.5CHF patients exhibit insufficient enthusiasm in CR programs, limited and low‐intensity daily activities/exercises, and prevalent kinesiophobia. Pairwise correlations exist among cardiac function, health status, ADL and kinesiophobia of CHF patients. The cardiac function not only directly impairs health status, but also indirectly through kinesiophobia and ADL, the mediational effects of which are equal. Kinesiophobia should be relieved and ADL enhanced to improve the health status and prolong the life expectancy of HF patients.Tang Yifan: Writing original draft, Investigation, Methodology, Formal analysis. Huang Yanling: Formal analysis, Investigation, Methodology. Wang Weiyun: Investigation, Data curation. Hu Xiaolin: Project administration, Supervision. Gu Zejuan: Project administration. Wang Rong: Project administration. Gao Chunhong: Writing—review and editing, Project administration, Conceptualization, Supervision.The authors declare no conflict of interest.


Patients with certain autoimmune conditions are at a reduced risk of developing breast cancer compared to the general population. Despite this, little is known about outcomes in patients with breast cancer who have a concurrent autoimmune diagnosis.This study compared differences in outcomes between women with breast cancer who had or did not have an autoimmune diagnosis. The SEER‐Medicare databases (2007–2014) were used to identify patients with breast cancer and diagnosis codes were used to identify those with an autoimmune disorder.p < 0.0001). After controlling for the effects of age, race, chronic kideny disease, chemotherapy, and radiation therapy autoimmune disease was still predictive of improved OS  and CSM . By contrast, in patients with stage I–III breast cancer, the presence of an autoimmune diagnosis was associated with a lower OS , compared to patients without autoimmune disease.The studied autoimmune diseases had a prevalence of 27% among the 137,324 patients with breast cancer. Autoimmune disease was associated with significantly longer overall survival (OS) and significantly lower cancer‐specific mortality (CSM) among stage IV breast cancer patients (We found a higher prevalence of rheumatoid arthritis, Crohn's disease, ulcerative colitis, and systemic lupus erythematosus in patients with breast cancer compared to age matched cohorts in the general population. The presence of an autoimmune diagnosis was associated with a lower OS in stages I–III breast cancer and improved OS and CSM in patients with stage IV disease. These results suggest that anti‐tumor immunity plays an important role in late stage breast cancer and could potentially be exploited to improve the effectiveness of immunotherapy. This study investigated the impact of autoimmune disease on survival in women with breast cancer using a national database of cancer patients. Our analysis showed that women with breast cancer have higher rates of certain autoimmune conditions than previously reported including rheumatoid arthritis and inflammatory bowel disease. Patients with early stage breast cancer and autoimmune disease were found to have slightly worse survival, although this did not seem to be due to their breast cancer but instead may have been due to older age and a higher rate of kidney disease in those with an autoimmune diagnosis. However, we also found that in women with advanced breast cancer, having an autoimmune diagnosis, correlated with longer survival. This study raises the possibility that the immune system plays an important role in late stage breast cancer. More research is needed to clarify why autoimmune disease was linked to improved outcomes. There are conflicting reports in the published literature reporting both improved and inferior outcomes in patients with cancer and a concomitant diagnosis of an autoimmune rheumatic disease, which suggests a dynamic and bidirectional relationship between the two.Several studies have shown a lower risk of developing breast cancer in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared to the general population.2Materials and methods were adapted from a previous study also published by this research group.2.1SEER‐MedicareBaseline characteristics included age, sex, race, level of urban development, poverty rates within the census tract, chronic kidney disease (CKD), hormone receptor status, breast cancer stage, and oncologic treatment history including chemotherapy and any surgery or radiation treatment within 4 months of cancer diagnosis. A diagnosis of CKD by SEER‐Medicare required at least one or two (depending on the type of claim) insurance claims with a CKD diagnosis code.2.2Certain patients were excluded from the analysis: 1) individuals whose age was ≥90 years, as recorded on the date of breast cancer diagnosis; (2) individuals with health maintenance organization (HMO) insurance; (3) patients with an additional cancer diagnosis of any kind during their lifetime; and (4) those first identified as having breast cancer during autopsy . Death from a non‐cancer related cause was a competing risk. OS was defined as the time that elapsed between the date of breast cancer diagnosis and date of death. OS was censored for survivors at the date of last recorded follow up. CSM was defined as the time that elapsed between the date of cancer diagnosis and the date of death from a non‐cancer cause. CSM was censored for those still alive at the date of last recorded follow up.2.4p‐values ≤0.05 were considered statistically significant. Analyses were conducted using R statistical software  and SAS statistical software .Descriptive statistics were used to assess baseline patient and cancer characteristics, additionally the chi square test with continuity correction was used as the test of significance for differences in categorical variables between the groups of patients who have and do not have an autoimmune diagnosis. The Kaplan‐Meier method was used to estimate the OS with log rank test used for the difference between groups.33.1n = 242,480) were identified from the SEER‐Medicare database. A total of 105,156 patients did not meet eligibility criteria and were excluded, the remaining 137,324 patients were included in the analysis , had stage I disease. Approximately 26% , 9% , and 5% (n = 6681) had stage II–IV disease, respectively, while in approximately 4% of patients (n = 5185) stage was unknown. The overall prevalence of the included autoimmune diagnoses in the 137,324 eligible patients was approximately 27%. Table Patients with breast cancer (p < 0.001). This was also seen but was less pronounced in the stage IV breast cancer cohort . Disease stage at the time of diagnosis did differ significantly between both groups (p < 0.001), but absolute differences on a percentage basis were small. A similar trend was also noted with both hormone receptor and HER2 status . Patients with an autoimmune diagnosis were more likely to have CKD . The difference in the prevalence of CKD was even larger in those with stage IV breast cancer . The percentages of those treated with radiation were similar in both groups. Significantly fewer women with an autoimmune diagnosis were treated with surgery (p < 0.001); however, this difference was small. With respect to chemotherapy, patients with autoimmune disease in the overall cohort were significantly more likely to have received chemotherapy . This difference was even more pronounced in patients with stage IV breast cancer .Patient characteristics which included: sex, race, urban versus rural locality, and poverty rates, were numerically similar when comparing the cohorts of patients with and without an autoimmune diagnosis Table . As expe3.2p = 0.0014), which was statistically significant when compared to patients without an autoimmune diagnosis, however, the difference was very modest and the survival curves were almost completely overlapping  between them and those, with a diagnosis of a different non‐RA autoimmune diagnosis, and patients without a diagnosis of an autoimmune disease , with CSM being lower in the patients with a diagnosis of an autoimmune disease, whether RA or a different autoimmune diagnosis, than in patients without an autoimmune diagnosis. There was not a statistically significant difference in CSM between the two autoimmune subsets, RA versus other autoimmune diagnosis  than patients without an autoimmune diagnosis, with the greatest separation of OS curves occurring after 60 months    Figure . Howevery) Table .p < 0.0001)  and CSM  Figure . The estp < 0.0001; II, HR: 1.06, 95% CI: 1.06–1.06, p < 0.0001; III, HR: 1.03, 95% CI: 1.03–1.04, p < 0.0001; IV, HR: 1.036, 95% CI: 1.03–1.04, p < 0.0001). Patients with breast cancer who self‐identify as Black had significantly worse OS than patients self‐identifying as White across all stages, even after adjusting for the impact of an autoimmune diagnosis, age, CKD and oncologic treatment, : 1.23–1.43, p < 0.0001; II, HR: 1.34, 95% CI: 1.25–1.43, p < 0.0001; III, HR: 1.42, 95% CI: 1.32–1.54, p < 0.0001; IV, HR: 1.32, 95% CI: 1.23–1.43, p < 0.0001). We also observed that patients with breast cancer who self‐identified as other than Black or White had significantly improved OS compared to patients who self‐identified as White for all stages : 0.51–0.65, p < 0.0001; II, HR: 0.73, 95% CI: 0.66–0.82, p < 0.0001; III, HR: 0.86, 95% CI: 0.75–0.97, p = 0.018; IV, HR: 0.72, 95% CI: 0.63–0.82, p < 0.0001). CKD was predictive of lower OS even when age, race, autoimmune disease and oncologic treatment are adjusted for across all stages : 0.33–0.36, p < 0.0001; II, HR: 0.39, 95% CI: 0.38–0.40, p < 0.0001; III, HR: 0.47, 95% CI: 0.44–0.50, p < 0.0001; IV, HR: 0.76, 95% CI: 0.71–0.8, p < 0.0001) : 1.09–1.10, ) Tables .4This study used a national cohort to investigate the impact of common autoimmune disorders on breast cancer survival outcomes. Our survival analysis showed a statistically significant improvement in OS among patients with breast cancer and a diagnosis of an autoimmune disease compared to those without autoimmune disease. This improvement in OS was primarily driven by patients with an RA diagnosis who were found to have a significantly higher OS, compared both to patients with a different non‐RA autoimmune diagnosis and patients without an autoimmune diagnosis. Patients with a different non‐RA autoimmune diagnosis had lower OS than patients without an autoimmune diagnosis. Given that patients with a diagnosis of an autoimmune disease other than RA had a significantly lower CSM than patients without any autoimmune diagnosis, the lower OS in patients with a non‐RA autoimmune diagnosis is likely not cancer related, and is possibly due to differences in baseline characteristics such as older age and higher rates of CKD among patients with autoimmune disease.In patients with non‐metastatic breast cancer (stages I–III) we observed that an autoimmune diagnosis was associated with worse OS. However, in these patients we observed no difference in CSM in those with or without an autoimmune diagnosis. Furthermore, when controlling for differences in age, race, CKD, chemotherapy, and radiation therapy, we found that an autoimmune diagnosis was actually associated with a significantly improved OS compared to patients with non‐metastatic breast cancer without an autoimmune diagnosis. Interestingly, in patients with stage IV breast cancer, we found the inverse relationship; a diagnosis of an autoimmune disorder was correlated with significantly improved OS and a mOS that was approximately 6 months longer than patients with stage IV breast cancer without an autoimmune diagnosis. This improved OS was at least in part due to a reduction in CSM associated with an autoimmune diagnosis and was not wholly explained by differences in baseline patient characteristics.The improvement in OS identified in this study in patients with breast cancer and an autoimmune diagnosis both in the overall cohort, among patients with RA specifically and in stage IV breast cancer contrasts with most previously reported studies.The overall prevalence of autoimmune disease in this population was approximately 27%. Several autoimmune diagnoses were enriched in this breast cancer cohort Table . Most noThere were several notable findings in terms of baseline patient characteristics of this cohort related to race and CKD. Whites were overrepresented in this breast cancer cohort compared to the general population of the United States (81% vs. 72%) while Blacks were underrepresented in this cohort compared to the general population (11% vs. 13%).The large size of this cohort decreases the possibility that the observed differences in OS are due to random chance. The 7‐year period data was collected over allowed for long term follow up. Furthermore, the years included in this study predate the use of immune checkpoint inhibitors in breast cancer, removing a possible confounder, and isolating the effect of autoimmune disease on outcomes from that of treatment with immunotherapy. There were also certain limitations to this analysis. SEER‐Medicare data largely includes patients >65 years old. Therefore, the findings of this study may not be generalizable to a younger population with breast cancer. Using diagnosis codes to identify patients with autoimmune disease could potentially allow patients that do not meet clinical and laboratory criteria for an autoimmune diagnosis to be included in the autoimmune group. Similarly, those with a true history of autoimmune disease that was not captured by the SEER‐Medicare database may not have been included. A small percentage of patients in the autoimmune cohort (13%) had their earliest autoimmune diagnosis code applied after their cancer diagnosis. This raises the possibility of survivor bias, longer lived patients are more likely to have the opportunity to develop new conditions. However, in these patients who had an autoimmune disease that developed or came to medical attention relatively soon after their cancer diagnosis, the autoimmune process could reasonably have influenced the disease course. It is difficult to discern if an autoimmune disease diagnosed after a cancer diagnosis was simply not documented until the patient was receiving closer monitoring due to their cancer treatment or if the patient truly did not develop an autoimmune disease until they reached advanced age.Furthermore, the Charlson comorbidity and National Cancer Institute indices were excluded from the multivariate regressions due to collinearity between comorbidity indices and autoimmune disease Tables . Both in5This study observed a higher prevalence of specific common autoimmune diseases such as RA, CD, UC, and SLE in patients with breast cancer compared to cohorts of similar age ranges in the general population. A diagnosis of an autoimmune disease in patients with stage I–III breast cancer was associated with a significantly worse OS, which disappeared when controlling for differences in age, race, CKD, chemotherapy, and radiation therapy. Moreover, in patients with non‐metastatic breast cancer the presence of an autoimmune diagnosis was not associated with any significant differences in CSM. By contrast, in patients with advanced stage breast cancer, a diagnosis of an autoimmune disease was associated with significantly improved OS and CSM even when controlling for differences in age, race, CKD, and oncologic treatments. This study raises the possibility that anti‐tumor immunity plays an important role in late stage breast cancer. Additional investigation is required to further elucidate the interplay between autoimmune diseases and breast cancer; as well as to explore the relationship between autoimmune disorders and outcomes in other cancer types. Pathways found to be contributing to the development of an elevated antitumor immune response in patients with autoimmune disease could be exploited to improve the effectiveness of immunotherapy and for future drug development.Demitrios Dedousis: Conceptualization (lead); data curation ; formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); supervision ; validation (lead); visualization ; writing – original draft (lead); writing – review and editing . Annie L Zhang: Project administration ; validation ; visualization ; writing – original draft ; writing – review and editing . Anastasia N Vassiliou: Data curation ; formal analysis ; investigation ; software ; visualization ; writing – original draft . Shufen Cao: Data curation ; formal analysis ; methodology ; software ; validation ; visualization ; writing – original draft . Deepthi Yammani: Data curation ; formal analysis ; software ; validation . Ravi K. Kyasaram: Data curation (supporting); software (supporting). John Shanahan: Resources ; supervision (supporting); writing – original draft (supporting). Melissa C Keinath: Visualization . Melinda L Hsu: Supervision ; writing – review and editing . Pingfu Fu: Data curation ; formal analysis ; methodology ; resources ; software ; supervision ; validation ; visualization . Afshin Dowlati: Conceptualization ; formal analysis ; funding acquisition (lead); methodology ; resources ; supervision ; validation ; writing – review and editing . Alberto J Montero: Conceptualization ; supervision (lead); validation ; writing – review and editing .This work was supported by the Division of Hematology and Oncology at University Hospitals.Dr. Dowlati reports personal fees from Seattle Genetics, AstraZeneca, Jazz Pharmaceuticals, Bristol Myers Squibb, Ipsen, Eli Lilly, Merck, Abbvie, and G1 Therapeutics outside the submitted work. The remaining authors declare no conflict of interest.This study was reviewed by the University Hospitals Institutional Review Board and determined to be an exempt study. All standard ethical guidelines were followed.Data S1:Click here for additional data file.


Reactive lymphocytes are substantial components of germinoma, which are believed to be related to the favorable prognosis of this intracranial tumor and better response to immunotherapy. However, the mechanisms managing the recruitment of lymphocytes are poorly understood. High endothelial venules (HEVs) are specialized blood vessels that play key roles in lymphocyte trafficking in Lymph nodes. These vessels are associated with lymphocyte infiltration in chronic inflammatory diseases and various malignant tumors, but their distribution and implications in germinoma are unknown. This study aimed to investigate the distribution and implications of HEVs in intracranial germinomas.We investigated the presence and distribution of HEVs in 42 germinomas by immunohistochemical staining of peripheral node addressin (PNAd) and transmission electron microscopic examination. The correlation of the densities of HEVs with the extent of T and B lymphocyte infiltration and several clinicopathological characteristics were also analyzed to determine whether HEVs are responsible for lymphocyte recruitment and their roles in anti‐tumor immunity in germinoma.p = 0.0410, 0.0023, and 0.0061, respectively). Higher HEVs density was also correlated with several clinicopathological parameters, which are recognized indicators for favorable prognosis in germinomas, including typical tumor location (p = 0.0093), lower tumor cell content (p = 0.0428), and younger age at diagnosis (p = 0.0121). Furthermore, bioinformatics analysis showed HEVs‐associated genes mainly enriched in immune‐related Gene Ontology terms, including innate immune response, inflammatory response, and B cell receptor signaling pathway. The xCell analysis revealed that germinomas with higher HEVs enrichment scores had increased levels of the immune score, microenvironment score, dendritic cells, CD8+ central memory T‐cells, CD4+ memory T‐cells, and B‐cells.PNAd‐positive HEVs were detected in 31% (13/42) of germinomas, and their presence correlated with abundant infiltrating CD3+ T cells, CD20 + B cells and CD8+ cytotoxic T lymphocytes (Our findings indicate that HEVs could contribute to lymphocyte recruitment in germinomas, thus may serve as a predictor of favorable prognosis and better response to immunotherapy in this intracranial tumor. The incidence of this tumor in eastern Asia is more than triple thaImmunotherapy for several kinds of tumors has achieved great success in the last decade. While various strategies, such as adoptive lymphocyte transfers, vaccines, and checkpoint blockade, have little to no effect if there are insufficient lymphocytes in the tumor microenvironment. At the same time, reactive lymphocytes are substantial components besides large primordial germ cells in germinoma.High endothelial venules (HEVs) are specialized post‐capillary venules that efficiently recruit lymphocytes from blood circulation into lymph nodes. The distinctive features of HEVs include the cuboidal endothelial cells and positive expression of peripheral node addressin (PNAd) on the apical surface of these cells. These characteristics can distinguish HEVs from other types of vessels under light microscopic examination. Besides lymph nodes and other secondary lymphoid organs, blood vessels with HEV‐like characteristics can also present in various kinds of chronic inflammatory diseases, including rheumatoid arthritis,This study aimed to investigate the distribution and implications of HEVs in intracranial germinomas. The presence of HEVs was analyzed using immunohistochemical staining, immunofluorescence staining, and transmission electron microscopic examination. The correlation between the density of HEVs with the extent of T‐ and B‐cell infiltration and several clinicopathological characteristics were also analyzed to determine whether HEVs are responsible for lymphocyte recruitment and their roles in anti‐tumor immunity in germinoma. Furthermore, we conducted a preliminary bioinformatic analysis on a series of transcriptome data to assess HEV‐related biological processes and enriched cell subsets.22.1We retrospectively identified 42 patients with pure intracranial germinoma who received surgical resection or stereotactic biopsy between 2016 and 2020 in the Department of Neurosurgery at Beijing Tiantan Hospital, China. The histopathological diagnoses were determined by routine evaluation of Formalin‐fixed paraffin‐embedded samples with hematoxylin and eosin (H&E) staining. All the 42 cases showing pure germinoma features without any other germ cell components were checked by at least two experienced neuropathologists. Formalin‐fixed paraffin‐embedded tissue blocks with adequate tumor tissue of these cases were obtained from the Department of Neuropathology. One patient's freshly resected tumor specimen was conducted for the transmission electron microscopic examination.Clinical and pathological information, including age, gender, tumor volume, and tumor location, were collected and summarized in Table Tumor cell content was determined by measuring the proportion of neoplastic elements, which were marked by immunohistochemical staining of organic cation/carnitine transporter4 (OCT4) among all kinds of cells in the tumor microenvironment. The measurement was conducted in the slides with the most abundant tumor cells.2.2Immunohistochemistry and immunofluorescence were performed on 4‐μm thick sections from the tissue blocks. The primary antibodies utilized in this study were as follows: PNAd Carbohydrate Epitope ; CD3 , CD8 , CD20 , and OCT4 . For Immunohistochemical staining, slides were incubated in an automated IHC Staining System  and visualized by Bond Polymer Refine Detection Kit . For immunofluorescence staining, slides were incubated with secondary antibodies and counterstained with DAPI.2.32 tumor area. In accord with the cutoff points for tumor HEVs density previously described,2 were designated as HEV‐high germinomas. In contrast, those with HEVs less than 0.20/mm2 were defined as HEV‐low germinomas.For HEVs, we first counted the number of MECA‐79‐positive vessels in each tumor slide under a light microscope. Then we determined HEVs density by calculating them as counts per mmThe amounts of CD3+ T cells, CD20+ B cells, and CD8+ cytotoxic T lymphocytes (CTLs) were determined by automatically counting these cells in five representative high‐power fields (HPF) with the most prominent tumor‐infiltrating lymphocytes (TILs). The counting was conducted using Image J Software (NIH).2.43) was fixed in 2.5% glutaraldehyde and 2% polyformaldehyde at 4°C for 2 hours. After rinsing with PBS, the tumor tissue was fixed in 1% osmium acid, followed by acetone dehydration, soaking, and epoxy resin embedding. One‐micrometer semi‐thin sections were then prepared and stained using the Azure‐methylene Blue Dye for localization analysis. Next, we prepared 80‐nm ultrathin sections and stained them with uranium dioxyacetate‐lead citrate. The ultrathin sections were observed by a Hitachi H‐7650 transmission electron microscope (Hitachi).Fresh germinoma tissue  and SPSS (IBM). Differences in HEVs density among groups categorized by gender, age, and tumor location were analyzed using the chi‐square test. Correlations of HEVs density with tumor volume, tumor cell content, and the amount of infiltrating CD3+ T cells, CD20 + B cells, and CD8+ CTLs were calculated using the Mann–Whitney 2.6t‐tests . Gene Ontology (GO) analysis was applied on the HEVs‐associated highly expressed genes. Meanwhile, the xCell algorithm was used to calculate the cell‐type enrichment scores.The transcriptome data were obtained from the Gene Expression Omnibus Datasets (GSE19348).33.12 to 21.13/mm2 with a median value of 0.20/mm2. We failed to observe any HEVs in 29 cases, mainly due to the small sizes of the resected tumor specimens. Six cases with HEVs density less than 0.10/mm2 only showed few locally presented MECA‐79‐positive vessels. These 35 cases were designated as HEV‐Low germinomas. HEVs density in the rest of the seven cases ranged from 0.20/mm2 to 21.13/mm2. HEVs density of these cases was higher than the median value, so they were designated as HEV‐high germinomas. The grouping was in line with the cutoff point for HEVs density previously described in several kinds of tumors.In total, 42 cases of pure intracranial germinoma patients who received craniotomy were enrolled in this study. We found MECA‐79‐positive HEVs in 13 (31%) cases. HEVs density range from 0.01/mm3.2p = 0.041). The same situation existed for CD8+ cytotoxic T cells . Furthermore, HEV‐high germinomas contain more abundant CD20+ B cells than HEV‐low germinomas . Representative images of immunohistochemical staining for lymphocytes are shown in Figure To investigate the role of HEVs in the process of lymphocyte trafficking in germinoma, we analyzed the association of HEVs density with immune infiltration. The mean amount of CD3+ T cells, CD20 + B cells, and CD8+ CTLs in all the germinomas in this study were 1077.6/HPF, 923.4/HPF, and 678.3/HPF, respectively. The amount of CD3+ T cells in HEV‐high tumors  was higher than that in HEV‐low tumors . The difference was statistically significant .Infiltrating lymphocytes have been proved to be directly cytotoxic to tumor cells in germinoma,3.3p = 0.0121). The age of HEV‐low germinoma patients was evenly balanced . In other words, 29.2% (7/24) of the younger cases were HEV high, while none of the older cases were HEV high.Clinicopathological characteristics of 42 germinoma patients are presented in Table p = 0.0013): Six out of 14 (42.8%) female patients were HEV high, whereas only one out of 28 male patients (3.6%) were HEV high.There were 14 female patients and 28 male patients in the present study. We observed significantly higher HEVs density in female patients : the proportion of HEV‐high tumors was 40% in germinomas in the suprasellar compartment, 7% in germinomas in the pineal region, and 0% in germinomas in other locations, respectively.Besides, HEVs density differed significantly between different tumor locations  was potentially higher than that of HEV‐high tumors , even though the difference did not reach statistical significance   Figure .4Germinoma is an intracranial tumor with abundant lymphocytes. However, the mechanisms managing the recruitment of lymphocytes remains poorly understood. In this study, we demonstrated the presence of HEVs in germinoma for the first time. We found that HEVs density was correlated with infiltrating immune cells, including CD3+ T cells, CD8+ cytotoxic T cells, and CD20+ B cells in this intracranial tumor. We also found HEVs were associated with smaller tumor volume and less content of malignant cells in germinoma. Furthermore, bioinformatics analysis showed HEVs‐associated genes mainly enriched in immune‐related biological processes and cell subsets.Light and electron microscopic examination revealed a plump or cuboidal appearance of the endothelial cells in these HEVs, unlike the flat appearance of endothelial cells in other postcapillary venules Figure . Besides3. Together with MECA‐79 negative cases, these cases were classified into the HEV‐Low group, according to the cutoff point defined by a previous study conducted on HEVs in multiple human malignancies.MECA‐79‐positive HEVs were observed in the major proportion of several distinct tumor types.To clarify whether HEVs play a role in lymphocyte recruitment in germinomas, the association of HEVs density with the level of infiltrating lymphocytes was tested Figure . We founHence, HEVs may be essential for effective antitumor immune responses in germinoma and improve patient outcomes. Due to the excellent long‐term survival of pure germinoma, all patients remained disease‐free until the last follow‐up (data not shown), making it difficult to evaluate the correlation between HEVs density and outcomes. That would be the limitation of this study. However, several factors have been shown to possess prognosis significance for germinoma in previous studies. Lower tumor cell content, typical location , and abundant infiltrating lymphocytes are significantly associated with a better prognosis of germinoma.Interestingly, we found that most of the cases in the HEV‐high group were female Figure . It has Bioinformatic analysis showed that HEV‐associated genes were mainly involved in the innate immune response. In accordance with the roles of HEVs in lymphocyte trafficking, HEV‐high germinomas possessed higher levels of the immune score, microenvironment score, B cells, and certain subtypes of T cells. Besides, we observed elevated enrichment scores of dendritic cells and conventional dendritic cells in the HEV‐high group. DCs and DC‐produced lymphotoxin are associated with HEVs formation by downstream LT‐βR signaling in lymph nodes and solid human tumors,Although malignant, pure germinoma has a significant favorable prognosis due to its sensitivity to radiotherapy and chemotherapy.5In summary, this study demonstrated the presence of HEVs in germinoma. Higher HEVs density was correlated with abundant infiltrating lymphocytes and multiple clinicopathological features, which are recognized indicators for favorable prognosis in germinomas. Besides, the bioinformatic analysis showed HEV‐associated genes mainly enriched in immune‐related biological processes and cell subsets. Our findings indicate that HEVs could contribute to lymphocyte recruitment in germinomas, and thus may serve as a predictor of favorable prognosis and better response to immunotherapy in this intracranial tumor.Huiyuan Chen: Conceptualization (lead); formal analysis ; investigation (lead); project administration ; software ; validation ; visualization (lead); writing – original draft (lead); writing – review and editing . Guilin Li: Funding acquisition ; methodology ; resources ; supervision . Yun Cui: Investigation ; methodology ; resources . Qi Zhang: Methodology ; resources ; visualization . Bo Li: Data curation ; supervision . Xing Liu: Conceptualization ; data curation ; funding acquisition ; investigation ; software ; supervision ; validation ; writing – review and editing (lead).The authors declare that they have no conflict of interest.The study protocol was approved by the Institutional Review Board and Ethics Committee of Beijing Tiantan Hospital.Written informed consent was obtained from all patients.Appendix S1Click here for additional data file.


Female head‐porters are a cohort of women who have migrated from their rural communities into commercial cities in search of better economic opportunities. These young women are vulnerable to untoward reproductive experiences. The study assesses the reproductive experiences of women and the factors influencing contraceptive use among them.n = 280). The study included 280 female head‐porters within the reproductive age of 15–49 years. Convenience sampling and consecutive recruitment were used to obtain the needed sample size. All statistical significance was declared at a p‐value of <0.05.A cross‐sectional study was conducted from January to May 2021 in the Kumasi Metropolis (p < 0.0001), parity (p < 0.0001), number of sexual partners post‐migration (p = 0.008), and age of first sex (p = 0.033) to be associated with contraceptive use among female head‐porters.Forty‐two percent of respondents had a history of contraceptive use . The study found gravidity  have a significant influence on the use of contraception. SDGs 3.7 and 5.6 place a premium on ensuring that everyone has access to family planning, information, and education, as well as other sexual and reproductive health services.Migration has become an essential part of human social life due to economic disparities across the globe, regions, or nations. Even in the same country, inequitable distribution of socioeconomic and educational opportunities causes people to move from one part of the country to the other.Female head‐porters are a cohort of women who have migrated from their rural towns into commercial/urban cities in search of better economic opportunities. This internal migration involves young women moving from their places of origin or livelihood to destinations across geographical boundaries within the same country.Kayayei (Kayayo for singular). “Kaya” in Hausa means luggage, load, or goods, while “yei” in Ga language means females. Their name literally means a young woman who carries luggage, goods, or a load for a fee. They usually help carry heavy loads of goods from bus stations and markets to various destinations for a negotiated fee.Female head‐porters are locally referred to as Movement of these young women has both positive and negative effects on their personal lives and the society at large. Even though some may have an improvement in their personal incomes, they may suffer from many dire consequences which may affect their future and health.Kayayei, sexual violence was a common phenomenon, and even for consensual sex, their partners often object to the use of contraception.Female head‐porters may not have access to modern contraceptives as they are usually beyond the reach of reproductive health advocacy programs. Previous studies show that among 22.1This was a cross‐sectional analytical study that took place from January to May 2021. This was the most feasible and cost‐effective design to answer study questions and evaluate study objectives within the given time period. The study determined both independent and dependent factors at the same time.2.2This study was conducted in the Asokore Mampong Municipality and Kumasi Metropolis. Kumasi is the second largest city in the middle half of Ghana. Market centers in the metropolis used for this study included Bantama market, Kejetia market, and Racecourse market. The residencies of female head‐porters are in the slums and deprived areas of the city. Some of these areas do not have potable water, access roads, health facilities, or security. Their living and sleeping places do not provide privacy for these girls. The majority of the places of residence of female head‐porters have no access to mass media. The market centers where they trade have information centers. These information centers, however, hardly provide information on reproductive health but are usually used for advertisement of goods sold at the market and for religious activities.2.3The study's population included female head‐porters whose ages were between 15 and 49. They are also referred to as “Kayayei” or “paa‐o‐paa” in the local dialect. They have low socioeconomic status and thus migrate to the cities in search of greener pastures. In their bid to make some income, they are compelled to carry head‐loads of goods from place to place around markets and bus stations for a fee.2.4This study included female head‐porters within the reproductive age of 15–49 years.However, female head‐porters who speak languages that cannot be understood by the research team, those who have spent less than a year in the city post‐migration, and those unable to give informed consent to participate in the study were excluded from the study. Also excluded from the study are those not certain of their age.2.5n is the minimum sample size, P is the estimated value for the proportion of contraceptive use among Ghanaian females = 18.3%,Q = 1 − P, Z = z value at 95% confidence (1.96), and d is the margin of error (0.05).Sample size was obtained using the formula:Taking into consideration 15% nonresponse rate and to increase statistical power, 280 participants were recruited for the study.2.6Ethical approval for this study was granted by the Committee on Human Research, Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology (CHRPE/SMS/KNUST) and Komfo Anokye Teaching Hospital (KATH). Consent was also sought from the Kumasi Metropolitan Assembly and the Metro Health Directorate. Moreover, individual consent was sought from each client before enrolling them as study participants.2.7Convenience sampling was used to select certain markets and residential places for the study. This sampling technique helped us locate market centers with the most intense head porterage activities. Convenience sampling also enabled the research team to identify residencies with high numbers of female head‐porters. Since there was no sampling frame for female head‐porters for random sampling, respondents were recruited consecutively till we achieved the required sample size of 280.Data were collected with semi‐structured questionnaire  for analysis and interpretation. The electronic data were strictly kept under passwords available to the principal investigator and ethics committee chairman alone to ensure confidentiality.p‐value of <0.05 was considered statistically significant.Data were summarized by descriptive statistics; for continuous variables, mean and median were determined. Proportions and percentages were used to estimate the prevalence of discrete predictors and outcome measures. Chi‐square or Fischer's square was used to determine the level of significant associations between the study variables and contraceptive use. A 33.1Of the 280 study participants included in the statistical analysis, most were between the ages of 15–19 years (37.1%). Majority of participants earned above the minimum daily wage of ≥Gh₵12.00. Most (84.3%) had no extra income activities. Half of the participants had no formal education (48.9%); 53.9% were married; 54.2% resided at Racecourse; and 45.0% had migrated from Northern, North East, or Savannah region. Moreover, the majority belonged to the Islamic religion (65.0%) and had indoor shelters  had history of contraceptive use while more than half (57.9%) had no history of contraceptive use  were somehow satisfied and 21% were unsatisfied , marital status (p < 0.0001), and religion (p = 0.029) to be significantly associated with contraceptive use among female head‐porters , income level (p = 0.201), type of shelter (p = 0.693), hospital available at area (p = 0.479), local hospital education programs (p = 0.319), and media access at home (p = 0.310) were not significantly associated with contraceptive use among female head‐porters.However, the educational level (3.4p < 0.0001), parity of participants (p < 0.0001), number of sexual partners post‐migration (p = 0.008), and age of first sex (p = 0.033) to be significantly associated with contraceptive use among female head‐porters.Table p = 0.569) and nonconsensual sexual debut (p = 0.237) were not significantly associated with contraceptive use among female head‐porters.On the contrary, sexual exploitation (4p = 0.005), marital status (p < 0.0001), and religion (p = 0.029) to be significantly associated with contraceptive use among female head‐porters. Furthermore, gravidity (p < 0.0001), parity (p < 0.0001), number of sexual partners post‐migration (p = 0.008), and age of first sex (p = 0.033) were significantly associated with contraceptive use among female head‐porters.Of the 280 participants included in this study, 202 (72%) were aware of contraception which is lower than the 99% awareness in the country according to the 2014 Ghana Demographic and Health Survey. The study found the age group of female head‐porters  who had ever used contraception were satisfied with its use. Fourteen percent had experienced sexual exploitation post‐migration. The first sexual encounter of about one‐third was nonconsensual. Nineteen percent had sex at or before 16 years. Awareness of contraception was 72%.Factors such as gravidity, parity, the number of sexual partners post‐migration, and age of first sex significantly influenced the use of contraceptives among female head‐porters.Seth Amponsah‐Tabi: Conceptualization; investigation; methodology; project administration; resources; supervision; writing—review and editing. Kwadwo Sarbeng: Supervision. Edward Dassah: Supervision; validation. Amponsah Peprah: Conceptualization; project administration; validation; writing—original draft. Gerald Owusu Asubonteng: Supervision; writing—review and editing. Frank Ankobea: Supervision; validation. Stephen Opoku: Data curation; formal analysis; investigation; writing—review and editing. Ebenezer Senu: Formal analysis; methodology; writing—review and editing. Emmanuel S. K. Morhe: Supervision; validation; writing—review and editing. Kwabena Antwi Danso: Supervision; visualization; writing—original draft.The authors declare no conflict of interest.The study was approved by the Committee on Human Research, Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology (CHRPE/SMS/KNUST) and Komfo Anokye Teaching Hospital (KATH). Consent was also sought from the Kumasi Metropolitan Assembly and the Metro Health Directorate. Moreover, individual consent was sought from each client before enrolling them as study participants.The lead author Seth Amponsah‐Tabi affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.Supporting information.Click here for additional data file.